Search results for: recombinant protein expression

Authors: Dilip Nath

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A study has been conducted on the available aquatic invertebrates in the river Dhansiri at Dimapur site. The study confirmed that the river body composed of aquatic macroinvertebrate community under two phyla viz., Arthropods and Molluscs. Total 10 species have been identified from there as the source of alternative protein food for the common people. Not only the protein source, they are also the component of aquatic food chain and indicators of aquatic ecosystem. Proper management and strategies to promote the edible invertebrates can be considered as the alternative protein and alternative income source for the common people for sustainable livelihood improvement.

Keywords: Dhansiri, Dimapur, invertebrates, livelihood improvement, protein

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Authors: Sri Rahayu, M. Dwi Susan, Aris Soewondo, W. M. Agung Pramana

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Semen is an organic fluid (seminal plasma) that contain spermatozoa. Proteins are one of the major seminal plasma components that modulate sperm functionality, influence sperm capacitation and maintaining the stability of the membrane. Semen freezing is a procedure to preserve sperm cells. The process causes decrease in sperm viability due to temperature shock and oxidation stress. Oxidation stress is a disturbance on phosphorylation that increases ROS concentration, and it produces lipid peroxide in spermatozoa membrane resulted in high MDA (malondialdehyde) concentration. The objective of this study was to examine the effect of freezing on protein and MDA profile of bovine sperm cell and seminal plasma after freezing. Protein and MDA of sperm cell and seminal plasma were isolated from 10 sample. Protein profiles was analyzed by SDS PAGE with separating gel 12,5 %. The concentration of MDA was measured by spectrophotometer. The results of the research indicated that freezing of semen cause lost of the seminal plasma proteins with molecular with 20, 10, and 9 kDa. In addition, the result research showed that protein of the sperm (26, 10, 9, 7, and 6 kDa) had been lost. There were difference MDA concentration of seminal plasma and sperm cell were increase after freezing. MDA concentration of seminal plasma before and after freezing were 2.2 and 2.4 nmol, respectively. MDA concentration of sperm cell before and after freezing were 1,5 and 1.8 nmol, respectively. In conclusion, there were differences protein profiles of spermatozoa before and after semen freezing and freezing cause increasing of the MDA concentration.

Keywords: MDA, semen freezing, SDS PAGE, protein profile

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Authors: Ali Asghar Saki, Sayed Ali Hosseini Siyar, Abbass Ashoori

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This study was conducted to evaluate the effect of Echinacea purpurea on the expression of cyclooxygenase-2 (COX-2), interleukin-17F (IL-17F) in seven-day-old broiler chickens. Four groups were fed with concentration of 0 g/kg, 5 g/kg, 10 g/kg and 20 g/kg from the root of E. purpurea in the basal diet and two other groups were only fed with the basal diet for 21 days. At the 28^th day, lipopolysaccharide (LPS, 2 mg/kg diet) was injected in four groups and the basal diet group was injected by saline as control. The chickens’ spleen RNA expression was measured for the COX-2 and IL-17F genes by Real-Time PCR. The results have shown that chickens which were fed E. purpurea had a lower COX-2 and IL-17F mRNA expression. The chickens who have received LPS only, lymphocyte was lower than other treatments. Vital organ weights were not significantly different, but body weight loss was recovered by dietary herbs inclusion. The results of this study have shown the positive effect of an anti-inflammatory herb to prevent the undesirable effect of inflammation.

Keywords: broiler chickens, Echinacea purporea, gene expression, lipopolysaccharide

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Authors: Reshu Saxena, R. K. Tripathi

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HIV-1 transmission and spread involves significant host-virus interaction. Potential targets for prevention of HIV-1 lies at the site of mucosal barriers. Thus a better understanding of how HIV-1 infects target cells at such sites and lead their invasion is required, with prime focus on the host determinants regulating HIV-1 spread. HIV-1 Nef is important for viral infectivity and pathogenicity. It promotes HIV-1 replication, facilitating immune evasion by interacting with various host factors and altering cellular pathways via multiple protein-protein interactions. In this study nef was sequenced from HIV-1 patients, and showed specific mutations revealing sequence variability in nef. To explore the difference in Nef functionality based on sequence variability we have studied the effects of HIV-1 Nef in human SupT1 T cell line and (THP-1) monocyte-macrophage cell lines through proteomics approach. 2D-Gel Electrophoresis in control and Nef-transfected SupT1 cells demonstrated several differentially expressed proteins with significant modulation of alpha-enolase. Through further studies, effects of Nef on alpha-enolase regulation were found to be cell lineage-specific, being stimulatory in macrophages/monocytes, inhibitory in T cells and without effect in HEK-293 cells. Cell migration and invasion studies were employed to determine biological function affected by Nef mediated regulation of alpha-enolase. Cell invasion was enhanced in THP-1 cells but was inhibited in SupT1 cells by wildtype nef. In addition, the modulation of enolase and cell invasion remained unaffected by a unique nef variant. These results indicated that regulation of alpha-enolase expression and invasive property of host cells by Nef is sequence specific, suggesting involvement of a particular motif of Nef. To precisely determine this site, we designed a heptapeptide including the suggested alpha-enolase regulating sequence of nef and a nef mutant with deletion of this site. Macrophages/monocytes being the major cells affected by HIV-1 at mucosal barriers, were particularly investigated by the nef mutant and peptide. Both the nef mutant and heptapeptide led to inhibition of enhanced enolase expression and increased invasiveness in THP-1 cells. Together, these findings suggest a possible mechanism of host invasion by HIV-1 through Nef mediated regulation of alpha-enolase and identifies a potential therapeutic target for HIV-1 entry at mucosal barriers.

