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Sharath Belame Shivakumar, Dinesh Bharti, Raghavendra Baregundi Subbarao, Ju-Mi Park, Young-Bum Son, Imran Ullah, Yong-Ho Choe, Hyeong-Jeong Lee, Bong-Wook Park, Sung-Lim Lee, Gyu-Jin Rho
Following success of pancreatic islet transplantation in the treatment of Type I diabetes mellitus, there is a growing interest in using cell-based treatment approaches. However, severe shortage of donor islets-pancreas impeded the growth, and made researchers to search for an alternative treatment approaches. In this context, recently, stem cell-based therapy has gained more attention. The current study demonstrated that epigenetic modification improves the in vitro differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs) into pancreatic endocrine-like cells...
October 21, 2018: Journal of Cellular Physiology
Gyeong Joon Moon, Ji Hee Sung, Dong Hee Kim, Eun Hee Kim, Yeon Hee Cho, Jeong Pyo Son, Jae Min Cha, Oh Young Bang
Mesenchymal stem cells (MSCs) exert their therapeutic capability through a variety of bioactive substances, including trophic factors, microRNAs, and extracellular vesicles (EVs) in infarcted tissues. We therefore hypothesized that MSC-derived EVs (MSC-EVs) possess therapeutic molecules similar to MSCs. Moreover, given their nature as nanosized and lipid-shielded particles, the intravenous infusion of MSC-EVs would be advantageous over MSCs as a safer therapeutic approach. In this study, we investigated the biodistribution, therapeutic efficacy, and mode of action of MSC-EVs in a rat stroke model...
October 19, 2018: Translational Stroke Research
Bahareh Rajaei, Mohammad Massumi, Michael Wheeler
The International Diabetic Federation estimated that 415 million adults currently have diabetes and 318 million adults had impaired glucose tolerance, putting them at high risk of developing diabetes in the future. In Type 1 Diabetes (T1D), the β cells are lost because of autoimmune reactions. Although islet transplantation has been a promising therapy for T1D, it is greatly limited by pancreatic donors. Here, we describe a protocol to generate glucose- responsive pancreatic β-like (GRPβ-L) cells from human-induced pluripotent stem (iPS) cells...
October 18, 2018: Current Protocols in Human Genetics
Shailima Rampogu, Ayoung Baek, Rohit S Bavi, Minky Son, Guang Ping Cao, Raj Kumar, Chanin Park, Amir Zeb, Rabia Mukthar Rana, Seok Ju Park, Keun Woo Lee
Aromatase inhibitors with an IC50 value ranging from 1.4 to 49.7uM are known to act as antiepileptic drugs besides being potential breast cancer inhibitors. The aim of the present study is to identify novel antiepileptic aromatase inhibitors with higher activity exploiting the ligand-based pharmacophore approach utilizing the experimentally known inhibitors. The resultant Hypo1 consists of four features and was further validated by using three different strategies. Hypo1 was allowed to screen different databases to identify lead molecules and were further subjected to Lipinski' Rule of Five and ADMET to establish their drug-like properties...
October 12, 2018: IEEE/ACM Transactions on Computational Biology and Bioinformatics
Aran Son, Namju Kang, Ki Woo Kim, Yu-Mi Yang, Dong Min Shin
Mechanical stress plays an important role in the regulation of bone turnover. However, the mechanism underlying hypo-osmotic stress-induced cellular response in osteoblasts remains poorly understood. In this study, we investigated the effect of hypotonic stress on the expression of bone remodeling factors, including the receptor activator of nuclear factor-kappa B ligand (RANKL) and the nuclear factor of activated T cells type c1 (NFATc1) in primary mouse osteoblasts and MC3T3-E1 cells. Hypo-osmotic stress induced significant increases in RANKL mRNA expression and intracellular Ca2+ concentration ([Ca2+]i) from the extracellular space...
October 15, 2018: Journal of Molecular Endocrinology
Kaori Nakatani, Toshimitsu Yamaoka, Motoi Ohba, Ken-Ichi Fujita, Satoru Arata, Sojiro Kusumoto, Iori Taki-Takemoto, Daisuke Kamei, Shinichi Iwai, Junji Tsurutani, Tohru Ohmori
The critical T790M mutation in epidermal growth factor receptor (EGFR), which mediates resistance to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs; gefitinib, erlotinib, and afatinib), has facilitated the development of third-generation mutation-selective EGFR TKIs (rociletinib and osimertinib). We previously reported heterogeneous afatinib-resistance mechanisms, including emergence of T790M-EGFR, and responses to third-generation EGFR-TKIs. Here, we used afatinib-resistant lung adenocarcinoma cells (AfaR [formerly AFR3] cells), carrying exon 19 deletion/T790M in EGFR...
