Sample records for kit micro typing
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NEMO educational kit on micro-optics at the secondary school
NASA Astrophysics Data System (ADS)
Flores-Arias, M. T.; Bao-Varela, Carmen
2014-07-01
NEMO was the "Network of Excellence in Micro-Optics" granted in the "Sixth Framework Program" of the European Union. It aimed at providing Europe with a complete Micro-Optics food-chain, by setting up centers for optical modeling and design; measurement and instrumentation; mastering, prototyping and replication; integration and packaging and reliability and standardization. More than 300 researchers from 30 groups in 12 countries participated in the project. One of the objectives of NEMO was to spread excellence and disseminate knowledge on micro-optics and micro-photonics. To convince pupils, already from secondary school level on, about the crucial role of light and micro-optics and the opportunities this combination holds, several partners of NEMO had collaborate to create this Educational Kit. In Spain the partner involved in this aim was the "Microoptics and GRIN Optics Group" at the University of Santiago of Compostela (USC). The educational kits provided to the Secondary School were composed by two plastic cards with the following microoptical element: different kinds of diffractive optical elements or DOES and refractive optical elements or ROEs namely arrays of micro-lenses. The kit also included a DVD with a handbook for performing the experiments as well as a laser pointer source. This kit was distributed free of charge in the countries with partners in NEMO. In particular in Spain was offered to around 200 Secondary School Centers and only 80 answered accepting evaluate the kit.
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Data on quantification of signaling pathways activated by KIT and PDGFRA mutants.
Bahlawane, Christelle; Schmitz, Martine; Letellier, Elisabeth; Arumugam, Karthik; Nicot, Nathalie; Nazarov, Petr V; Haan, Serge
2016-12-01
The present data are related to the article entitled "Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling" (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016) [1]. Constitutive and ligand-derived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST) were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells). Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf). We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and micro-array analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Kit Ex11 deletion mutant was able to elicit an effective STATs response whereas all PDGFRA were able to do so.
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Orbital Maneuvering Vehicle (OMV) remote servicing kit
NASA Technical Reports Server (NTRS)
Brown, Norman S.
1988-01-01
With the design and development of the Orbital Maneuvering Vehicle (OMV) progressing toward an early 1990 initial operating capability (IOC), a new era in remote space operations will evolve. The logical progression to OMV front end kits would make available in situ satellite servicing, repair, and consummables resupply to the satellite community. Several conceptual design study efforts are defining representative kits (propellant tanks, debris recovery, module servicers); additional focus must also be placed on an efficient combination module servicer and consummables resupply kit. A remote servicer kit of this type would be designed to perform many of the early maintenance/resupply tasks in both nominal and high inclination orbits. The kit would have the capability to exchange Orbital Replacement Units (ORUs), exchange propellant tanks, and/or connect fluid transfer umbilicals. Necessary transportation system functions/support could be provided by interfaces with the OMV, Shuttle (STS), or Expendable Launch Vehicle (ELV). Specific remote servicer kit designs, as well as ground and flight demonstrations of servicer technology are necessary to prepare for the potential overwhelming need. Ground test plans should adhere to the component/system/breadboard test philosophy to assure maximum capability of one-g testing. The flight demonstration(s) would most likely be a short duration, Shuttle-bay experiment to validate servicer components requiring a micro-g environment.
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Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling.
Ding, Meng; Wang, Cheng; Lu, Xiaolan; Zhang, Cuiping; Zhou, Zhen; Chen, Xi; Zhang, Chen-Yu; Zen, Ke; Zhang, Chunni
2018-06-01
Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. Here, we analyzed the advantages and weaknesses of four commonly used commercial kits for exosomal miRNA profiling and their application to the sample of serum and/or plasma, respectively. NanoSight and Western blotting were conducted to evaluate the efficiency and purity of the isolated exosomes. In our conditions, the size distribution of the isolated particles was appropriate (40-150 nm), and ExoQuick™ Exosome Precipitation Solution (EXQ) generated a relatively high yield of exosomes. Nevertheless, albumin impurity was ubiquitous for all the four kits, and Total Exosome Isolation for serum or plasma (TEI) yielded a relatively pure isolation. We further performed Illumina sequencing combined with RT-qPCR to determine the ability of these kits for miRNA profiling. There was significant correlation of the exosomal miRNA profile and specific miRNAs between kits, but with differences depending on methods. exoRNeasy Serum/Plasma Midi Kit (EXR) and EXQ performed better in the specific exosomal miRNAs recovery. Intraassay CVs for specific miRNA measurement were 0.88-3.82, 1.19-3.77, 0-2.70, and 1.23-9.11% for EXR, TEI, EXQ, and RIBO™ Exosome Isolation Reagent (REI), respectively. In each kit, serum yielded a higher abundance of exosomes and exosomal miRNAs than plasma, yet with more albumin impurity. In conclusion, our data provide some valuable guidance for the methodology of disease biomarker identification of circulation exosomal miRNAs. Graphical abstract Circulating exosomal microRNAs (miRNAs) are valuable biomarker candidates; however, information on the characterization and mutual agreement of commercial kits for circulating exosomal miRNA profiling is scarce. In this study, we compared four commonly used commercially available kits for exosomal miRNAsextraction and analyzed the advantages and weaknesses of each kit and their application to the sample ofserum and/or plasma.
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Siemens, Helge; Jackstadt, Rene; Kaller, Markus; Hermeking, Heiko
2013-01-01
The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit. However, the underlying mechanism has remained unknown. Here, we show that the p53-induced miR-34 microRNA family mediates repression of c-Kit by p53 via a conserved seed-matching sequence in the c-Kit 3'-UTR. Ectopic miR-34a resulted in a decrease in Erk signaling and transformation, which was dependent on the down-regulation of c-Kit expression. Furthermore, ectopic expression of c-Kit conferred resistance of colorectal cancer (CRC) cells to treatment with 5-fluorouracil (5-FU), whereas ectopic miR-34a sensitized the cells to 5-FU. After stimulation with c-Kit ligand/stem cell factor (SCF) Colo320 CRC cells displayed increased migration/invasion, whereas ectopic miR-34a inhibited SCF-induced migration/invasion. Activation of a conditional c-Kit allele induced several stemness markers in DLD-1 CRC cells. In primary CRC samples elevated c-Kit expression also showed a positive correlation with markers of stemness, such as Lgr5, CD44, OLFM4, BMI-1 and β-catenin. On the contrary, activation of a conditional miR-34a allele in DLD-1 cells diminished the expression of c-Kit and several stemness markers (CD44, Lgr5 and BMI-1) and suppressed sphere formation. MiR-34a also suppressed enhanced sphere-formation after exposure to SCF. Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells. Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches. PMID:24009080
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Wall, Jason; Conrad, Rick; Latham, Kathy; Liu, Eric
2014-03-01
Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time to results compared to traditional culture methods. The addition of a recirculating pooled immunomagnetic separation method prior to real-time PCR analysis increases processing output while reducing both cost and labor. This AOAC Research Institute method modification study validates the MicroSEQ® Salmonella spp. Detection Kit [AOAC Performance Tested Method (PTM) 031001] linked with the Pathatrix® 10-Pooling Salmonella spp. Kit (AOAC PTM 090203C) in diced tomatoes, chocolate, and deli ham. The Pathatrix 10-Pooling protocol represents a method modification of the enrichment portion of the MicroSEQ Salmonella spp. The results of the method modification were compared to standard cultural reference methods for diced tomatoes, chocolate, and deli ham. All three matrixes were analyzed in a paired study design. An additional set of chocolate test portions was analyzed using an alternative enrichment medium in an unpaired study design. For all matrixes tested, there were no statistically significant differences in the number of positive test portions detected by the modified candidate method compared to the appropriate reference method. The MicroSEQ Salmonella spp. protocol linked with the Pathatrix individual or 10-Pooling procedure demonstrated reliability as a rapid, simplified, method for the preparation of samples and subsequent detection of Salmonella in diced tomatoes, chocolate, and deli ham.
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High-throughput and reliable protocols for animal microRNA library cloning.
Xiao, Caide
2011-01-01
MicroRNAs are short single-stranded RNA molecules (18-25 nucleotides). Because of their ability to silence gene expressions, they can be used to diagnose and treat tumors. Experimental construction of microRNA libraries was the most important step to identify microRNAs from animal tissues. Although there are many commercial kits with special protocols to construct microRNA libraries, this chapter provides the most reliable, high-throughput, and affordable protocols for microRNA library construction. The high-throughput capability of our protocols came from a double concentration (3 and 15%, thickness 1.5 mm) polyacrylamide gel electrophoresis (PAGE), which could directly extract microRNA-size RNAs from up to 400 μg total RNA (enough for two microRNA libraries). The reliability of our protocols was assured by a third PAGE, which selected PCR products of microRNA-size RNAs ligated with 5' and 3' linkers by a miRCat™ kit. Also, a MathCAD program was provided to automatically search short RNAs inserted between 5' and 3' linkers from thousands of sequencing text files.