Keywords: HIV-1 Nef, nef variants, host-virus interaction, tissue invasion

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Authors: Rabah Iratni, Husain El Hasasna, Khawlah Athamneh, Halima Al Sameri, Nehla Benhalilou, Asma Al Rashedi

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Background: Cancer therapies have witnessed great advances in the recent past, however, cancer continues to be a leading cause of death, with colorectal cancer being the fourth cause of cancer-related deaths. Colorectal cancer affects both sexes equally with poor survival rate once it metastasizes. Phytochemicals, which are plant derived compounds, have been on a steady rise as anti-cancer drugs due to the accumulation of evidences that support their potential. Here, we investigated the anticancer effect of Rhus coriaria on colon cancer cells. Material and Method: Human colon cancer HT-29 cell line was used. Protein expression and protein phosphorylation were examined using Western blotting. Transcription activity was measure using Quantitative RT-PCR. Human tumoral clonogenic assay was used to assess cell survival. Senescence was assessed by the senescence-associated beta-galactosidase assay. Results: Rhus coriaria extract (RCE) was found to significantly inhibit the viability and colony growth of human HT-29 colon cancer cells. RCE induced senescence and cell cycle arrest at G1 phase. These changes were concomitant with upregulation of p21, p16, downregulation of cyclin D1, p27, c-myc and expression of Senescence-associated-β-Galactosidase activity. Moreover, RCE induced non-canonical beclin-1independent autophagy and subsequent apoptotic cell death through activation of activation caspase 8 and caspase 7. The blocking of autophagy by 3-methyladenine (3-MA) or chloroquine (CQ) reduced RCE-induced cell death. Further, RCE induced DNA damage, reduced mutant p53 protein level and downregulated phospho-AKT and phospho-mTOR, events that preceded autophagy. Mechanistically, we found that RCE inhibited the AKT and mTOR pathway, a regulator of autophagy, by promoting the proteasome-dependent degradation of both AKT and mTOR proteins. Conclusion: Our findings provide strong evidence that Rhus coriaria possesses strong anti-colon cancer activity through induction of senescence and autophagic cell death, making it a promising alternative or adjunct therapeutic candidate against colon cancer.

Keywords: autophagy, proteasome degradation, senescence, mTOR, apoptosis, Beclin-1

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Authors: Aakansha Gupta, Neha Vadnere, Tapasvi Soni, M. Anbarsi

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Protein secondary structure prediction is one of the most important goals pursued by bioinformatics and theoretical chemistry; it is highly important in medicine and biotechnology. Some aspects of protein functions and genome analysis can be predicted by secondary structure prediction. This is used to help annotate sequences, classify proteins, identify domains, and recognize functional motifs. In this paper, we represent protein secondary structure as a mathematical model. To extract and predict the protein secondary structure from the primary structure, we require a set of parameters. Any constants appearing in the model are specified by these parameters, which also provide a mechanism for efficient and accurate use of data. To estimate these model parameters there are many algorithms out of which the most popular one is the EM algorithm or called the Expectation Maximization Algorithm. These model parameters are estimated with the use of protein datasets like RS126 by using the Bayesian Probabilistic method (data set being categorical). This paper can then be extended into comparing the efficiency of EM algorithm to the other algorithms for estimating the model parameters, which will in turn lead to an efficient component for the Protein Secondary Structure Prediction. Further this paper provides a scope to use these parameters for predicting secondary structure of proteins using machine learning techniques like neural networks and fuzzy logic. The ultimate objective will be to obtain greater accuracy better than the previously achieved.

Keywords: model parameters, expectation maximization algorithm, protein secondary structure prediction, bioinformatics

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Authors: Marawan Abd Elbaset Mohamed, Hanan A. Ogaly, Rehab F. Abdel-Rahman, Ahmed-Farid O.A., Marwa S. Khattab, Reham M. Abd-Elsalam

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Liver fibrosis is a severe worldwide health concern due to various chronic liver disorders. Hepatic encephalopathy (HE) is one of its most common complications affecting liver and brain cognitive function. Beta-Carotene (B-Car) is an organic, strongly colored red-orange pigment abundant in fungi, plants, and fruits. The study attempted to know B-Car neuroprotective potential against thioacetamide (TAA)-induced neurotoxicity and cognitive decline in HE in rats. Hepatic encephalopathy was induced by TAA (100 mg/kg, i.p.) three times per week for two weeks. B-Car was given orally (10 or 20 mg/kg) daily for two weeks after TAA injections. Organ body weight ratio, Serum transaminase activities, liver’s antioxidant parameters, ammonia, and liver histopathology were assessed. Also, the brain’s mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NF-κB), antioxidant parameters, adenosine triphosphate (ATP), adenosine monophosphate (AMP), norepinephrine (NE), dopamine (DA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) cAMP response element-binding protein (CREB) expression and B-cell lymphoma 2 (Bcl-2) expression were measured. The brain’s cognitive functions (Spontaneous locomotor activity, Rotarod performance test, Object recognition test) were assessed. B-Car prevented alteration of the brain’s cognitive function in a dose-dependent manner. The histopathological outcomes supported these biochemical evidences. Based on these results, it could be established that B-Car could be assigned to treat the brain’s neurotoxicity consequences of HE via downregualtion of MAPK/NF-κB signaling pathways.

Keywords: beta-carotene, liver injury, MAPK, NF-κB, rat, thioacetamide

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Authors: Raju Ranjha, Surbhi Aggarwal

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Background: Inflammation plays an important role in infectious and non-infectious diseases. MiRNA is also reported to play role in inflammation and associated cancers. Chemokine CXCL12 is also known to play role in inflammation and various cancers. CXCL12/CXCR4 chemokine axis was involved in pathogenesis of IBD specially UC. Supplementation of CXCL12 induces homing of dendritic cells to spleen and enhances control of plasmodium parasite in BALB/c mice. We looked at the regulation of CXCL12β by miRNA in UC colitis. Prolonged inflammation of colon in UC patient increases the risk of developing colorectal cancer. We looked at the expression differences of CXCl12β and its targeting miRNA in cancer susceptible area of colon of UC patients. Aim: Aim of this study was to find out the expression regulation of CXCL12β by miRNA in inflammation. Materials and Methods: Biopsy samples and blood samples were collected from UC patients and non-IBD controls. mRNA expression was analyzed using microarray and real-time PCR. CXCL12β targeting miRNA were looked by using online target prediction tools. Expression of CXCL12β in blood samples and cell line supernatant was analyzed using ELISA. miRNA target was validated using dual luciferase assay. Results and conclusion: We found miR-200a regulate the expression of CXCL12β in UC. Expression of CXCL12β was increased in cancer susceptible part of colon and expression of its targeting miRNA was decreased in the same part of colon. miR-200a regulate CXCL12β expression in inflammation and may be an important therapeutic target in inflammation associated cancer.

