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FACS replacement

Jörn Lakowski, Emily Welby, Dimitri Budinger, Fabiana Di Marco, Valentina Di Foggia, James W B Bainbridge, Kyle Wallace, David M Gamm, Robin R Ali, Jane C Sowden
Loss of photoreceptor cells due to retinal degeneration is one of the main causes of blindness in the developed world. Although there is currently no effective treatment, cell replacement therapy using stem-cell-derived photoreceptor cells may be a feasible future treatment option. In order to ensure safety and efficacy of this approach, robust cell isolation and purification protocols must be developed. To this end, we previously developed a biomarker panel for the isolation of mouse photoreceptor precursors from the developing mouse retina and mouse embryonic stem cell cultures...
January 12, 2018: Stem Cells
Peng Jin, De-Zhi Wang, Chen-Xi Lyu, Ya-Ting Wang, Yu-Qi He, Jian-Qiu Sheng, Xuhang Li
BACKGROUND: Epidemiological studies suggest a protective role of estrogen against colon carcinogenesis; this effect appears to be dependent on mismatch repair (MMR) status. However, the underlying mechanism remains unclear. This study investigated the role of MMR proteins in apoptosis of colon cancer cells in the presence or absence of estrogen. METHODS: Two major MMR proteins, human mutL homolog 1 (hMLH1) and mutS homolog 2 (hMSH2), as well as estrogen receptor-β (ERβ), were transiently expressed in either hMLH1-deficient HCT116 cells or hMSH2-deficient LoVo cells...
2017: Journal of Cancer
Cora Delling, Matthias Lendner, Uwe Müller, Arwid Daugschies
Cryptosporidium parvum has been suggested as a suitable target for in vitro efficacy testing of disinfectants. To improve validity of a method based on exposure of HCT-8 monolayers to C. parvum oocysts we here critically evaluate and we propose certain procedural steps needed for the validation of disinfectants. Within a range of 0.02% to 0.4%, sodium taurocholate at 0.2% stimulated infection most efficiently while preserving host cell integrity. The course of invasion was monitored for periods of 30-240min post infection (p...
October 15, 2017: Veterinary Parasitology
Dahe Li, Wenwen Tong, Denghui Liu, Yuming Zou, Chen Zhang, Weidong Xu
BACKGROUND: With the re-popularity of metal-on-metal (MoM) bearing in recent years, the cobalt toxicity has been a cause for concern in the total hip replacement surgery by both physicians and patients. METHODS: MG-63 cell line was cultured in vitro and incubated with cobalt (II) chloride (CoCl2) and/or with astaxanthin (ASX) for 24 h. MTT assay was conducted to evaluate the cell viability after cobalt exposure and ASX treatment. Fluorescence-activated cell sorting (FACS) analysis was performed to examine the reactive oxygen species (ROS) level...
July 24, 2017: BMC Pharmacology & Toxicology
Antonio Shegani, Charalampos Triantis, Berthold A Nock, Theodosia Maina, Christos Kiritsis, Vassilis Psycharis, Catherine Raptopoulou, Ioannis Pirmettis, Francesco Tisato, Minas S Papadopoulos
In the present work, we investigated potential means to obtain neutral tricarbonyl mixed-ligand fac-[M(CO)3L(1)L(2)] complexes (M = Re, (99m)Tc) containing the (2-hydroxyphenyl)diphenylphosphine (POH) bidentate ligand (L(1)H) and a series of monodentate ligands (L(2)). First, fac-[Re(CO)3(PO)(H2O)], 1, was synthesized by reaction of POH and [Et4N]2[Re(CO)3Br3] in equimolar amounts in MeOH at room temperature. Interestingly, with excess of POH this reaction afforded fac-[Re(CO)3(PO)(POH)], 2, with POH operating both as a bidentate and as a monodentate ligand...
July 17, 2017: Inorganic Chemistry
Saixiang Feng, Chenggang Xu, Kaijie Yang, Haihong Wang, Huiying Fan, Ming Liao
In Haemophilus parasuis, the genes HAPS_0217 and HAPS_1695 are predicted to encode long-chain fatty acid-CoA ligases (FACSs). These proteins contain ATP/AMP signature motifs and FACS conserved motifs that are homologous to those in Escherichia coli FadD (EcFadD). In this study, we demonstrate that HAPS_0217 and HAPS_1695 can functionally replace EcFadD in the E. coli fadD mutant JW1794, and were thus designated fadD1 and fadD2, respectively. An evaluation of kinetic parameters indicated that FadD1 and FadD2 have a substrate preference for long-chain fatty acids...