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ERIC Educational Resources Information Center
San Mateo County Office of Education, Redwood City, CA. Career Preparation Centers.
This thirteenth of fifteen sets of Adult Competency Education (ACE) Competency Based Job Descriptions in the ACE kit contains job descriptions for Secretary, Keypunch Operator, Electronics Assembler, Printed Circuit Assembler, Micro Electronincs Assembler, Chassis Assembler, and Machinist Apprentice. Each begins with a fact sheet that includes…
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Portable thin layer chromatography for field detection of explosives and propellants
NASA Astrophysics Data System (ADS)
Satcher, Joe H.; Maienschein, Jon L.; Pagoria, Philip F.; Racoveanu, Ana; Carman, M. Leslie; Whipple, Richard E.; Reynolds, John G.
2012-06-01
A field deployable detection kit for explosives and propellants using thin layer chromatography (TLC) has been developed at Lawrence Livermore National Laboratory (LLNL). The chemistry of the kit has been modified to allow for field detection of propellants (through propellant stabilizers), military explosives, peroxide explosives, nitrates and inorganic oxidizer precursors. For many of these target analytes, the detection limit is in the μg to pg range. A new miniaturized, bench prototype, field portable TLC (Micro TLC) kit has also been developed for the detection and identification of common military explosives. It has been demonstrated in a laboratory environment and is ready for field-testing. The kit is comprised of a low cost set of commercially available components specifically assembled for rapid identification needed in the field and identifies the common military explosives: HMX, RDX, Tetryl, Explosive D or picric acid, and TNT all on one plate. Additional modifications of the Micro TLC system have been made with fluorescent organosilicon co-polymer coatings to detect a large suite of explosives.
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Liu, Chang; Guo, Zhide; Zhang, Pu; Song, Manli; Zhao, Zuoquan; Wu, Xiaowei; Zhang, Xianzhong
2014-08-01
Specific targeting of galactose-carrying molecule to ASGP-R in normal hepatocytes has been demonstrated before. In this study, galactosyl polystyrene was synthesized from controllable ratio of functional monomers and radio-labelled with (99m)Tc by formulated kit for SPECT imaging of hepatic function. p(VLA-co-VNI)(46:54) was synthesized by free-radical copolymerization initiated by AIBN, purified by dialysis, lyophilized to kit with Tricine and TPPTS as co-ligands for (99m)Tc labeling. Radiotracer (99m)Tc-p(VLA-co-VNI)(46:54)(Tricine)(TPPTS) was prepared and evaluated by in vitro stability, in vivo metabolism, ex vivo biodistribution and microSPECT/CT imaging in normal KM mice. MicroSPECT/CT and microMRI imaging were also performed in C57BL/b6 mice with xenograft hepatic carcinoma for hepatic function evaluation. (99m)Tc-p(VLA-co-VNI)(46:54)(Tricine)(TPPTS) was obtained in high radio chemical purity (RCP) (>99%) by using instant kit without further purification and excellent in vitro and in vivo stability. The result of biodistribution showed that liver had high uptake (90.49±10.68 ID%/g) at 30 min after injection and was blocked significantly by cold copolymer. MicroSPECT imaging in normal KM mice at 1h and 4h after injection showed good liver retention and targeting properties. Significant defect of activity was observed in the tumor site which was confirmed by MRI imaging. (99m)Tc-p(VLA-co-VNI)(46:54)(Tricine)(TPPTS) with lower ratio of targeting moiety has no observable effect on the specific binding affinity and liver uptake. This makes it possible to introduce more imaging units for multi-modality imaging. Furthermore, the instant kit preparation of (99m)Tc-labeling provides great potential for the evaluation of hepatocyte function in clinical application. Copyright © 2014 Elsevier Inc. All rights reserved.
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miR-137 downregulates c-kit expression in acute myeloid leukemia.
Hu, Yanping; Dong, Xiaolong; Chu, Guoming; Lai, Guangrui; Zhang, Bijun; Wang, Leitong; Zhao, Yanyan
2017-06-01
The oncogene c-kit plays a vital role in the pathogenesis of acute myeloid leukemia (AML). However, the mechanism of microRNAs targeting c-kit in AML has not been determined in detail. Moreover, the role miR-137 in tumor cell proliferation remains controversial. The aim of this work was to verify whether miR-137 targets c-kit and to research the biological effects of restoring miR-137 expression in leukemia cells. We found that miR-137 binds specifically to the 3'-UTR of c-kit and suppresses the expression and activities of c-kit. There is a negative correlation between miR-137 and c-kit expression in both patients and cell lines determined by screening large clinical samples. We found that miR-137 can inhibit proliferation, promote apoptosis, and induce differentiation of c-kit+ AML cells. We determined that miR-137 can participate in the leukemogenesis by regulating c-kit, which could be used as a therapeutic target for acute myeloid leukemia. Copyright © 2017 Elsevier Ltd. All rights reserved.
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No GIST-type c-kit gain of function mutations in neuroblastic tumours
Korja, M; Finne, J; Salmi, T T; Haapasalo, H; Tanner, M; Isola, J
2005-01-01
Aims: Neuroblastic tumours (NTs) have been shown to respond to imatinib treatment in vivo and in vitro, possibly via inactivating the c-kit receptor. The purpose of this study was to identify gastrointestinal stromal tumour (GIST)-type c-kit gene associated mutations in exons 9, 11, 13, and 17 in NTs to recognise a subset of tumours that would probably respond to imatinib treatment. Methods: Expression of the c-kit protein was detected immunohistochemically in a total of 37 archival paraffin wax embedded NTs using polyclonal rabbit antihuman c-kit antibody. After immunohistochemistry, c-kit gene associated chromosomal mutations in all cases of NT were detected with denaturing high performance liquid chromatography (HPLC). Results: Denaturing HLPC analysis did not reveal GIST-type mutations in four immunohistochemically detected c-kit positive or in 33 c-kit negative NTs. Conclusions: c-kit receptor expression and GIST-type c-kit gene mutations are rare events in NTs. Oncogenic activation of c-kit in NTs presumably differs from that of GISTs, which may influence their responsiveness to imatinib treatment. Whether c-kit has an essential role in the pathogenesis of NTs remains to be investigated. PMID:15976348
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Kajimoto, Noriko; Nakai, Norihiro; Ohkouchi, Mizuka; Hashikura, Yuka; Liu-Kimura, Ning-Ning; Isozaki, Koji; Hirota, Seiichi
2015-01-01
Sporadic mast cell neoplasms and gastrointestinal stromal tumors (GISTs) often have various types of somatic gain-of-function mutations of the c-kit gene which encodes a receptor tyrosine kinase, KIT. Several types of germline gain-of-function mutations of the c-kit gene have been detected in families with multiple GISTs. All three types of model mice for the familial GISTs with germline c-kit gene mutations at exon 11, 13 or 17 show development of GIST, while they are different from each other in skin mast cell number. Skin mast cell number in the model mice with exon 17 mutation was unchanged compared to the corresponding wild-type mice. In the present study, we characterized various types of mast cells derived from the model mice with exon 17 mutation (KIT-Asp818Tyr) corresponding to human familial GIST case with human KIT-Asp820Tyr to clarify the role of the c-kit gene mutation in mast cells. Bone marrow-derived cultured mast cells (BMMCs) derived from wild-type mice, heterozygotes and homozygotes were used for the experiments. Immortalized BMMCs, designated as IMC-G4 cells, derived from BMMCs of a homozygote during long-term culture were also used. Ultrastructure, histamine contents, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-kit gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We demonstrated that KIT-Asp818Tyr did not affect ultrastructure and proliferation profiles but did histamine contents in BMMCs. IMC-G4 cells had an additional novel c-kit gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell line to investigate mast cell biology. PMID:26722383
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Accuracy of user-friendly blood typing kits tested under simulated military field conditions.
Bienek, Diane R; Charlton, David G
2011-04-01
Rapid user-friendly ABO-Rh blood typing kits (Eldon Home Kit 2511, ABO-Rh Combination Blood Typing Experiment Kit) were evaluated to determine their accuracy when used under simulated military field conditions and after long-term storage at various temperatures and humidities. Rates of positive tests between control groups, experimental groups, and industry standards were measured and analyzed using the Fisher's exact chi-square method to identify significant differences (p < or = 0.05). When Eldon Home Kits 2511 were used in various operational conditions, the results were comparable to those obtained with the control group and with the industry standard. The performance of the ABO-Rh Combination Blood Typing Experiment Kit was adversely affected by prolonged storage in temperatures above 37 degrees C. The diagnostic performance of commercial blood typing kits varies according to product and environmental storage conditions.