Keywords: inflammation, miRNA, regulation, CXCL12

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Authors: Mohammed Hakim Jafferali, Balaje Vijayaraghavan, Ricardo A. Figueroa, Ellinor Crafoord, Veronica J. Larsson, Einar Hallberg, Santhosh Gudise

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The nuclear envelope which surrounds the chromatin of eukaryotic cells contains more than a hundred transmembrane proteins. Mutations in some genes encoding nuclear envelope proteins give rise to human diseases including neurological disorders. The function of many nuclear envelope proteins is not well established. This is partly because nuclear envelope proteins and their interactions are difficult to study due to the inherent resistance to extraction of nuclear envelope proteins. We have developed a novel method called MCLIP, to identify interacting partners of nuclear envelope proteins in live cells. Using MCLIP, we found three new binding partners of the inner nuclear membrane protein Samp1: the intermediate filament protein Lamin B1, the LINC complex protein Sun1 and the G-protein Ran. Furthermore, using in vitro studies, we show that Samp1 binds both Emerin and Ran directly. We have also studied the interaction between Samp1 and Ran in detail. The results show that the Samp1 binds stronger to RanGTP than RanGDP. Samp1 is the first transmembrane protein known to bind Ran and it is tempting to speculate that Samp1 may provide local binding sites for RanGTP at membranes.

Keywords: MCLIP, nuclear envelope, ran, Samp1

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Authors: Sehmus Aslan, Sibel Karacaoglu, Cengiz Sevgin, Ummuhan Bas Aslan

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Objective: Anger expression style is an important moderator of the effects on the person and person’s environment. Anger and anger expression have become important constructs in identifying individuals at high risk for psychological difficulties. To our knowledge, there is no information about anger and anger expression of adolescents with visual impairment. The aim of this study was to compare anger and anger expression among adolescents with and without visual impairment. Methods: Thirty-eight adolescents with visual impairment (18 female, 20 male) and 44 adolescents without visual impairment (22 female, 24 male), in totally 84 adolescents aged between 12 to 15 years, participated in the study. Anger and anger expression of the participants assessed with The State-Trait Anger Scale (STAS). STAS, a self-report questionnaire, is designed to measure the experience and expression of anger. STAS has four subtitles including continuous anger, anger in, anger out and anger control. Reliability and validity of the STAS have been well established among adolescents. Mann-Whitney U Test was used for statistical analysis. Results: No significant differences were found in the scores of continuous anger and anger out between adolescents with and without visual impairment (p < 0.05). On the other hand, there were differences in scores of anger control and anger in between adolescents with and without visual impairment (p>0.05). The score of anger control in adolescents with visual impairment were higher compared with adolescents without visual impairment. Meanwhile, the adolescents with visual impairment had lower score for anger in compared with adolescents without visual impairment. Conclusions: The results of this study suggest that there is no difference in anger level among adolescents with and without visual impairment meanwhile there is difference in anger expression.

Keywords: adolescent, anger, impaired, visual

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Authors: Yi-Hui Lai, Chih-Hsiang Yang, Li-Chung Hsu

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The inflammasome is a protein complex that modulates caspase-1 activity, resulting in proteolytic cleavage of proinflammatory cytokines such as IL-1β and IL-18, into their bioactive forms. It has been shown that the inflammasomes play a crucial role in the clearance of pathogenic infection and tissue repair. However, dysregulated inflammasome activation contributes to a wide range of human diseases such as cancers and auto-inflammatory diseases. Yet, regulation of NLRP3 inflammasome activation remains largely unknown. We discovered a novel DEAD box protein, whose biological function has not been reported, not only negatively regulates NLRP3 inflammasome activation by interfering NLRP3 inflammasome assembly and cellular localization but also mitigate pyroptosis upon pathogen evasion. The DEAD-box protein is the first DEAD-box protein gets involved in modulation of the inflammasome activation. In our study, we found that caspase-1 activation and mature IL-1β production were largely enhanced upon LPS challenge in the DEAD box-containing protein- deleted THP-1 macrophages and bone marrow-derived macrophages (BMDMs). In addition, this DEAD box-containing protein migrates from the nucleus to the cytoplasm upon LPS stimulation, which is required for its inhibitory role in NLRP3 inflammasome activation. The DEAD box-containing protein specifically interacted with the LRR motif of NLRP3 via its DEAD domain. Furthermore, due to the crucial role of the NLRP3 LRR domain in the recruitment of NLRP3 to mitochondria and binding to its adaptor ASC, we found that the interaction of NLRP3 and ASC was downregulated in the presence of the DEAD box-containing protein. In addition to the mechanical study, we also found that this DEAD box protein protects host cells from inflammasome-triggered cell death in response to broad-ranging pathogens such as Candida albicans, Streptococcus pneumoniae, etc., involved in nosocomial infections and severe fever shock. Collectively, our results suggest that this novel DEAD box molecule might be a key therapeutic strategy for various infectious diseases.

Keywords: inflammasome, inflammation, innate immunity, pyroptosis

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Authors: Samina Jam Nazeer Ahmad, Samia Yasin, Ijaz Ahmad, Muhammad Tahir, Jam Nazeer Ahmad

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Phytoplasmas are phloem-inhibited plant pathogenic bacteria that are transferred by insect vectors. Among biotic factors, Phytoplasma infection induces abnormality influencing the physiology as well as morphology of plants. In 16Sr-IX group phytoplasma-infected brassica compestris, flower abnormalities have been associated with changes in the expression of floral development genes. To determine whether methylation was involved in down-regulation of flower development, the process of DNA methylation and Demethylation was investigated as a possible mechanism for regulation of floral gene expression in phytoplasma infected Brassica transmitted by Orosious orientalis vector by using RT-PCR, MSRE-PCR, Southern blotting, Bisulfite Sequencing, etc. Transcriptional expression of methylated genes was found to be globally down-regulated in plants infected with phytoplasma, but not severely in those infested by insect vectors and variation in expression was found in genes involved in methylation. These results also showed that genes particularly orthologous to Arabidopsis APETALA3 involved in petal formation and flower development was down-regulated severely in phytoplasma-infected brassica and with the fact that phytoplasma and insect induce variation in developmental gene expression. The DNA methylation status of flower developmental gene in phytoplasma infected plants with 5-azacytidine restored gene expression strongly suggesting that DNA methylation was involved in down-regulation of floral development genes in phytoplasma infected brassica.