2017: Frontiers in Cellular and Infection Microbiology
Matilda H-C Sheng, Kin-Hing William Lau, Ram Lakhan, Abu Shufian Ishtiaq Ahmed, Charles H Rundle, Patra Biswanath, David J Baylink
The present study was undertaken to determine the mechanism whereby calcitropic hormones and mesenchymal stem cell progeny changes are involved in bone repletion, a regenerative bone process that restores the bone lost to calcium deficiency. To initiate depletion, weanling mice with a mixed C57BL/6 (75%) and CD1 (25%) genetic background were fed a calcium-deficient diet (0.01%) for 14 days. For repletion, the mice were fed a control diet containing 1.2% calcium for 14 days. Depletion decreased plasma calcium and increased plasma parathyroid hormone, 1,25(OH)2D (calcitriol), and C-terminal telopeptide of type I collagen...
April 1, 2017: Endocrinology
John K Nganga, Christian R Samanamu, Joseph M Tanski, Carlos Pacheco, Cesar Saucedo, Victor S Batista, Kyle A Grice, Mehmed Z Ertem, Alfredo M Angeles-Boza
A series of rhenium tricarbonyl complexes coordinated by asymmetric diimine ligands containing a pyridine moiety bound to an oxazoline ring were synthesized, structurally and electrochemically characterized, and screened for CO2 reduction ability. The reported complexes are of the type Re(N-N)(CO)3Cl, with N-N = 2-(pyridin-2-yl)-4,5-dihydrooxazole (1), 5-methyl-2-(pyridin-2-yl)-4,5-dihydrooxazole (2), and 5-phenyl-2-(pyridin-2-yl)-4,5-dihydrooxazole (3). The electrocatalytic reduction of CO2 by these complexes was observed in a variety of solvents and proceeds more quickly in acetonitrile than in dimethylformamide (DMF) and dimethyl sulfoxide (DMSO)...
March 9, 2017: Inorganic Chemistry
Deborah A Sarkes, Margaret M Hurley, Dimitra N Stratis-Cullum
Peptide capture agents have become increasingly useful tools for a variety of sensing applications due to their ease of discovery, stability, and robustness. Despite the ability to rapidly discover candidates through biopanning bacterial display libraries and easily mature them to Protein Catalyzed Capture (PCC) agents with even higher affinity and selectivity, an ongoing challenge and critical selection criteria is that the peptide candidates and final reagent be selective enough to replace antibodies, the gold-standard across immunoassay platforms...
November 9, 2016: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Thibaut Mirlicourtois, Tche Hong Loquineau, Pierre Yves Chagnon, Thomas Rondepierre, Serge Mirlicourtois, Jean Christophe Bonnardel
OBJECTIVE: The replacement of a mechanical knee with prosthetic class I foot fitting by an electronic knee with prosthetic class II foot was proposed to a 54-years-old schizophrenic patient, active, with right femoral and left tibial traumatic amputation. This fitting evolution was assessed both quantitatively and qualitatively. OBSERVATIONS/METHOD: Compared evaluation of electronic knee with prosthetic class II foot versus mechanical polycentric knee with prosthetic class I foot (fitting unchanged moreover) in: - functional abilities and quality of life using standardized scores (Functional Ambulation Classification, Houghton, Functional Independence Measurement, B...
September 2016: Annals of Physical and Rehabilitation Medicine
Christine A Marsh, Jenny Browne, Jan Taylor, Deborah Davis
BACKGROUND: The conduct of research regardless of the subject or methods employed brings responsibilities and challenges. These are greater when dealing with sensitive topics and vulnerable groups and therefore researchers must navigate a range of complex issues and make choices in relation to practical, ethical and philosophical concerns. While literature dealing with research methodologies and research design may assist to some degree, it cannot provide a clear pathway or template as each research project must respond to a unique set of circumstances...
February 2017: Women and Birth: Journal of the Australian College of Midwives
Tali Benromano, Chaim G Pick, Joav Merick, Ruth Defrin
Objective: As individuals with intellectual disability (ID) due to cerebral palsy (CP) are at high risk of experiencing pain, measuring their pain is crucial for adequate treatment. While verbal reports are the gold standard in pain measurements, they may not be sufficient in ID. The aim was to detect behavioral/autonomic responses that may indicate the presence and intensity of pain in individuals with CP and ID, using calibrated stimuli, here for the first time. Subjects: Thirteen adults with CP and ID (CPID), 15 healthy controls (HC), and 5 adults with CP with no ID (CPNID)...
March 1, 2017: Pain Medicine: the Official Journal of the American Academy of Pain Medicine
Christos S Karamitros, Manfred Konrad
Immunogenicity is one of the most common complications occurring during therapy making use of protein drugs of nonhuman origin. A notable example of such a case is bacterial l-asparaginases (L-ASNases) used for the treatment of acute lymphoblastic leukemia (ALL). The replacement of the bacterial enzymes by human ones is thought to set the basis for a major improvement of antileukemic therapy. Recently, we solved the crystal structure of a human enzyme possessing L-ASNase activity, designated hASNase-3. This enzyme is expressed as an inactive precursor protein and post-translationally undergoes intramolecular processing leading to the generation of two subunits which remain noncovalently, yet tightly associated and constitute the catalytically active form of the enzyme...