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FES kinase participates in KIT-ligand induced chemotaxis
DOE Office of Scientific and Technical Information (OSTI.GOV)
Voisset, Edwige, E-mail: Edwige.Voisset@inserm.fr; Institut Paoli-Calmettes, Marseille; Universite de la Mediterranee, Aix-Marseille II
2010-02-26
FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicatedmore » in cell migration.« less
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Stability of user-friendly blood typing kits stored under typical military field conditions.
Bienek, Diane R; Chang, Cheow K; Charlton, David G
2009-10-01
To help preserve in-theater strength within deployed military units, commercially available, rapid, user-friendly ABO-Rh blood typing kits were evaluated to determine their stability in storage conditions commonly encountered by the warfighter. Methods for environmental exposure testing were based on MIL-STD-810F. When Eldon Home Kits 2511 were exposed to various temperature/relative humidity conditions, the results were comparable to those obtained with the control group and those obtained with industry-standard methods. For the ABO-Rh Combination Blood Typing Experiment Kits, 2 of the exposure treatments rendered them unusable. In addition, a third set of exposure treatments adversely affected the kits, resulting in approximately 30% blood type misclassifications. Collectively, this evaluation of commercial blood typing kits revealed that diagnostic performance can vary between products, lots, and environmental storage conditions.
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Experimental investigation of a supersonic micro turbine running with hexamethyldisiloxane
NASA Astrophysics Data System (ADS)
Weiß Andreas, P.; Josef, Hauer; Tobias, Popp; Markus, Preißinger
2017-09-01
Experimentally determined efficiency characteristics of a supersonic micro turbine are discussed in the present paper. The micro turbine is a representative of a "micro-turbine-generator-construction-kit" for ORC small scale waste heat recovery. The isentropic total-to-static efficiency of the 12 kW turbine reaches an excellent design point performance of 73.4 %. Furthermore, its off-design operating behavior is very advantageous for small waste heat recovery plants: the turbine efficiency keeps a high level over a wide range of pressure ratio and rotational speed.
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Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation
Alsaweed, Mohammed; Hepworth, Anna R.; Lefèvre, Christophe; Hartmann, Peter E.; Geddes, Donna T.
2015-01-01
ABSTRACT MicroRNA have been recently discovered in human milk signifying potentially important functions for both the lactating breast and the infant. Whilst human milk microRNA have started to be explored, little data exist on the evaluation of sample processing, and analysis to ensure that a full spectrum of microRNA can be obtained. Human milk comprises three main fractions: cells, skim milk, and lipids. Typically, the skim milk fraction has been measured in isolation despite evidence that the lipid fraction may contain more microRNA. This study aimed to standardize isolation of microRNA and total RNA from all three fractions of human milk to determine the most appropriate sampling and analysis procedure for future studies. Three different methods from eight commercially available kits were tested for their efficacy in extracting total RNA and microRNA from the lipid, skim, and cell fractions of human milk. Each fraction yielded different concentrations of RNA and microRNA, with the highest quantities found in the cell and lipid fractions, and the lowest in skim milk. The column‐based phenol‐free method was the most efficient extraction method for all three milk fractions. Two microRNAs were expressed and validated in the three milk fractions by qPCR using the three recommended extraction kits for each fraction. High expression levels were identified in the skim and lipid milk factions for these microRNAs. These results suggest that careful consideration of both the human milk sample preparation and extraction protocols should be made prior to embarking upon research in this area. J. Cell. Biochem. 116: 2397–2407, 2015. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. PMID:25925799
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Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation.
Alsaweed, Mohammed; Hepworth, Anna R; Lefèvre, Christophe; Hartmann, Peter E; Geddes, Donna T; Hassiotou, Foteini
2015-10-01
MicroRNA have been recently discovered in human milk signifying potentially important functions for both the lactating breast and the infant. Whilst human milk microRNA have started to be explored, little data exist on the evaluation of sample processing, and analysis to ensure that a full spectrum of microRNA can be obtained. Human milk comprises three main fractions: cells, skim milk, and lipids. Typically, the skim milk fraction has been measured in isolation despite evidence that the lipid fraction may contain more microRNA. This study aimed to standardize isolation of microRNA and total RNA from all three fractions of human milk to determine the most appropriate sampling and analysis procedure for future studies. Three different methods from eight commercially available kits were tested for their efficacy in extracting total RNA and microRNA from the lipid, skim, and cell fractions of human milk. Each fraction yielded different concentrations of RNA and microRNA, with the highest quantities found in the cell and lipid fractions, and the lowest in skim milk. The column-based phenol-free method was the most efficient extraction method for all three milk fractions. Two microRNAs were expressed and validated in the three milk fractions by qPCR using the three recommended extraction kits for each fraction. High expression levels were identified in the skim and lipid milk factions for these microRNAs. These results suggest that careful consideration of both the human milk sample preparation and extraction protocols should be made prior to embarking upon research in this area. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.
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Sequence-specific inhibition of microRNA-130a gene by CRISPR/Cas9 system in breast cancer cell line
NASA Astrophysics Data System (ADS)
Ainina Abdollah, Nur; Das Kumitaa, Theva; Yusof Narazah, Mohd; Razak, Siti Razila Abdul
2017-05-01
MicroRNAs (miRNAs) are short stranded noncoding RNA that play important roles in apoptosis, cell survival, development and cell proliferation. However, gene expression control via small regulatory RNA, particularly miRNA in breast cancer is still less explored. Therefore, this project aims to develop an approach to target microRNA-130a using the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system in MCF7, breast cancer cell line. The 20 bp sequences target at stem loop, 3ʹ and 5ʹ end of miR130a were cloned into pSpCas9(BB)-2A-GFP (PX458) plasmid, and the positive clones were confirmed by sequencing. A total of 5 μg of PX458-miR130a was transfected to MCF7 using Lipofectamine® 3000 according to manufacturer’s protocol. The transfected cells were maintained in the incubator at 37 °C under humidified 5% CO2. After 48 hours, cells were harvested and total RNA was extracted using miRNeasy Mini Kit (Qiagen). cDNAs were synthesised specific to miR-130a using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Then, qRT-PCR was carried out using TaqMan Universal Master Mix (Applied Biosystems) to quantify the knockdown level of mature miRNAs in the cells. Result showed that miR-130a-5p was significantly downregulated in MCF7 cell line. However, no significant changes were observed for sequences targeting miR-130a-3p and stem loop. Thus, this study showed that the expression of miR-130a-5p was successfully down-regulated using CRISPR silencing system. This technique may be useful to manipulate the level of miRNA in various cell types to answer clinical questions at the molecular level.
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46 CFR 160.054-2 - Type and size.
Code of Federal Regulations, 2011 CFR
2011-10-01
...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-2 Type and size. (a) Type. First-aid kits covered by this specification shall be of the water-tight type... special consideration. (b) Size. First-aid kits shall be of a size adequate for packing 12 standard single...
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46 CFR 160.054-2 - Type and size.
Code of Federal Regulations, 2014 CFR
2014-10-01
...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-2 Type and size. (a) Type. First-aid kits covered by this specification shall be of the water-tight type... special consideration. (b) Size. First-aid kits shall be of a size adequate for packing 12 standard single...
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46 CFR 160.054-2 - Type and size.
Code of Federal Regulations, 2013 CFR
2013-10-01
...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-2 Type and size. (a) Type. First-aid kits covered by this specification shall be of the water-tight type... special consideration. (b) Size. First-aid kits shall be of a size adequate for packing 12 standard single...
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46 CFR 160.054-2 - Type and size.
Code of Federal Regulations, 2012 CFR
2012-10-01
...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-2 Type and size. (a) Type. First-aid kits covered by this specification shall be of the water-tight type... special consideration. (b) Size. First-aid kits shall be of a size adequate for packing 12 standard single...
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46 CFR 160.054-2 - Type and size.
Code of Federal Regulations, 2010 CFR
2010-10-01
...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-2 Type and size. (a) Type. First-aid kits covered by this specification shall be of the water-tight type... special consideration. (b) Size. First-aid kits shall be of a size adequate for packing 12 standard single...