Keywords: genes expression, phytoplasma, DNA methylation, flower development

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Authors: Jamerson Felipe Pereira Lima, Jeane Cecília Bezerra de Melo

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The difficulty and cost related to obtaining of protein tertiary structure information through experimental methods, such as X-ray crystallography or NMR spectroscopy, helped raising the development of computational methods to do so. An approach used in these last is prediction of tridimensional structure based in the residue chain, however, this has been proved an NP-hard problem, due to the complexity of this process, explained by the Levinthal paradox. An alternative solution is the prediction of intermediary structures, such as the secondary structure of the protein. Artificial Intelligence methods, such as Bayesian statistics, artificial neural networks (ANN), support vector machines (SVM), among others, were used to predict protein secondary structure. Due to its good results, artificial neural networks have been used as a standard method to predict protein secondary structure. Recent published methods that use this technique, in general, achieved a Q3 accuracy between 75% and 83%, whereas the theoretical accuracy limit for protein prediction is 88%. Alternatively, to achieve better results, support vector machines prediction methods have been developed. The statistical evaluation of methods that use different AI techniques, such as ANNs and SVMs, for example, is not a trivial problem, since different training sets, validation techniques, as well as other variables can influence the behavior of a prediction method. In this study, we propose a prediction method based on artificial neural networks, which is then compared with a selected SVM method. The chosen SVM protein secondary structure prediction method is the one proposed by Huang in his work Extracting Physico chemical Features to Predict Protein Secondary Structure (2013). The developed ANN method has the same training and testing process that was used by Huang to validate his method, which comprises the use of the CB513 protein data set and three-fold cross-validation, so that the comparative analysis of the results can be made comparing directly the statistical results of each method.

Keywords: artificial neural networks, protein secondary structure, protein structure prediction, support vector machines

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Authors: H. Haseena Banu, D. Karthick, R. Stalin, E. Nandha Kumar, T. P. Sachidanandam, P. Shanthi

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Background of the study: Type 2 diabetes is emerging as the predominant metabolic disorder in the world among adults characterized mainly by the resistance of the insulin sensitive tissues towards insulin followed by the decrease in the insulin secretion. The treatment for this disease usually involves treatment with oral synthetic drugs which are known to cause several side effects. Therefore, identification of new biomarkers as therapeutic target is the need of the hour. miRNAs are small, non–protein-coding RNAs that negatively regulate gene expression by promoting degradation and/or inhibit the translation of target mRNAs and have emerged as biomarkers in predicting diabetes mellitus. Objective of the study: To elucidate the therapeutic role of gallic acid in modulating the alterations in glucose metabolism induced by miRNAs 194 and 135a in Type 2 diabetic rats. Materials and Methods: T2D was induced in rats by feeding them with a high fat diet for 2 weeks followed by intraperitoneal injection of 35 mg/kg/body weight (b.wt.) of streptozotocin. Microarrays were used to assess the expression of miRNAs in control, diabetic and gallic acid treated rats. Gene expression studies were carried out by RT PCR analysis. Results: Forty one miRNAs were differentially expressed in Type 2 diabetic rats. Among these, the expression of miRNA 194 was significantly decreased whereas miRNA 135a was significantly increased in Type 2 diabetic rats. The glucose metabolism was also altered significantly in skeletal muscle of Type 2 diabetic rats. Conclusion: T2D is associated with alterations in the expression of miRNAs in skeletal muscle. Both these miRNAs 194 and 135a play an important role in glucose metabolism in skeletal muscle of diabetic rats. Gallic acid effectively ameliorated the alterations in glucose metabolism. Hence, both these miRNAs can serve as biomarkers and therapeutic targets in diabetes mellitus. The study also establishes the role of gallic acid as therapeutic agent. Acknowledgment: The financial assistance provided in the form of ICMR women scientist by ICMR DHR INDIA is gratefully acknowledged here.

Keywords: gallic acid, high fat diet, type 2 diabetes mellitus, miRNAs

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Authors: Viviane A. O. Silva, Angela M. Costa, Glaucia N. M. Hajj, Ana Preto, Aline Tansini, Martin Roffé, Peter Jordan, Rui M. Reis

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Autophagy is a recycling and degradative system suggested to be a major cell death pathway in cancer cells. Autophagy pathway is interconnected with the endocytosis pathways sharing the same ultimate lysosomal destination. Lysosomes are crucial regulators of cell homeostasis, responsible to downregulate receptor signalling and turnover. It seems highly likely that derailed endocytosis can make major contributions to several hallmarks of cancer. WNK2, a member of the WNK (with-no-lysine [K]) subfamily of protein kinases, had been found downregulated by its promoter hypermethylation, and has been proposed to act as a specific tumour-suppressor gene in brain tumors. Although some contradictory studies indicated WNK2 as an autophagy modulator, its role in cancer cell death is largely unknown. There is also growing evidence for additional roles of WNK kinases in vesicular traffic. Aim: To evaluate the role of WNK2 in autophagy and endocytosis on glioma context. Methods: Wild-type (wt) A172 cells (WNK2 promoter-methylated), and A172 transfected either with an empty vector (Ev) or with a WNK2 expression vector, were used to assess the cellular basal capacities to promote autophagy, through western blot and flow-cytometry analysis. Additionally, we evaluated the effect of WNK2 on general endocytosis trafficking routes by immunofluorescence. Results: The re-expression of ectopic WNK2 did not interfere with autophagy-related protein light chain 3 (LC3-II) expression levels as well as did not promote mTOR signaling pathway alteration when compared with Ev or wt A172 cells. However, the restoration of WNK2 resulted in a marked increase (8 to 92,4%) of Acidic Vesicular Organelles formation (AVOs). Moreover, our results also suggest that WNK2 cells promotes delay in uptake and internalization rate of cholera toxin B and transferrin ligands. Conclusions: The restoration of WNK2 interferes in vesicular traffic during endocytosis pathway and increase AVOs formation. This results also suggest the role of WNK2 in growth factor receptor turnover related to cell growth and homeostasis and associates one more time, WNK2 silencing contribution in genesis of gliomas.

Keywords: autophagy, endocytosis, glioma, WNK2

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Authors: Hirowati Ali, Aisyah Ellyanti, Dewi Rusnita, Septelia Inawati Wanandi

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Stem cell has been known for its potency to be differentiated in many cells. Recently stem cell has been used for many treatment of degenerative medicine. It is still controversy whether stem cell can be used for therapy or these cells can activate cancer stem cell. SCUBE2 is a novel secreted and membrane-anchored protein which has been reported to its role in better prognosis and inhibition of cancer cell proliferation. Our study aims to observe whether stem cell can up-regulate SCUBE2 gene in MCF7 breast cancer cell line. We used in vitro study using MCF-7 cell treated with stem cell derived from placenta Wharton's jelly which has been known for its stemness and widely used. Our results showed that MCF-7 cell line grows up rapidly in 6-well culture dish. Stem cell was cultured in 6-well dish. After 50%-60% MCF-7 confluence, we co-cultured these cells with stem cells for 24 hours and 48 hours. We hypothesize SCUBE2 gene which is previously known for its higher expression in better prognosis of breast cancer, is up-regulated after stem cells addition in MCF7 culture dishes.