October 15, 2018: Molecular Cancer Therapeutics
Seung-Hyun Noh, Shin-Woong Kim, Jae-Won Kim, Tae-Hoon Lee, Jae-Woon Nah, Young-Gi Lee, Mi-Kyung Kim, Yoshihiro Ito, Tae-Il Son
This study demonstrated the anti-adhesion and wound healing effect of a visible light curable anti-adhesion agent using an alginate derivative modified with a furfuryl moiety. Visible light-curable furfuryl alginate (F-Alg) was prepared in conjugation with alginate and furfurylamine by an amide coupling reaction, and the conjugated F-Alg was characterized by 1 H NMR analysis. The cytotoxicity, cell adhesion, and cell permeability of the F-Alg were evaluated for use in anti-adhesion applications. Drug immobilization and protein release were assessed to verify whether the alginate derivatives and drugs were photo-immobilized...
October 9, 2018: International Journal of Biological Macromolecules
Ling Fu, Keke Liu, Renan B Ferreira, Kate S Carroll, Jing Yang
Oxidation of a protein cysteinyl thiol (Cys-SH) to S-sulfenic acid (Cys-SOH) by a reactive oxygen species (e.g., hydrogen peroxide), which is termed protein S-sulfenylation, is a reversible post-translational modification that plays a crucial role in redox regulation of protein function in various biological processes. Due to its intrinsically labile nature, protein S-sulfenylation cannot be directly detected or analyzed. Chemoselective probing has been the method of choice for analyzing S-sulfenylated proteins either in vitro or in situ, as it allows stabilization and direct detection of this transient oxidative intermediate...
October 12, 2018: Current Protocols in Protein Science
Daniel Medina-Cano, Ekin Ucuncu, Lam Son Nguyen, Michael Nicouleau, Joanna Lipecka, Jean-Charles Bizot, Christian Thiel, François Foulquier, Nathalie Lefort, Catherine Faivre-Sarrailh, Laurence Colleaux, Ida Chiara Guerrera, Vincent Cantagrel
Proper brain development relies highly on protein N-glycosylation to sustain neuronal migration, axon guidance and synaptic physiology. Impairing the N-glycosylation pathway at early steps produces broad neurological symptoms identified in congenital disorders of glycosylation. However, little is known about the molecular mechanisms underlying these defects. We generated a cerebellum specific knockout mouse for Srd5a3 , a gene involved in the initiation of N-glycosylation. In addition to motor coordination defects and abnormal granule cell development, Srd5a3 deletion causes mild N-glycosylation impairment without significantly altering ER homeostasis...
October 12, 2018: ELife
Dae Woong Ham, Tae-Il Son, Tae Jin Lee, Kwang-Sup Song
The osteogenetic potential of photo-immobilized azdiophenyl (Az)-natural polymers as a carrier of bone morphogenetic protein-2 (BMP-2) was assessed in 56 rats randomized to four groups. The control group comprised implanted collagen sheet with BMP-2. In the three experimental groups, the implant comprised collagen sheet with photo-immobilized BMP-2 on Az-gelatin (Az-Gel), Az-O-carboxymethyl chitosan (Az-OMC), or Az‑O‑carboxymethyl low molecular chitosan (Az-LMC). Micro-computed tomography analysis revealed more regenerated bone in Az-Gel at 8weeks...
October 6, 2018: International Journal of Biological Macromolecules
Shin-Bi Oh, Min Sun Kim, SuJi Park, HyunJu Son, Seog-Young Kim, Min-Seon Kim, Dong-Gyu Jo, Eunyoung Tak, Joo-Yong Lee
While clusterin is reportedly involved in Alzheimer's disease (AD) pathogenesis, how clusterin interacts with amyloid-β (Aß) to cause Aß neurotoxicity remains unclear in vivo. Using 5×FAD transgenic mice, which develop robust AD pathology and memory deficits when very young, we detected interactions between clusterin and Aß in the mouse brains. The two proteins were concurrently upregulated and bound or colocalized with each other in the same complexes or in amyloid plaques. Neuropathology and cognitive performance were assessed in the progeny of clusterin-null mice crossed with 5×FAD mice, yielding clu-/- ;5×FAD and clu+/+ ;5×FAD...
October 8, 2018: Brain Pathology
Sang Yeon Cho, Sungha Kim, Mi-Ju Son, Woo Sun Rou, Seok Hyun Kim, Hyuk Soo Eun, Byung Seok Lee
BACKGROUND: Oxidative stress occurs due to the excessive generation of cellular reactive oxygen species and antioxidant system dysfunction. The thioredoxin (TXN) system and TXN-domain-containing protein (TXNDC) family form networks maintaining the cellular reducing environment. Recently, the importance of these genes in the tumor environment has been emphasized. AIM: To investigate the clinical significance of TXNs and TXNDC family members in HCC. METHODS: Genomic data from 367 hepatocellular carcinoma (HCC) patients who underwent hepatic resections were analyzed to determine genetic alterations in mRNA and protein levels between patients and healthy controls...
October 4, 2018: Digestive Diseases and Sciences
Koen Kole, Tansu Celikel
The heterogeneous organization of the mammalian neocortex poses a challenge for elucidating the molecular mechanisms underlying its physiological processes. Although high-throughput molecular methods are increasingly deployed in neuroscience, their anatomical specificity is often lacking. In this unit, we introduce a targeted microdissection technique that enables extraction of high-quality RNA and proteins at high anatomical resolution from acutely prepared brain slices. We exemplify its utility by isolating single cortical columns and laminae from the mouse primary somatosensory (barrel) cortex...