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A meta-analysis of prognostic value of KIT mutation status in gastrointestinal stromal tumors
Jiang, Zhiqiang; Zhang, Jian; Li, Zhi; Liu, Yingjun; Wang, Daohai; Han, Guangsen
2016-01-01
Numerous types of KIT mutations have been reported in gastrointestinal stromal tumors (GISTs); however, controversy still exists regarding their clinicopathological significance. In this study, we reviewed the publicly available literature to assess the data by a meta-analysis to characterize KIT mutations and different types of KIT mutations in prognostic prediction in patients with GISTs. Twenty-eight studies that included 4,449 patients were identified and analyzed. We found that KIT mutation status was closely correlated with size of tumors and different mitosis indexes, but not with tumor location. KIT mutation was also observed to be significantly correlated with tumor recurrence, metastasis, as well as the overall survival of patients. Interestingly, there was higher risk of progression in KIT exon 9-mutated patients than in exon 11-mutated patients. Five-year relapse-free survival (RFS) rate was significantly higher in KIT exon 11-deleted patients than in those with other types of KIT exon 11 mutations. In addition, RFS for 5 years was significantly worse in patients bearing KIT codon 557–558 deletions than in those bearing other KIT exon 11 deletions. Our results strongly support the hypothesis that KIT mutation status is another evaluable factor for prognosis prediction in GISTs. PMID:27350754
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Matsuo, Toshihiro; Miyagawa, Jun-Ichiro; Kusunoki, Yoshiki; Miuchi, Masayuki; Ikawa, Takashi; Akagami, Takafumi; Tokuda, Masaru; Katsuno, Tomoyuki; Kushida, Akira; Inagaki, Takashi; Namba, Mitsuyoshi
2016-05-01
The aims of the present study were to investigate the performance of a novel sandwich enzyme-linked immunosorbent assay (ELISA) for measuring glucagon (1-29) with monoclonal antibodies against both the C- and N-terminal regions of glucagon (1-29), and to analyze the differences in plasma levels and responses of glucagon (1-29) to oral glucose loading in normal glucose tolerance (NGT) subjects and patients with type 2 diabetes mellitus. The cross-reactivity against proglucagon fragments using the ELISA kit and two types of conventional radioimmunoassay (RIA) kits was evaluated. A 75-g oral glucose tolerance test was carried out with NGT subjects and patients with type 2 diabetes mellitus, and the glucagon (1-29) concentration was measured using three types of kit. The ELISA kit clearly had the lowest cross-reactivity against miniglucagon (19-29) and glicentin (1-61). The oral glucose tolerance test was carried out with 30 NGT and 17 patients with type 2 diabetes mellitus. The glucagon (1-29) levels measured by the ELISA kit after glucose loading were significantly higher at all time-points in the type 2 diabetes mellitus group than in the NGT group. However, the glucagon (1-29) levels measured by one RIA kit were significantly higher in the NGT group, and those measured with the other RIA kit were approximately the same among the groups. The novel sandwich ELISA accurately determines plasma glucagon (1-29) concentrations with much less cross-reactivity against other proglucagon fragments than conventional RIA kits.
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46 CFR 160.041-2 - Type and size.
Code of Federal Regulations, 2011 CFR
2011-10-01
...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Merchant Vessels § 160.041-2 Type and size. (a) Type. First-aid kits covered by this specification shall be of the water-tight cabinet carrying... consideration. (b) Size. First-aid kits shall be of a size (approximately 9″×9″×21/2″ inside) adequate for...
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46 CFR 160.041-2 - Type and size.
Code of Federal Regulations, 2012 CFR
2012-10-01
...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Merchant Vessels § 160.041-2 Type and size. (a) Type. First-aid kits covered by this specification shall be of the water-tight cabinet carrying... consideration. (b) Size. First-aid kits shall be of a size (approximately 9″×9″×21/2″ inside) adequate for...
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46 CFR 160.041-2 - Type and size.
Code of Federal Regulations, 2013 CFR
2013-10-01
...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Merchant Vessels § 160.041-2 Type and size. (a) Type. First-aid kits covered by this specification shall be of the water-tight cabinet carrying... consideration. (b) Size. First-aid kits shall be of a size (approximately 9″ × 9″ × 21/2″ inside) adequate for...
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46 CFR 160.041-2 - Type and size.
Code of Federal Regulations, 2014 CFR
2014-10-01
...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Merchant Vessels § 160.041-2 Type and size. (a) Type. First-aid kits covered by this specification shall be of the water-tight cabinet carrying... consideration. (b) Size. First-aid kits shall be of a size (approximately 9″ × 9″ × 21/2″ inside) adequate for...
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46 CFR 160.041-2 - Type and size.
Code of Federal Regulations, 2010 CFR
2010-10-01
...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Merchant Vessels § 160.041-2 Type and size. (a) Type. First-aid kits covered by this specification shall be of the water-tight cabinet carrying... consideration. (b) Size. First-aid kits shall be of a size (approximately 9″×9″×21/2″ inside) adequate for...
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Investigating the isolation and amplification of microRNAs for forensic body fluid identification.
Glynn, Claire L; O Leary, Kelsie R
2018-04-30
The discovery of forensic DNA typing evolved molecular biology far beyond what could have been expected in terms of its forensic application, and now there exists other developments in molecular biology which are ready for application to forensic challenges. One such challenge is the identification of the body fluid source of stains recovered from evidence items and crime scenes. Currently there are significant efforts in the research field to develop novel methods for the molecular identification of body fluids, with microRNAs (miRNAs) revealing great potential. MiRNAs have been shown to have high tissue specificity and are less susceptible to degradation as a result of their small size, which infers great advantages to their potential role for identifying forensically relevant body fluids. This study investigated the isolation and amplification of miRNAs from forensically relevant body fluids. Venous blood, menstrual blood, semen, saliva, and vaginal material samples were extracted using; miRNeasy® mini kit (Qiagen), mirVana™ miRNA isolation kit (Ambion), and a modified mirVana™ method, and the quality/quantity of isolated miRNA was determined. miRNAs previously identified to show specificity for particular forensically relevant body fluids were examined. Real Time-Quantitative PCR (RT-qPCR) was performed targeting 5 miRNAs of interest, miR-451, miR-412, miR-891a, miR-205 and miR-124a. This study identified the miRNeasy® mini kit as the optimal method of the three methods investigated for the extraction of miRNAs from body fluids and further validates a selection of miRNAs previously suggested as potential biomarkers. This research highlights the potential of miRNAs as novel markers for the identification of forensically relevant body fluids. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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Sun, Yuning; Xu, Rongfeng; Huang, Jia; Yao, Yuyu; Pan, Xiaodong; Chen, Zhongpu; Ma, Genshan
2018-02-21
C-kit-positive cardiac stem cells (CSCs) have been shown to be a promising candidate treatment for myocardial infarction and heart failure. Insulin-like growth factor (IGF)-1 is an anabolic growth hormone that regulates cellular proliferation, differentiation, senescence, and death in various tissues. Although IGF-1 promotes the migration and proliferation of c-kit-positive mouse CSCs, the underlying mechanism remains unclear. Cells were isolated from adult mouse hearts, and c-kit-positive CSCs were separated using magnetic beads. The cells were cultured with or without IGF-1, and c-kit expression was measured by Western blotting. IGF-1 induced CSC proliferation and migration, as measured through Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The miR-193a expression was measured by quantitative real-time PCR (qPCR) assays. IGF-1 enhanced c-kit expression in c-kit-positive CSCs. The activities of the phosphoinositol 3-kinase (PI3K)/AKT signaling pathway and DNA methyltransferases (DNMTs) were enhanced, and their respective inhibitors LY294002 and 5-azacytidine (5-AZA) blunted c-kit expression. Based on the results of quantitative real-time PCR (qPCR) assays, the expression of miR-193a, which is embedded in a CpG island, was down-regulated in the IGF-1-stimulated group and negatively correlated with c-kit expression, whereas c-kit-positive CSCs infected with lentivirus carrying micro-RNA193a displayed reduced c-kit expression, migration and proliferation. IGF-1 upregulated c-kit expression in c-kit-positive CSCs resulting in enhanced CSC proliferation and migration by activating the PI3K/AKT/DNMT signaling pathway to epigenetically silence miR-193a, which negatively modifies the c-kit expression level.
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Immunohistochemical study of C-kit expression in subtypes of renal cell carcinoma.