Keywords: breast cancer cells, inhibition of cancer cells, mesenchymal stem cells, SCUBE2

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Authors: Ekta Yadav, Sukanta Bhattacharya, Brijesh Yadav, Ariel Hus, Jagjit Yadav

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Background and Significance: Respiratory exposure to toxicants (chemicals or particulates) causes disruption of lung homeostasis leading to lung toxicity/injury manifested as pulmonary inflammation, edema, and/or other effects depending on the type and extent of exposure. This emphasizes the need for investigating toxicant type-specific mechanisms to understand therapeutic targets. Aquaporins, aka water channels, are known to play a role in lung homeostasis. Particularly, the two major lung aquaporins AQP5 and AQP1 expressed in alveolar epithelial and vasculature endothelia respectively allow for movement of the fluid between the alveolar air space and the associated vasculature. In view of this, the current study is focused on understanding the regulation of lung aquaporins and other targets during inhalation exposure to toxic chemicals (Cigarette smoke chemicals) versus toxic particles (Carbon nanoparticles) or co-exposures to understand their relevance as markers of injury and intervention. Methodologies: C57BL/6 mice (5-7 weeks old) were used in this study following an approved protocol by the University of Cincinnati Institutional Animal Care and Use Committee (IACUC). The mice were exposed via oropharyngeal aspiration to multiwall carbon nanotube (MWCNT) particles suspension once (33 ugs/mouse) followed by housing for four weeks or to Cigarette smoke Extract (CSE) using a daily dose of 30µl/mouse for four weeks, or to co-exposure using the combined regime. Control groups received vehicles following the same dosing schedule. Lung toxicity/injury was assessed in terms of homeostasis changes in the lung tissue and lumen. Exposed lungs were analyzed for transcriptional expression of specific targets (AQPs, surfactant protein A, Mucin 5b) in relation to tissue homeostasis. Total RNA from lungs extracted using TRIreagent kit was analyzed using qRT-PCR based on gene-specific primers. Total protein in bronchoalveolar lavage (BAL) fluid was determined by the DC protein estimation kit (BioRad). GraphPad Prism 5.0 (La Jolla, CA, USA) was used for all analyses. Major findings: CNT exposure alone or as co-exposure with CSE increased the total protein content in the BAL fluid (lung lumen rinse), implying compromised membrane integrity and cellular infiltration in the lung alveoli. In contrast, CSE showed no significant effect. AQP1, required for water transport across membranes of endothelial cells in lungs, was significantly upregulated in CNT exposure but downregulated in CSE exposure and showed an intermediate level of expression for the co-exposure group. Both CNT and CSE exposures had significant downregulating effects on Muc5b, and SP-A expression and the co-exposure showed either no significant effect (Muc5b) or significant downregulating effect (SP-A), suggesting an increased propensity for infection in the exposed lungs. Conclusions: The current study based on the lung toxicity mouse model showed that both toxicant types, particles (CNT) versus chemicals (CSE), cause similar downregulation of lung innate defense targets (SP-A, Muc5b) and mostly a summative effect when presented as co-exposure. However, the two toxicant types show differential induction of aquaporin-1 coinciding with the corresponding differential damage to alveolar integrity (vascular permeability). Interestingly, this implies the potential of AQP1 as a differential marker of toxicant type-specific lung injury.

Keywords: aquaporin, gene expression, lung injury, toxicant exposure

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Authors: J. Suwanprateeb, F. Thammarakcharoen

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Calcium phosphate coating (CaP) has been employed for protein delivery, but the typical direct protein adsorption on the coating led to low incorporation content and fast release of the protein from the coating. By using bovine serum albumin (BSA) as a model protein, rapid biomimetic co-precipitation between calcium phosphate and BSA was employed to control the distribution of BSA within calcium phosphate coating during biomimetic formation on titanium surface for only 6 h at 50 oC in an accelerated calcium phosphate solution. As a result, the amount of BSA incorporation and release duration could be increased by using a rapid biomimetic co-precipitation technique. Up to 43 fold increases in the BSA incorporation content and the increase from 6 h to more than 360 h in release duration compared to typical direct adsorption technique were observed depending on the initial BSA concentration used during co-precipitation (1, 10, and 100 microgram/ml). From X-ray diffraction and Fourier transform infrared spectroscopy studies, the coating composition was not altered with the incorporation of BSA by this rapid biomimetic co-precipitation and mainly comprised octacalcium phosphate and hydroxyapatite. However, the microstructure of calcium phosphate crystals changed from straight, plate-like units to curved, plate-like units with increasing BSA content.

Keywords: biomimetic, Calcium Phosphate Coating, protein, titanium

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Authors: Alireza Naseri, Susan L. Holdt, Charlotte Jacobsen

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In 2014, the global amount of carrageenan production was 60,000 ton with a value of US$ 626 million. From this number, it can be estimated that the total dried seaweed consumption for this production was at least 300,000 ton/year. The protein content of these types of seaweed is 5 – 25%. If just half of this total amount of protein could be extracted, 18,000 ton/year of a high-value protein product would be obtained. The overall aim of this study was to develop a technology that will ensure further utilization of the seaweed that is used only as raw materials for carrageenan production as single extraction at present. More specifically, proteins should be extracted from the seaweed either before or after extraction of carrageenan with focus on maintaining the quality of carrageenan as a main product. Different mechanical, chemical and enzymatic technologies were evaluated. The optimized process was implemented in lab scale and based on its results; the new experiments were done a pilot and larger scale. In order to calculate the efficiency of the new upstream multi-extraction process, protein content was tested before and after extraction. After this step, the extraction of carrageenan was done and carrageenan content and the effect of extraction on yield were evaluated. The functionality and quality of carrageenan were measured based on rheological parameters. The results showed that by using the new multi-extraction process (submitted patent); it is possible to extract almost 50% of total protein without any negative impact on the carrageenan quality. Moreover, compared to the routine carrageenan extraction process, the new multi-extraction process could increase the yield of carrageenan and the rheological properties such as gel strength in the final carrageenan had a promising improvement. The extracted protein has initially been screened as a plant protein source in typical food applications. Further work will be carried out in order to improve properties such as color, solubility, and taste.