October 4, 2018: Current Protocols in Neuroscience
Rachel A Coleman, Darci J Trader
Fluorescence resonance energy transfer (FRET) technology is a useful tool to monitor protein interactions as well as protease activity. We have recently reported a biochemical assay utilizing a FRET reporter peptide to monitor the activity of the 20S catalytic particle (20S CP) of the proteasome. This assay is designed specifically to have increased sensitivity to identify stimulators of the 20S CP, which may hold therapeutic potential to treat protein-accumulation diseases. The protocol described here details the necessary steps in synthesizing the FRET reporter peptide and performing the FRET assay with the 20S CP...
October 4, 2018: Current Protocols in Chemical Biology
Geeta Chaudhri, Georgina Kaladimou, Pratikshya Pandey, Gunasegaran Karupiah
Ectromelia virus (ECTV) is an orthopoxvirus that causes mousepox in mice. Members of the genus orthopoxvirus are closely related and include variola (the causative agent of smallpox in humans), monkeypox, and vaccinia. Common features of variola virus and ECTV further include a restricted host range and similar disease progression in their respective hosts. Mousepox makes an excellent small animal model for smallpox to investigate pathogenesis, vaccine and antiviral agent testing, host-virus interactions, and immune and inflammatory responses...
October 3, 2018: Current Protocols in Microbiology
Paschalis-Thomas Doulias, Neal S Gould
The wide reactivity of the thiol group enables the formation of a variety of reversible, covalent modifications on cysteine residues. S-nitrosylation, like many other post-translational modifications, is site selective, reversible, and necessary for a wide variety of fundamental cellular processes. The overall abundance of S-nitrosylated proteins and reactivity of the nitrosyl group necessitates an enrichment strategy for accurate detection with adequate depth. Herein, a method is presented for the enrichment and detection of endogenous protein S-nitrosylation from complex mixtures of cell or tissue lysate utilizing organomercury resin...
October 3, 2018: Current Protocols in Protein Science
Hong-Soo Lee, Yoo-Jin Park, Doo-Wan Cho, Su-Cheol Han, Soo Youn Jun, Gi Mo Jung, Woo-Jong Lee, Chi-Min Choi, Eun-Jung Park, Son-Il Pak
High-dose radiation-induced tissue damage is a major limiting factor in the medical application of nuclear technology. Herein, we tested 28-day repeated-dose toxicity of KMRC011, an agonist of toll-like receptor (TLR) 5, which is being developed as a medical countermeasure for radiation, using cynomolgus monkeys. KMRC011 (0.01, 0.02 or 0.04 mg/kg/day) was intramuscularly injected once daily for 4 weeks, and each two monkeys in both control and 0.04 mg/kg/day group were observed for an additional 2-week recovery period...
September 11, 2018: Journal of Applied Toxicology: JAT
Molly Hunter, Ping Yuan, Divya Vavilala, Mark Fox
Recombinant proteins, such as monoclonal antibodies, are produced in mammalian cell lines to introduce proper protein folding and post-translational modifications, which are essential for full biological activity. In both the industrial and academic environments, the use of recombinant proteins varies widely and, with it, the method of production. The amount of an antibody needed for a toxicity study is far greater than that needed by a research lab performing cellular assays, and the amount of effort put into the development of the protein will vary accordingly...
September 28, 2018: Current Protocols in Protein Science
Bridget L Menasche, Lauren Crisman, Daniel R Gulbranson, Eric M Davis, Haijia Yu, Jingshi Shen
About one-third of cellular proteins in eukaryotic cells are localized to membrane-enclosed organelles in the endomembrane system. Trafficking of these membrane proteins (including soluble lumenal proteins) among the organelles is mediated by small sac-like vesicles. Vesicle-mediated membrane trafficking regulates a broad range of biological processes, many of which are still poorly understood at the molecular level. A powerful approach to dissect a vesicle-mediated membrane trafficking pathway is unbiased genome-wide genetic screening, which only recently became possible in mammalian cells with the isolation of haploid human cell lines and the development of CRISPR-Cas9 genome editing...
September 28, 2018: Current Protocols in Cell Biology
Lisa Prevedel, Nancy Ruel, Paul Castellano, Carla Smith, Shaily Malik, Courtney Villeux, Morgane Bomsel, Susan Morgello, Eliseo A Eugenin
The major barrier to eradicating human immunodeficiency virus-1 (HIV) infection is the generation and extended survival of HIV reservoirs. In order to eradicate HIV infection, it is essential to detect, quantify, and characterize circulating and tissue-associated viral reservoirs in infected individuals. Currently, PCR-based technologies and Quantitative Viral Outgrowth Assays (Q-VOA) are the gold standards to detect viral reservoirs. However, these methods are limited to detecting circulating viral reservoirs, and it has been shown that they misrepresent the size of the reservoirs, largely because they detect only one component of the HIV life cycle and are unable to detect viral reservoirs in tissues...
September 28, 2018: Current Protocols in Cell Biology
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