Norouzinia, Farahnaz; Abbasi, Fariba; Dindarian, Sina; Mohammadi, Sedra; Meisami, Farid; Bagheri, Mahdi; Mohammadi, Hozan
2018-01-01
Renal cell carcinomas (RCCs) include about 2% of adult neoplasms and 90-95% of all renal tumors. Mostly, it is possible to distinguish RCC subtypes using hematoxylin-eosin staining. However, overlapping morphologic features cause some difficulties in making a precise diagnosis. In order to render an accurate diagnosis, additional methods such as immunohistochemical staining for c-kit have been recommended. In this study, we aimed to investigate c-kit gene expression in various subtypes of RCC. We reviewed 65 diagnosed RCC cases. Formalin- fixed, paraffin- embedded specimens were available for the cases. The expression of c-kit was evaluated using immunohistochemistry. The correlation between c-kit expression and clinicopathological parameters including patients' age and gender in addition to grade, stage, and size of the tumor were investigated. Six cases of 39 clear cell types (15.4%), 8 of 13 papillary types (61.5%), 11 of 12 chromophobe types (91.7%), and no sarcomatoid type were positive for c-kit expression. Based on chi-square test results, there was a significant relationship between RCC subtypes and c-kit expression (p=0.001). There was no significant correlation between age, sex, grade, stage, and size of the tumor and c-kit expression. The expression of c-kit in RCC may have diagnostic significance in subtypes of RCC especially papillary and chromophobe subtypes of RCC.
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NASA Technical Reports Server (NTRS)
Stow, D. A.; Estes, J. E.; Mertz, F. C.
1981-01-01
A learning kit is an essential part of any remote sensing workshop, course, or in-house training program to provide the "hands-on" experience of working with remotely sensed imagery. This is the objective of laboratory and field exercises as well as the reason behind the production of imagery/map kits. The way in which these learning kits (containing conventional remotely sensed and collateral data products) are put together is described and some concerns that influence the creation of learning kits are discussed. These include budgetary constraints, number of imagery types, and number of collateral data types.
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Kohiyama, Risa; Miyazawa, Takashi; Shibano, Nobuko; Inano, Koichi
2014-01-01
Because it is not easy to differentiate Influenza virus (Flu) from RS virus (RSV) just by clinical symptoms, to accurately diagnose those viruses in conjunction with patient's clinical symptoms, rapid diagnostic kits has been used separately for each of those viruses. In our new study, we have developed a new rapid diagnostic kit, QuickNavi™-Flu+RSV. The kit can detect Flu A, Flu B, and RSV antigens with a single sample collection and an assay. Total of 2,873 cases (including nasopharyngeal swabs and nasopharyngeal aspirates specimens) in 2010/2011 and 2011/2012 seasons were evaluated with QuickNavi™-Flu+RSV and a commercially available kit. Sensitivity, specificity, and accuracy of Flu type A, type B, and RSV were above 95% when compared to commercially available kits (QuickNavi™-Flu and QuickNavi™-RSV) and considered to be equivalent to the commercially available kits. In 2011/2012 season, RSV infections increased prior to Flu season and continued during the peak of the Flu season. The kit can contribute to accurate diagnosis of Flu and RSV infections since co-infection cases have also been reported during the 2011/2012 season. QuickNavi™-Flu+RSV is useful for differential diagnosis of respiratory infectious diseases since it can detect Flu type A, type B, and RSV virus antigens with a single sample collection.
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The clinical significance of c-Kit mutations in metastatic oral mucosal melanoma in China.
Ma, Xuhui; Wu, Yunteng; Zhang, Tian; Song, Hao; Jv, Houyu; Guo, Wei; Ren, Guoxin
2017-10-10
c-Kit mutations are frequently detected in mucosal melanomas, but their clinical significance in metastatic oral mucosal melanomas (OMM) remains unclear. The main purpose of this study was to investigate the clinical and pathological features of metastatic OMMs with c-Kit mutations and the efficiency of the tyrosine kinase inhibitor imatinib in treating metastatic OMMs. We found thatresidual primary lesion and neck lymph nodes could act as independent prognostic factors in metastatic OMM patients. c-Kit mutations were detected in 22 out of 139 (15.8%) metastatic OMM patients. Under chemotherapy, the overall survival (OS) of c-Kit mutant patients was significantly shorter than that of wild-type patients. The Ki67 expression was significantly higher in c-Kit mutant patients than in wild-type patients. In distant metastatic OMM patients with c-Kit mutations, the treatment with c-Kit inhibitor resulted in a better OS. In conclusion, residual primary lesion, cervical lymph nodes and c-Kit mutations act as adverse prognostic factors of metastatic OMMs. The Kit inhibitor imatinib could benefit metastatic OMM patients with c-Kit mutations.
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Wong, Y-P; Othman, S; Lau, Y-L; Radu, S; Chee, H-Y
2018-03-01
Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease. © 2017 The Society for Applied Microbiology.
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ERIC Educational Resources Information Center
School Science Review, 1983
1983-01-01
Provided are reviews of science equipment/apparatus. Items reviewed include: Harris Micro-ecology tubes; Harris chromosome investigation kit; Harris trycult slides; a pressure cooker with thermometer; digital pH meter; digital scaler timer; electrical compensation calorimeter; and Mains alternating current ammeter. (JN)
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Defective bone repair in mast cell-deficient Cpa3Cre/+ mice.
Ramirez-GarciaLuna, Jose Luis; Chan, Daniel; Samberg, Robert; Abou-Rjeili, Mira; Wong, Timothy H; Li, Ailian; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Henderson, Janet E; Martineau, Paul A
2017-01-01
In the adult skeleton, cells of the immune system interact with those of the skeleton during all phases of bone repair to influence the outcome. Mast cells are immune cells best known for their pathologic role in allergy, and may be involved in chronic inflammatory and fibrotic disorders. Potential roles for mast cells in tissue homeostasis, vascularization and repair remain enigmatic. Previous studies in combined mast cell- and Kit-deficient KitW-sh/W-sh mice (KitW-sh) implicated mast cells in bone repair but KitW-sh mice suffer from additional Kit-dependent hematopoietic and non- hematopoietic deficiencies that could have confounded the outcome. The goal of the current study was to compare bone repair in normal wild type (WT) and Cpa3Cre/+ mice, which lack mast cells in the absence of any other hematopoietic or non- hematopoietic deficiencies. Repair of a femoral window defect was characterized using micro CT imaging and histological analyses from the early inflammatory phase, through soft and hard callus formation, and finally the remodeling phase. The data indicate 1) mast cells appear in healing bone of WT mice but not Cpa3Cre/+ mice, beginning 14 days after surgery; 2) re-vascularization of repair tissue and deposition of mineralized bone was delayed and dis-organised in Cpa3Cre/+ mice compared with WT mice; 3) the defects in Cpa3Cre/+ mice were associated with little change in anabolic activity and biphasic alterations in osteoclast and macrophage activity. The outcome at 56 days postoperative was complete bridging of the defect in most WT mice and fibrous mal-union in most Cpa3Cre/+ mice. The results indicate that mast cells promote bone healing, possibly by recruiting vascular endothelial cells during the inflammatory phase and coordinating anabolic and catabolic activity during tissue remodeling. Taken together the data indicate that mast cells have a positive impact on bone repair.
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Defective bone repair in mast cell-deficient Cpa3Cre/+ mice
Chan, Daniel; Samberg, Robert; Abou-Rjeili, Mira; Wong, Timothy H.; Li, Ailian; Feyerabend, Thorsten B.; Rodewald, Hans-Reimer; Henderson, Janet E.; Martineau, Paul A.