Keywords: carrageenan, extraction, protein, seaweed

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Authors: Ishani Majumdar, Jaishree Paul

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Introduction: Ulcerative Colitis (UC) is an inflammatory disease of the colon resulting from an autoimmune response towards individual’s own microbiota. Excessive inflammation is characterized by hyper-activation of NFkB, a transcription factor regulating expression of various pro-inflammatory genes. The ubiquitin editing complex consisting of TNFAIP3, ITCH, RNF11 and TAX1BP1 maintains homeostatic levels of active NFkB through feedback inhibition and assembles in response to various stimuli that activate NFkB. TNFAIP3 deubiquitinates key signaling molecules involved in NFkB activation pathway. ITCH, RNF11 and TAX1BP1 provide substrate specificity, acting as adaptors for TNFAIP3 function. Aim: This study aimed to find expression of members of the ubiquitin editing complex at the transcript level in inflamed colon tissues of UC patients. Materials and Methods: Colonic biopsy samples were collected from 30 UC patients recruited at Department of Gastroenterology, AIIMS (New Delhi). Control group (n= 10) consisted of individuals undergoing examination for functional disorders. Real Time PCR was used to determine relative expression with GAPDH as housekeeping gene. Results: Expression of members of the ubiquitin editing complex was significantly altered during active disease. Expression of TNFAIP3 was upregulated while concomitant decrease in expression of ITCH, RNF11, TAX1BP1 was seen in UC patients. Discussion: This study reveals that increase in expression of TNFAIP3 was unable to control inflammation during active UC. Further, insufficient upregulation of ITCH, RNF11, TAX1BP1 may limit the formation of the ubiquitin complex and contribute to pathogenesis of UC.

Keywords: altered expression, inflammation, ubiquitin editing complex, ulcerative colitis

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Authors: Giorgio Bertolazzi, Panayiotis Benos, Michele Tumminello, Claudia Coronnello

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MicroRNAs are small non-coding RNAs that post-transcriptionally regulate the expression levels of messenger RNAs. MicroRNA regulation activity depends on the recognition of binding sites located on mRNA molecules. ComiR (Combinatorial miRNA targeting) is a user friendly web tool realized to predict the targets of a set of microRNAs, starting from their expression profile. ComiR incorporates miRNA expression in a thermodynamic binding model, and it associates each gene with the probability of being a target of a set of miRNAs. ComiR algorithms were trained with the information regarding binding sites in the 3’UTR region, by using a reliable dataset containing the targets of endogenously expressed microRNA in D. melanogaster S2 cells. This dataset was obtained by comparing the results from two different experimental approaches, i.e., inhibition, and immunoprecipitation of the AGO1 protein; this protein is a component of the microRNA induced silencing complex. In this work, we tested whether including coding region binding sites in the ComiR algorithm improves the performance of the tool in predicting microRNA targets. We focused the analysis on the D. melanogaster species and updated the ComiR underlying database with the currently available releases of mRNA and microRNA sequences. As a result, we find that the ComiR algorithm trained with the information related to the coding regions is more efficient in predicting the microRNA targets, with respect to the algorithm trained with 3’utr information. On the other hand, we show that 3’utr based predictions can be seen as complementary to the coding region based predictions, which suggests that both predictions, from 3'UTR and coding regions, should be considered in a comprehensive analysis. Furthermore, we observed that the lists of targets obtained by analyzing data from one experimental approach only, that is, inhibition or immunoprecipitation of AGO1, are not reliable enough to test the performance of our microRNA target prediction algorithm. Further analysis will be conducted to investigate the effectiveness of the tool with data from other species, provided that validated datasets, as obtained from the comparison of RISC proteins inhibition and immunoprecipitation experiments, will be available for the same samples. Finally, we propose to upgrade the existing ComiR web-tool by including the coding region based trained model, available together with the 3’UTR based one.

Keywords: AGO1, coding region, Drosophila melanogaster, microRNA target prediction

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Authors: N. Umasuthan, S. D. N. K. Bathige, W. S. Thulasitha, I. Whang, J. Lee

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The MyD88 is an evolutionarily conserved host-expressed adaptor protein that is essential for proper TLR/ IL1R immune-response signaling. A previously identified complete cDNA (1626 bp) of OfMyD88 comprised an ORF of 867 bp encoding a protein of 288 amino acids (32.9 kDa). The gDNA (3761 bp) of OfMyD88 revealed a quinquepartite genome organization composed of 5 exons (with the sizes of 310, 132, 178, 92 and 155 bp) separated by 4 introns. All the introns displayed splice signals consistent with the consensus GT/AG rule. A bipartite domain structure with two domains namely death domain (24-103) coded by 1st exon, and TIR domain (151-288) coded by last 3 exons were identified through in silico analysis. Moreover, homology modeling of these two domains revealed a similar quaternary folding nature between human and rock bream homologs. A comprehensive comparison of vertebrate MyD88 genes showed that they possess a 5-exonic structure. In this structure, the last three exons were strongly conserved, and this suggests that a rigid structure has been maintained during vertebrate evolution. A cluster of TATA box-like sequences were found 0.25 kb upstream of cDNA starting position. In addition, putative 5'-flanking region of OfMyD88 was predicted to have TFBS implicated with TLR signaling, including copies of NFB1, APRF/ STAT3, Sp1, IRF1 and 2 and Stat1/2. Using qPCR technique, a ubiquitous mRNA expression was detected in liver and blood. Furthermore, a significantly up-regulated transcriptional expression of OfMyD88 was detected in head kidney (12-24 h; >2-fold), spleen (6 h; 1.5-fold), liver (3 h; 1.9-fold) and intestine (24 h; ~2-fold) post-Fla challenge. These data suggest a crucial role for MyD88 in antibacterial immunity of teleosts.

Keywords: MyD88, innate immunity, flagellin, genomic analysis

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Authors: Shih-Ping Liu, Cheng-Hsuan Chang, Yu-Chuen Huang, Shih-Yin Chen, Woei-Cherng Shyu

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Embryonic stem cells (ES) and induced pluripotnet stem cells (iPS) are both pluripotent stem cells. For mouse stem cells culture technology, leukemia inhibitory factor (LIF) was used to maintain the pluripotency of stem cells in vitro. However, LIF is an expensive reagent. The goal of this study was to find out a pure compound extracted from Chinese herbal medicine that could maintain stem cells pluripotency to replace LIF and improve the iPS generation efficiency. From 20 candidates traditional Chinese medicine we found that Cordycepsmilitaris triggered the up-regulation of stem cells activating genes (Oct4 and Sox2) expression levels in MEF cells. Cordycepin, a major active component of Cordycepsmilitaris, also could up-regulate Oct4 and Sox2 gene expression. Furthermore, we used ES and iPS cells and treated them with different concentrations of Cordycepin (replaced LIF in the culture medium) to test whether it was useful to maintain the pluripotency. The results showed higher expression levels of several stem cells markers in 10 μM Cordycepin-treated ES and iPS cells compared to controls that did not contain LIF, including alkaline phosphatase, SSEA1, and Nanog. Embryonic body formation and differentiation confirmed that 10 μM Cordycepin-containing medium was capable to maintain stem cells pluripotency after four times passages. For mechanism analysis, microarray analysis indicated extracellular matrix and Jak/Stat signaling pathway as the top two deregulated pathways. In ECM pathway, we determined that the integrin αVβ5 expression levels and phosphorylated Src levels increased after Cordycepin treatment. In addition, the phosphorylated Jak2 and phosphorylated Sat3 protein levels were increased after Cordycepin treatment and suppressed with the Jak2 inhibitor, AG490. The expression of cytokines associated with Jak2/Stat3 signaling pathway were also up-regulated by Q-PCR and ELISA assay. Lastly, we used Oct4-GFP MEF cells to test iPS generation efficiency following Cordycepin treatment. We observed that 10 Μm Cordycepin significantly increased the iPS generation efficiency in day 21. In conclusion, we demonstrated Cordycepin could maintain the pluripotency of stem cells through both of ECM and Jak2/Stat3 signaling pathway and improved iPS generation efficiency.