September 16, 2016: ACS Chemical Biology
Saki Ito, Sorin V Pislaru, Wern Miin Soo, Runqing Huang, Kevin L Greason, Verghese Mathew, Gurpreet S Sandhu, Mackram F Eleid, Rakesh M Suri, Jae K Oh, Vuyisile T Nkomo
BACKGROUND: The impact of pre-procedural RV size and function on outcomes following transcatheter aortic valve replacement (TAVR) is not well established. The aim of this study was to investigate the role of right ventricular size and function on outcomes following TAVR. METHODS AND RESULTS: Between November 2008 and June 2013, 268 consecutive patients (age 80.5±7.9years, aortic valve area 0.79±0.16cm(2)) with symptomatic severe aortic stenosis (AS) undergoing TAVR at Mayo Clinic were included...
October 15, 2016: International Journal of Cardiology
Susan M Byrne, George M Church
CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0...
2015: Current Protocols in Stem Cell Biology
Hong Guo, Stacy Cooper, Alan D Friedman
The murine Cebpa gene contains a +37 kb, evolutionarily conserved 440 bp enhancer that directs high-level expression to myeloid progenitors in transgenic mice. The enhancer is bound and activated by Runx1, Scl, GATA2, C/EBPα, c-Myb, Pu.1, and additional Ets factors in myeloid cells. CRISPR/Cas9-mediated replacement of the wild-type enhancer with a variant mutant in its seven Ets sites leads to 20-fold reduction of Cebpa mRNA in the 32Dcl3 myeloid cell line. To determine the effect of deleting the enhancer in vivo, we now characterize C57BL/6 mice in which loxP sites flank a 688 bp DNA segment containing the enhancer...
2016: PloS One
Raphael Ledermann, Silvan Strebel, Clara Kampik, Hans-Martin Fischer
UNLABELLED: Analysis of bacterial gene function commonly relies on gene disruption or replacement followed by phenotypic characterization of the resulting mutant strains. Deletion or replacement of targeted regions is commonly achieved via two homologous recombination (HR) events between the bacterial genome and a nonreplicating plasmid carrying DNA fragments flanking the region to be deleted. The counterselection of clones that have integrated the entire plasmid in their genome via a single HR event is crucial in this procedure...
May 2016: Applied and Environmental Microbiology
Wenyan Xie, Hongling Wen, Fulu Chu, Shaofeng Yan, Wenli Xie, Bin Lin, Yuzhen Chen, Zhenmei Li, Guijie Ren, Yanyan Song, Li Zhao, Zhiyu Wang
OBJECTIVE: To investigate the effect of the leucine zipper-like motif between HRA and HRB of the human parainfluenza virus 3 fusion protein on fusion activity. METHODS: Site-directed mutagenesis was utilized to substitute the heptadic residues at 257, 264, 271, 278, 285, 292, and 299 in this motif with alanine. Additionally, 3 middle heptadic leucine residues at 271, 278, and 285 were replaced with alanine singly or in combination. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated fusion (F) proteins...
2015: Intervirology
M A Cleary, R Narcisi, K Focke, R van der Linden, P A J Brama, G J V M van Osch
OBJECTIVE: Total bone marrow-derived mesenchymal stem cell (BMSC) populations differ in their potential to undergo chondrogenesis, with individual BMSCs differing in their chondrogenic capacity. The aim of this study was to explore the use of CD105 as a marker to isolate a chondrogenic subpopulation of BMSCs from the total, heterogeneous population. DESIGN: BMSCs were isolated from patients undergoing total hip replacement and following expansion (Passage 1-Passage 5), CD105 expression was investigated by FACS analysis...
May 2016: Osteoarthritis and Cartilage
Xuan Liu, Lunzhi Yuan, Quan Yuan, Yali Zhang, Kun Wu, Tianying Zhang, Yong Wu, Wangheng Hou, Tengyun Wang, Pingguo Liu, James Wai Kuo Shih, Tong Cheng, Ningshao Xia
On the basis of its close phylogenetic relationship with primates, the development of Tupaia belangeri as an infection animal model and drug metabolism model could provide a new option for preclinical studies, especially in hepatitis virus research. As a replacement for primary human hepatocytes (PHHs), primary tupaia hepatocytes (PTHs) have been widely used. Similar to human serum albumin, tupaia serum albumin (TSA) is the most common liver synthesis protein and is an important biomarker for PTHs and liver function...
May 20, 2016: Experimental Animals
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