2017-01-01
In the adult skeleton, cells of the immune system interact with those of the skeleton during all phases of bone repair to influence the outcome. Mast cells are immune cells best known for their pathologic role in allergy, and may be involved in chronic inflammatory and fibrotic disorders. Potential roles for mast cells in tissue homeostasis, vascularization and repair remain enigmatic. Previous studies in combined mast cell- and Kit-deficient KitW-sh/W-sh mice (KitW-sh) implicated mast cells in bone repair but KitW-sh mice suffer from additional Kit-dependent hematopoietic and non- hematopoietic deficiencies that could have confounded the outcome. The goal of the current study was to compare bone repair in normal wild type (WT) and Cpa3Cre/+ mice, which lack mast cells in the absence of any other hematopoietic or non- hematopoietic deficiencies. Repair of a femoral window defect was characterized using micro CT imaging and histological analyses from the early inflammatory phase, through soft and hard callus formation, and finally the remodeling phase. The data indicate 1) mast cells appear in healing bone of WT mice but not Cpa3Cre/+ mice, beginning 14 days after surgery; 2) re-vascularization of repair tissue and deposition of mineralized bone was delayed and dis-organised in Cpa3Cre/+ mice compared with WT mice; 3) the defects in Cpa3Cre/+ mice were associated with little change in anabolic activity and biphasic alterations in osteoclast and macrophage activity. The outcome at 56 days postoperative was complete bridging of the defect in most WT mice and fibrous mal-union in most Cpa3Cre/+ mice. The results indicate that mast cells promote bone healing, possibly by recruiting vascular endothelial cells during the inflammatory phase and coordinating anabolic and catabolic activity during tissue remodeling. Taken together the data indicate that mast cells have a positive impact on bone repair. PMID:28350850
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Surveillance for Enteric Pathogens in a Case-Control Study of Acute Diarrhea in Western Kenya
2012-11-01
isolates was ascertained using the MicroScan WalkAway 40 Plus. An enzyme immunoassay kit was used to detect rotavirus , and ova and parasite...examination was conducted by micros- copy and an enzyme immunoassay. Results: Rotavirus (10.2% and 10.5%) and Shigella (11% and 8%) were isolated significantly...Keywords: Gastroenteritis, Protozoa, Rotavirus , Shigella, Escherichia coli, Antimicrobial resistance Introduction Diarrheal disease continues to be a
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Cataldi, Mauro; Gaudino, Annarita; Lariccia, Vincenzo; Russo, Michela; Amoroso, Salvatore; di Renzo, Gianfranco; Annunziato, Lucio
2004-04-01
The 2-phenylaminopyrimidine derivative imatinib-mesylate, a powerful protein tyrosine kinase (PTK) inhibitor that targets abl, c-kit, and the platelet-derived growth factor receptors, is rapidly gaining a relevant role in the treatment of several types of neoplasms. Because first generation PTK inhibitors affect the activity of a large number of voltage-dependent ion channels, the present study explored the possibility that imatinib-mesylate could interfere with the activity of T-type channels, a class of voltage-dependent Ca2+ channels that take part in the chain of events elicited by PTK activation. The effect of the drug on T-type channel activity was examined using the whole-cell patch-clamp technique with Ba2+ (10 mM) as the permeant ion in human embryonic kidney-293 cells, stably expressing the rat Ca(V)3.3 channels. Imatinib-mesylate concentrations, ranging from 30 to 300 microM, reversibly decreased Ca(V)3.3 current amplitude with an IC(50) value of 56.9 microM. By contrast, when imatinib-mesylate (500 microM) was intracellularly dialyzed with the pipette solution, no reduction in Ba2+ current density was observed. The 2-phenylaminopyrimidine derivative modified neither the voltage dependence of activation nor the steady-state inactivation of Ca(V)3.3 channels. The decrease in extracellular Ba2+ concentration from 10 to 2 mM and the substitution of Ca2+ for Ba2+ increased the extent of 30 microM imatinib-mesylate-induced percentage of channel blockade from 25.9 +/- 2.4 to 36.3 +/- 0.9% in 2 mM Ba2+ and 44.2 +/- 2.3% in 2 mM Ca2+. In conclusion, imatinib-mesylate blocked the cloned Ca(V)3.3 channels by a PTK-independent mechanism. Specifically, the drug did not affect the activation or the inactivation of the channel but interfered with the ion permeation process.
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A technical assessment of the porcine ejaculated spermatozoa for a sperm-specific RNA-seq analysis.
Gòdia, Marta; Mayer, Fabiana Quoos; Nafissi, Julieta; Castelló, Anna; Rodríguez-Gil, Joan Enric; Sánchez, Armand; Clop, Alex
2018-04-26
The study of the boar sperm transcriptome by RNA-seq can provide relevant information on sperm quality and fertility and might contribute to animal breeding strategies. However, the analysis of the spermatozoa RNA is challenging as these cells harbor very low amounts of highly fragmented RNA, and the ejaculates also contain other cell types with larger amounts of non-fragmented RNA. Here, we describe a strategy for a successful boar sperm purification, RNA extraction and RNA-seq library preparation. Using these approaches our objectives were: (i) to evaluate the sperm recovery rate (SRR) after boar spermatozoa purification by density centrifugation using the non-porcine-specific commercial reagent BoviPure TM ; (ii) to assess the correlation between SRR and sperm quality characteristics; (iii) to evaluate the relationship between sperm cell RNA load and sperm quality traits and (iv) to compare different library preparation kits for both total RNA-seq (SMARTer Universal Low Input RNA and TruSeq RNA Library Prep kit) and small RNA-seq (NEBNext Small RNA and TailorMix miRNA Sample Prep v2) for high-throughput sequencing. Our results show that pig SRR (~22%) is lower than in other mammalian species and that it is not significantly dependent of the sperm quality parameters analyzed in our study. Moreover, no relationship between the RNA yield per sperm cell and sperm phenotypes was found. We compared a RNA-seq library preparation kit optimized for low amounts of fragmented RNA with a standard kit designed for high amount and quality of input RNA and found that for sperm, a protocol designed to work on low-quality RNA is essential. We also compared two small RNA-seq kits and did not find substantial differences in their performance. We propose the methodological workflow described for the RNA-seq screening of the boar spermatozoa transcriptome. FPKM: fragments per kilobase of transcript per million mapped reads; KRT1: keratin 1; miRNA: micro-RNA; miscRNA: miscellaneous RNA; Mt rRNA: mitochondrial ribosomal RNA; Mt tRNA: mitochondrial transference RNA; OAZ3: ornithine decarboxylase antizyme 3; ORT: osmotic resistance test; piRNA: Piwi-interacting RNA; PRM1: protamine 1; PTPRC: protein tyrosine phosphatase receptor type C; rRNA: ribosomal RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; SRR: sperm recovery rate; tRNA: transfer RNA.
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Code of Federal Regulations, 2014 CFR
2014-10-01
... APPROVAL LIFESAVING EQUIPMENT Desalter Kits, Sea Water, for Merchant Vessels § 160.058-2 Type. (a) Desalter kits specified by this subpart shall be of the type described in the specification listed in § 160.058...
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Code of Federal Regulations, 2013 CFR
2013-10-01
... APPROVAL LIFESAVING EQUIPMENT Desalter Kits, Sea Water, for Merchant Vessels § 160.058-2 Type. (a) Desalter kits specified by this subpart shall be of the type described in the specification listed in § 160.058...
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Code of Federal Regulations, 2012 CFR
2012-10-01
... APPROVAL LIFESAVING EQUIPMENT Desalter Kits, Sea Water, for Merchant Vessels § 160.058-2 Type. (a) Desalter kits specified by this subpart shall be of the type described in the specification listed in § 160.058...
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Code of Federal Regulations, 2011 CFR
2011-10-01
... APPROVAL LIFESAVING EQUIPMENT Desalter Kits, Sea Water, for Merchant Vessels § 160.058-2 Type. (a) Desalter kits specified by this subpart shall be of the type described in the specification listed in § 160.058...
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Roskoski, Robert
2018-04-25
The Kit proto-oncogene was found as a consequence of the discovery of the feline v-kit sarcoma oncogene. Stem cell factor (SCF) is the Kit ligand and it mediates Kit dimerization and activation. The Kit receptor contains an extracellular segment that is made up of five immunoglobulin-like domains (D1/2/3/4/5), a transmembrane segment, a juxtamembrane segment, a protein-tyrosine kinase domain that contains an insert of 77 amino acid residues, and a carboxyterminal tail. Activating somatic mutations in Kit have been documented in various neoplasms including gastrointestinal stromal tumors (GIST), mast cell overexpression (systemic mastocytosis), core-binding factor acute myeloid leukemias (AML), melanomas, and seminomas. In the case of gastrointestinal stromal tumors, most activating mutations occur in the juxtamembrane segment and these mutants are initially sensitive to imatinib. As with many targeted anticancer drugs, resistance to Kit antagonists occurs in about two years and is the result of secondary KIT mutations. An activation segment exon 17 D816V mutation is one of the more common resistance mutations in Kit and this mutant is resistant to imatinib and sorafenib. Type I protein kinase inhibitors interact with the active enzyme form with DFG-D of the proximal activation segment directed inward toward the active site (DFG-D in ). In contrast, type II inhibitors bind to their target with the DFG-D pointing away from the active site (DFG-D out ). Based upon the X-ray crystallographic structures, imatinib, sunitinib, and ponatinib are Type II Kit inhibitors. We used the Schrödinger induced fit docking protocol to model the interaction of midostaurin with Kit and the result indicates that it binds to the DFG-D in conformation of the receptor and is thus classified as type I inhibitor. This medication inhibits the notoriously resistant Kit D816V mutant and is approved for the treatment of systemic mastocytosis and is effective against tumors bearing the D816V activation/resistance mutation. Copyright © 2018 Elsevier Ltd. All rights reserved.
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KIT Suppresses BRAFV600E-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling.