Keywords: cordycepin, iPS cells, Jak2/Stat3 signaling pathway, molecular biology

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Authors: Shenghu Feng, Jun Cheng

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The molecular mechanism underlying nonalcoholic fatty liver disease (NAFLD) progression to hepatocellular carcinoma (HCC) remains unknown. In this study, immunohistochemistry staining result showed that NS5ABP37 protein expression decreased as with increasing degree of HCC malignancy. In agreement, NS5ABP37 protein overexpression significantly suppressed cell proliferation, caused G1/S cell cycle arrest, and induced apoptosis by increasing caspase-3/7 activity and cleaved caspase-3 levels. In addition, NS5ABP37 overexpression resulted in decreased intracellular TG and TC contents, with level reduction in SREBPs and downstream effectors. Furthermore, NS5ABP37 overexpression decreased SREBP1c and SREBP2 levels by inducing their respective promoters. Finally, ROS levels and ER-stress were both induced by NS5ABP37 overexpression. These findings together demonstrate that NS5ABP37 inhibits cancer cell proliferation and promotes apoptosis, by altering SREBP-dependent lipogenesis and cholesterogenesis in HepG2 cells and inducing oxidative stress and ER stress.

Keywords: NS5ABP37, liver cancer, lipid metabolism, oxidative stress, ER stress

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Authors: Fernanda Gonçalves, Karla Alcântara, Vanessa Moura, Patrícia Nolasco, Jorge Kalil, Maristela Hernandez

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Introduction: Atherosclerosis is a chronic disease where two striking features are observed: retention of lipids and inflammation. Understanding the interaction between immune cells and lipoproteins involved in atherogenesis are urgent challenges, since cardiovascular diseases are the leading cause of death worldwide. Macrophages are critical to the development of atherosclerotic plaques and in the perpetuation of inflammation in these lesions. These cells are also directly involved in unstable plaque rupture. Recently different populations of macrophages are being identified in atherosclerotic lesions. Although the presence of M2 macrophages (macrophages activated by the alternative pathway, eg. The IL-4) has been identified, the function of these cells in atherosclerosis is not yet defined. M2 macrophages have a high endocytic capacity, they promote remodeling of tissues and to have anti-inflammatory activity. However, in atherosclerosis, especially unstable plaques, severe inflammatory reaction, accumulation of cellular debris and intense degradation of the tissue is observed. Thus, it is possible that the M2 macrophages have altered function (phenotype) in atherosclerosis. Objective: Our aim is to evaluate if the presence of oxidized LDL alters the phenotype and function of M2 macrophages in vitro. Methods: For this, we will evaluate whether the addition of lipoprotein in M2 macrophages differentiated in vitro with IL -4 induces 1) a reduction in the secretion of anti-inflammatory cytokines (CBA and ELISA), 2) secretion of inflammatory cytokines (CBA and ELISA), 3) expression of cell activation markers (Flow cytometry), 4) alteration in gene expression of molecules adhesion and extracellular matrix (Real-Time PCR) and 5) Matrix degradation (confocal microscopy). Results: In oxLDL stimulated M2 macrophages cultures we did not find any differences in the expression of the cell surface markers tested, including: HLA-DR, CD80, CD86, CD206, CD163 and CD36. Also, cultures stimulated with oxLDL had similar phagocytic capacity when compared to unstimulated cells. However, in the supernatant of these cultures an increase in the secretion of the pro-inflammatory cytokine IL-8 was detected. No significant changes where observed in IL-6, IL-10, IL-12 and IL-1b levels. The culture supernatant also induced massive extracellular matrix (produced by mouse embryo fibroblast) filaments degradation. When evaluating the expression of 84 extracellular matrix and adhesion molecules genes, we observed that the stimulation of oxLDL in M2 macrophages decreased 47% of the genes and increased the expression of only 3% of the genes. In particular we noted that oxLDL inhibit the expression of 60% of the genes constituents of extracellular matrix and collagen expressed by these cells, including fibronectin1 and collagen VI. We also observed a decrease in the expression of matrix protease inhibitors, such as TIMP 2. On the opposite, the matricellular protein thrombospondin had a 12 fold increase in gene expression. In the presence of native LDL 90% of the genes had no altered expression. Conclusion: M2 macrophages stimulated with oxLDL secrete the pro-inflammatory cytokine IL-8, have an altered extracellular matrix constituents gene expression, and promote the degradation of extracellular matrix. M2 macrophages may contribute to the perpetuation of inflammation in atherosclerosis and to plaque rupture.

Keywords: atherosclerosis, LDL, macrophages, m2

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Authors: Don Buddika Oshadi Malaweera, Lora Harris, Bruce Rathgeber, Chibuike C. Udenigwe, Kirsti Rouvinen-Watt

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Fatty liver disease is among the three most severe health concerns for mink and believed to occur through the same mechanism as nursing sickness. In North America, nursing sickness affects about 45% of mink farms and in Canada, approximately 50,000 mink females is affected annually. Orotic acid (OA) plays a critical role in lipid metabolism and can increase hepatic lipids by enhancing Sterol regulatory element binding protein-1c expression and decreasing Carnitine palmitoyl transferase I activity. This study was conducted to identify particular pathways and regulatory control points involved in fatty liver development, and evaluate the effectiveness of arginine and bioactive peptides for prevention and treatment of fatty liver disease in mink. A total of 45 mink were used in 9 treatments. The experimental diets consisted of 1% OA, 2% L-arginine and 5% of whey protein hydrolysates. At the end of 10 days of experimental period, the mink were anaesthetized, sampled for blood and euthanized, samples were obtained for histological, biochemical and molecular assays. The blood samples will be analyzed for clinical chemistry and triacylglycerol. The liver samples will be analyzed for total lipid content and analyzed for 6 genes of interest involved in adipogenic transformation, ER stress, and liver inflammation.

Keywords: fatty liver, L-arginine, mink, orotic acid, whey protein hydrolysates

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Authors: Sheng-Hui Lan, Shan-Ying Wu, Shu-Ching Lin, Wei-Chen Wang, Hsiao-Sheng Liu

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Autophagy is an essential mechanism to maintain cellular homeostasis through its degradation function, and the autophagy deficiency is related various diseases including tumorigenesis in several cancers. MicroRNAs (miRNAs) are small none coding RNAs, which regulate gene expression through degradation of mRNA or inhibition of translation. However, the relationship between autophagy deficiency and dysregulated miRNAs is still unclear. We revealed a mechanism that autophagy up-regulates miR-449a expression at the transcriptional level through activation of forkhead transcription factor family member FoxO1 and then suppresses tumorigenesis in CRC. Our data showed that the autophagic activity and miR-449a expression were lower in colorectal cancer (CRC) and has a positive correlation. We further reveal that autophagy degrades p300 expression and then suppresses acetylation of FoxO1. Under autophagic induction conditions, FoxO1 is transported from the cytoplasm to the nucleus and binds to the miR-449a promoter and then promotes miR-449a expression. In addition, either miR-449a overexpression or amiodarone-induced autophagy inhibits cell cycle progression, proliferation, colony formation migration, invasion, and tumor formation of SW480 cells. Our findings indicate that autophagy inducers may have the potential to be used for prevention and treatment of CRC through upregulation of miR-449a expression.

Keywords: autophagy, MiR-449a, FoxO1, colorectal cancer

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Authors: Ji-Hyon Kil, Kew-Cheol Shim, Kyoung-Ae Park, Kyoungho Kim

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Ambrosia trifida L. is designated as invasive alien species by the Act on the Conservation and Use of Biodiversity by the Ministry of Environment, Korea. The purpose of present paper was to investigate the inhibitory effects of aqueous extracts of A.trifida on the development of root hairs of Triticum aestivum L., and Allium tuberosum Rottler ex Spreng and the electrophoretic protein patterns of their radicles. The development of root hairs was inhibited by increasing of aqueous extract concentrations. Through SDS-PAGE, the electrophoretic protein bands of extracted proteins from their radicles were appeared in controls, but protein bands of specific molecular weight disappeared or weakened in treatments. In conclusion, inhibitory effects of A. trifida made two receptor species changed morphologically, and at the molecular level in early growth stage.

Keywords: Ambrosia trifida L., invasive alien species, inhibitory effect, root hair, electrophoretic protein, radicle

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Authors: Mohammed I.A. Al-Neemi, Mohammed S.B., Al-Hlawee, Ilham N. Ezaddin, Soz A. Faris, Omer E. Fakhry, Heemen S. Mageed

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This objective of this study was to measure the effect of replacing different levels of animal protein concentrate with Red Tiger shrimp meal (RTSM: 60 % crude protein, 2400 M.E kcal/kg and the source of RTSM was imported from china) in the broiler starter diets. A total 300 broiler chicks (Ross-308) were randomly assigned in treatments dietary contained three different levels of RTSM (0.00, 4.16 and 8.32 %) in experimental diet with a completely randomized design (CRD). Each treatment included four replicates (floor pens) and 25 broilers in each replication (Pen). Therefore, floor space for each boilers was 900 cm2. Initially, the broilers where exposed to a continues lighting of 23:30 hours and dark period of 30 minutes in each 24 hours. Feed and water were supplied ad libitum to the broilers throughout the experimental period (1-21 days). The results of this study indicated that body weight (B.W.), body weight gain (B.W.G), conversion ratio of feed, protein and energy (F.CR, P.C.R and E.C.R) were significantly (p ≤ 0.05) decreased by complete substituting (RTSM) for animal protein concentration (third treatment). Mortality percentage significantly (p ≤ 0.05) increased for third dietary treatment. No significant differences were found for feed, protein and energy intake among treatments during the experimental period (three weeks). In conclusion, (RTSM) could be included to 4.16% in the broiler starter diet or substitute the protein Red Tiger shrimp as alternative of protein animal protein concentrate as much as 50%.

Keywords: red tiger shrimp, broiler, starter diet, growth performance, animal protein concentrate

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Authors: Jienny Lee, In-Soo Cho, Sang-Ho Cha

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Adult mesenchymal stem cells (MSCs) have been investigated using preclinical approaches for tissue regeneration. Porcine MSCs (pMSCs) are capable of growing and attaching to plastic with a fibroblast-like morphology and then differentiating into bone, adipose, and cartilage tissues in vitro. This study was conducted to investigate the proliferating abilities, differentiation potentials, and multipotency of miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with or without long-term cryopreservation, considering that cryostorage has the potential for use in clinical applications. After confirming the characteristics of the mpAD-MSCs, we examined the effect of long-term cryopreservation (> 2 years) on expression of cell surface markers (CD34, CD90 and CD105), proliferating abilities (cumulative population doubling level, doubling time, colony-forming unit, and MTT assay) and differentiation potentials into mesodermal cell lineages. As a result, the expression of cell surface markers is similar between thawed and fresh mpAD-MSCs. However, long-term cryopreservation significantly lowered the differentiation potentials (adipogenic, chondrogenic, and osteogenic) of mpAD-MSCs. When compared with fresh mpAD-MSCs, thawed mpAD-MSCs exhibited lower expression of mesodermal cell lineage-related genes such as peroxisome proliferator-activated receptor-g2, lipoprotein lipase, collagen Type II alpha 1, osteonectin, and osteocalcin. Interestingly, long-term cryostoraged mpAD-MSCs exhibited significantly higher cell viability than the fresh mpAD-MSCs. Long-term cryopreservation induced a 30% increase in the cell viability of mpAD-MSCs when compared with the fresh mpAD-MSCs at 5 days after thawing. However, long-term cryopreservation significantly lowered expression of stemness markers such as Oct3/4, Sox2, and Nanog. Furthermore, long-term cryopreservation negatively affected expression of senescence-associated genes such as telomerase reverse transcriptase and heat shock protein 90 of mpAD-MSCs when compared with the fresh mpAD-MSCs. The results from this study might be important for the successful application of MSCs in clinical trials after long-term cryopreservation.

Keywords: mesenchymal stem cells, cryopreservation, stemness, senescence

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