Neiswender, James V; Kortum, Robert L; Bourque, Caitlin; Kasheta, Melissa; Zon, Leonard I; Morrison, Deborah K; Ceol, Craig J
2017-11-01
The receptor tyrosine kinase KIT promotes survival and migration of melanocytes during development, and excessive KIT activity hyperactivates the RAS/MAPK pathway and can drive formation of melanomas, most notably of rare melanomas that occur on volar and mucosal surfaces of the skin. The much larger fraction of melanomas that occur on sun-exposed skin is driven primarily by BRAF- or NRAS-activating mutations, but these melanomas exhibit a surprising loss of KIT expression, which raises the question of whether loss of KIT in these tumors facilitates tumorigenesis. To address this question, we introduced a kit(lf) mutation into a strain of Tg(mitfa:BRAF V600E ); p53(lf) melanoma-prone zebrafish. Melanoma onset was accelerated in kit(lf); Tg(mitfa:BRAF V600E ); p53(lf) fish. Tumors from kit(lf) animals were more invasive and had higher RAS/MAPK pathway activation. KIT knockdown also increased RAS/MAPK pathway activation in a BRAF V600E -mutant human melanoma cell line. We found that pathway stimulation upstream of BRAF V600E could paradoxically reduce signaling downstream of BRAF V600E , and wild-type BRAF was necessary for this effect, suggesting that its activation can dampen oncogenic BRAF V600E signaling. In vivo , expression of wild-type BRAF delayed melanoma onset, but only in a kit -dependent manner. Together, these results suggest that KIT can activate signaling through wild-type RAF proteins, thus interfering with oncogenic BRAF V600E -driven melanoma formation. Cancer Res; 77(21); 5820-30. ©2017 AACR . ©2017 American Association for Cancer Research.
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[Consistency study of PowerPlex 21 kit and Goldeneye 20A kit and forensic application].
Ren, He; Liu, Ying; Zhang, Qing-Xia; Jiao, Zhang-Ping
2014-06-01
To ensure the consistency of genotype results for PowerPlex 21 kit and Goldeneye 20A kit. The STR loci were amplified in DNA samples from 205 unrelated individuals in Beijing Han population. And consistency of 19 overlap STR loci typing were observed. The genetic polymorphism of D1S1656 locus was obtained. All 19 overlap loci typing showed consistent. The proportion of peak height of heterozygous loci in two kits showed no statistical difference (P > 0.05). The observed heterozygosis of D1S1656 was 0.878. The discrimination power was 0.949. The excluding probability of paternity of triplet was 0.751. The excluding probability of paternity of diploid was 0.506. The polymorphism information content was 0.810. PowerPlex 21 kit and Goldeneye 20A kit present a good consistency. The primer design is reasonable. The polymorphism of D1S1656 is good. The two kits can be used for human genetic analysis, paternity test, and individual identification in forensic practice.
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Zupanič Pajnič, Irena; Gornjak Pogorelc, Barbara; Balažic, Jože; Zupanc, Tomaž; Štefanič, Borut
2012-01-01
Aim To perform an efficiency study of three new amplification kits with the extended European Standard Set (ESS) of loci for autosomal short tandem repeat (STR) typing of skeletal remains excavated from the World War II mass graves in Slovenia. Methods In the beginning of the 2011, we analyzed 102 bones and teeth using the PowerPlex ESX 17 System (Promega), AmpFiSTR NGM PCR Amplification Kit (Applied Biosystems), and Investigator ESSplex Kit (Qiagen). We cleaned the bones and teeth, removed surface contamination, and ground them into a powder using liquid nitrogen. Prior to DNA isolation with Biorobot EZ1 (Qiagen), 0.5 g bone or tooth powder was decalcified. Nuclear DNA of the samples was quantified using real-time polymerase chain reaction. All three kits used the same extract with the amplification conditions recommended by the manufacturers. Results We extracted up to 131 ng DNA/g of powder from the bones and teeth. All three amplification kits showed very similar efficiency, since DNA typing was successful with all amplification kits in 101 out of 102 bones and teeth, which represents a 99% success rate. Conclusion The commercially available ESX 17, ESSplex, and NGM kits are highly reliable for STR typing of World War II skeletal remains with the DNA extraction method optimized in our laboratory. PMID:22351574
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Brahimi-Adouane, Sabrina; Bachet, Jean-Baptiste; Tabone-Eglinger, Séverine; Subra, Frédéric; Capron, Claude; Blay, Jean-Yves; Emile, Jean-François
2013-06-01
Gain of function mutations of KIT are frequent in some human tumors, and are sensible to tyrosine kinase inhibitors. In most tumors, oncogenic mutations are heterozygous, however most in vitro data of KIT activation have been obtained with hemizygous mutation. This study aimed to investigate the maturation and activation of wild-type (WT) and mutant (M) forms of KIT in hemizygous and heterozygous conditions. WT and two types of exon 11 deletions M forms of human KIT were expressed in NIH3T3 cell lines. Membrane expression of KIT was quantified by flow cytometry. Quantification of glycosylated forms of KIT and phosphorylated forms of AKT and ERK were performed by western blot. Simultaneous activation of WT KIT and treatment with endoplasmic reticulum (ER) inhibitors, tunicamycin or brefeldin A induced a complete inhibition of membrane expression of the 145 kDa form of KIT. By contrast activation or ER inhibitors alone, only partly inhibited this form. ER inhibitors also inhibited KIT activation-dependent phosphorylation of AKT and ERK1/2. Brefeldin A induced a complete down regulation of the 145 kDa form in hemizygous M, and induced an intra-cellular accumulation of the 125 kDa form in WT but not in hemizygous M. Heterozygous cells had glycosylation and response to ER inhibitors patterns more similar to WT than to hemizygous M. Phosphorylated AKT was reduced in hemizygous cells in comparison to WT KIT cells and heterozygous cells, and in the presence of brefeldin A in all cell lines. Effects of ER inhibitors are significantly different in hemizygous and heterozygous mutants. Differences in intra-cellular trafficking of KIT forms result in differences in downstream signaling pathways, and activation of PI3K/AKT pathway appears to be tied to the presence of the mature 145 kDa form of KIT at the membrane surface. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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Antonescu, Cristina R; Viale, Agnes; Sarran, Lisa; Tschernyavsky, Sylvia J; Gonen, Mithat; Segal, Neil H; Maki, Robert G; Socci, Nicholas D; DeMatteo, Ronald P; Besmer, Peter
2004-05-15
Gastrointestinal stromal tumors (GISTs) are specific KIT expressing and KIT-signaling driven mesenchymal tumors of the human digestive tract, many of which have KIT-activating mutations. Previous studies have found a relatively homogeneous gene expression profile in GIST, as compared with other histological types of sarcomas. Transcriptional heterogeneity within clinically or molecularly defined subsets of GISTs has not been previously reported. We tested the hypothesis that the gene expression profile in GISTs might be related to KIT genotype and possibly to other clinicopathological factors. An HG-U133A Affymetrix chip (22,000 genes) platform was used to determine the variability of gene expression in 28 KIT-expressing GIST samples from 24 patients. A control group of six intra-abdominal leiomyosarcomas was also included for comparison. Statistical analyses (t tests) were performed to identify discriminatory gene lists among various GIST subgroups. The levels of expression of various GIST subsets were also linked to a modified version of the growth factor/KIT signaling pathway to analyze differences at various steps in signal transduction. Genes involved in KIT signaling were differentially expressed among wild-type and mutant GISTs. High gene expression of potential drug targets, such as VEGF, MCSF, and BCL2 in the wild-type group, and Mesothelin in exon 9 GISTs were found. There was a striking difference in gene expression between stomach and small bowel GISTs. This finding was validated in four separate tumors, two gastric and two intestinal, from a patient with familial GIST with a germ-line KIT W557R substitution. GISTs have heterogeneous gene expression depending on KIT genotype and tumor location, which is seen at both the genomic level and the KIT signaling pathway in particular. These findings may explain their variable clinical behavior and response to therapy.
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Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I.; Dwyer, Karen M.; Saffery, Richard
2018-01-01
Aim To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. Background DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. Methods QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Results Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0–0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0–9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0–17.7μg/mL and 0–1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. Conclusion High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease. PMID:29462136
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High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos
Ruocco, Nadia; Costantini, Susan; Zupo, Valerio; Romano, Giovanna; Ianora, Adrianna; Fontana, Angelo; Costantini, Maria
2017-01-01
The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this established model marine organism. PMID:28199408
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Lecamwasam, Ashani; Sexton-Oates, Alexandra; Carmody, Jake; Ekinci, Elif I; Dwyer, Karen M; Saffery, Richard
2018-01-01
To characterise the genomic DNA (gDNA) yield from urine and quality of derived methylation data generated from the widely used Illuminia Infinium MethylationEPIC (HM850K) platform and compare this with buffy coat samples. DNA methylation is the most widely studied epigenetic mark and variations in DNA methylation profile have been implicated in diabetes which affects approximately 415 million people worldwide. QIAamp Viral RNA Mini Kit and QIAamp DNA micro kit were used to extract DNA from frozen and fresh urine samples as well as increasing volumes of fresh urine. Matched buffy coats to the frozen urine were also obtained and DNA was extracted from the buffy coats using the QIAamp DNA Mini Kit. Genomic DNA of greater concentration than 20μg/ml were used for methylation analysis using the HM850K array. Irrespective of extraction technique or the use of fresh versus frozen urine samples, limited genomic DNA was obtained using a starting sample volume of 5ml (0-0.86μg/mL). In order to optimize the yield, we increased starting volumes to 50ml fresh urine, which yielded only 0-9.66μg/mL A different kit, QIAamp DNA Micro Kit, was trialled in six fresh urine samples and ten frozen urine samples with inadequate DNA yields from 0-17.7μg/mL and 0-1.6μg/mL respectively. Sufficient genomic DNA was obtained from only 4 of the initial 41 frozen urine samples (10%) for DNA methylation profiling. In comparison, all four buffy coat samples (100%) provided sufficient genomic DNA. High quality data can be obtained provided a sufficient yield of genomic DNA is isolated. Despite optimizing various extraction methodologies, the modest amount of genomic DNA derived from urine, may limit the generalisability of this approach for the identification of DNA methylation biomarkers of chronic diabetic kidney disease.
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Statistical modeling of dental unit water bacterial test kit performance.
Cohen, Mark E; Harte, Jennifer A; Stone, Mark E; O'Connor, Karen H; Coen, Michael L; Cullum, Malford E
2007-01-01
While it is important to monitor dental water quality, it is unclear whether in-office test kits provide bacterial counts comparable to the gold standard method (R2A). Studies were conducted on specimens with known bacterial concentrations, and from dental units, to evaluate test kit accuracy across a range of bacterial types and loads. Colony forming units (CFU) were counted for samples from each source, using R2A and two types of test kits, and conformity to Poisson distribution expectations was evaluated. Poisson regression was used to test for effects of source and device, and to estimate rate ratios for kits relative to R2A. For all devices, distributions were Poisson for low CFU/mL when only beige-pigmented bacteria were considered. For higher counts, R2A remained Poisson, but kits exhibited over-dispersion. Both kits undercounted relative to R2A, but the degree of undercounting was reasonably stable. Kits did not grow pink-pigmented bacteria from dental-unit water identified as Methylobacterium rhodesianum. Only one of the test kits provided results with adequate reliability at higher bacterial concentrations. Undercount bias could be estimated for this device and used to adjust test kit results. Insensitivity to methylobacteria spp. is problematic.
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XK-related protein 5 (XKR5) is a novel negative regulator of KIT/D816V-mediated transformation.
Sun, Jianmin; Thingholm, Tine; Højrup, Peter; Rönnstrand, Lars
2018-06-18
In order to investigate the molecular mechanisms by which the oncogenic mutant KIT/D816V causes transformation of cells, we investigated proteins that selectively bind KIT/D816V, but not wild-type KIT, as potential mediators of transformation. By mass spectrometry several proteins were identified, among them a previously uncharacterized protein denoted XKR5 (XK-related protein 5), which is related to the X Kell blood group proteins. We could demonstrate that interaction between XKR5 and KIT/D816V leads to phosphorylation of XKR5 at Tyr 369, Tyr487, and Tyr 543. Tyrosine phosphorylated XKR5 acts as a negative regulator of KIT signaling, which leads to downregulation of phosphorylation of ERK, AKT, and p38. This led to reduced proliferation and colony forming capacity in semi-solid medium. Taken together, our data demonstrate that XKR5 is a novel type of negative regulator of KIT-mediated transformation.
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Tekin, Kemal; Albay, Ali; Simsek, Hulya; Sig, Ali Korhan; Guney, Mustafa
2017-01-01
Objective: The present study aimed to evaluate the performances of the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test for detecting second-line antituberculosis drug resistance in Multidrug-resistant TB (MDR-TB) cases. Materials and Methods: Forty-six MDR-TB strains were studied. Second-line antituberculosis drug resistances were detected using the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test. The Middlebrook 7H10 agar proportion method was used as the reference test. Results: The sensitivity and specificity values for the BACTEC MGIT 960 SL DST kit were both 100% for amikacin, kanamycin, capreomycin (4 µg/mL), and ofloxacin; 100% and 95.3%, respectively, for capreomycin (10 µg/mL); and 85.7% and 100%, respectively, for moxifloxacin (0.5 µg/mL). The sensitivity and specificity values for the GenoType MTBDRsl test to detect fluoroquinolone and aminoglycoside/cyclic peptide resistance were 88.9% and 100%, respectively, for ofloxacin and 85.7% and 94.9%, respectively, for moxifloxacin (0.5 µg/mL). The accuracy of the GenoType MTBDRsl assay for kanamycin, capreomycin, ofloxacin, and moxifloxacin was lower than that of the BACTEC MGIT 960 SL DST. Conclusion: The BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl were successful in detecting second-line antituberculosis drug resistance. Preliminary results of the GenoType MTBDRsl are very valuable for early treatment decisions, but we still recommend additional BACTEC MGIT 960 SL DST kit usage in the routine evaluation of drug-resistant tuberculosis. PMID:29123441
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Tekin, Kemal; Albay, Ali; Simsek, Hulya; Sig, Ali Korhan; Guney, Mustafa
2017-10-01
The present study aimed to evaluate the performances of the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test for detecting second-line antituberculosis drug resistance in Multidrug-resistant TB (MDR-TB) cases. Forty-six MDR-TB strains were studied. Second-line antituberculosis drug resistances were detected using the BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl test. The Middlebrook 7H10 agar proportion method was used as the reference test. The sensitivity and specificity values for the BACTEC MGIT 960 SL DST kit were both 100% for amikacin, kanamycin, capreomycin (4 µg/mL), and ofloxacin; 100% and 95.3%, respectively, for capreomycin (10 µg/mL); and 85.7% and 100%, respectively, for moxifloxacin (0.5 µg/mL). The sensitivity and specificity values for the GenoType MTBDRsl test to detect fluoroquinolone and aminoglycoside/cyclic peptide resistance were 88.9% and 100%, respectively, for ofloxacin and 85.7% and 94.9%, respectively, for moxifloxacin (0.5 µg/mL). The accuracy of the GenoType MTBDRsl assay for kanamycin, capreomycin, ofloxacin, and moxifloxacin was lower than that of the BACTEC MGIT 960 SL DST. The BACTEC MGIT 960 SL DST kit and the GenoType MTBDRsl were successful in detecting second-line antituberculosis drug resistance. Preliminary results of the GenoType MTBDRsl are very valuable for early treatment decisions, but we still recommend additional BACTEC MGIT 960 SL DST kit usage in the routine evaluation of drug-resistant tuberculosis.
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Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT
Dubreuil, Patrice; Letard, Sébastien; Ciufolini, Marco; Gros, Laurent; Humbert, Martine; Castéran, Nathalie; Borge, Laurence; Hajem, Bérengère; Lermet, Anne; Sippl, Wolfgang; Voisset, Edwige; Arock, Michel; Auclair, Christian; Leventhal, Phillip S.; Mansfield, Colin D.; Moussy, Alain; Hermine, Olivier
2009-01-01
Background The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. Methodology/Principal Findings In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. Conclusions Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity. PMID:19789626
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Zandi, Keivan; Roostaee, Mohammad Hassan; Sadeghizadeh, Majid; Rasaee, Mohammad Javad; Sajedi, Reza Hassan; Soleimanjahi, Hoorieh
2007-08-01
The herpes simplex viruses are important causes of disease worldwide. Herpes simplex virus type 1 (HSV-1) is the primary cause of oral-facial and pharyngeal infections and may cause herpetic whitlow, eye infections as well as severe and sometimes dangerous infections of the eyes and brain. HSV-1 also accounts for 10-15% of all genital herpetic infections. Therefore, laboratory diagnosis of this virus and development of diagnostic serological techniques for HSV-1 is of particular importance. In the present study, pTrc His2A-gG1 plasmid, containing the full-length glycoprotein G (gG) protein, was produced in a prokaryotic system for the first time. Upon confirmation of a 37-kDa gG-1 protein production in a prokaryotic system based on western blotting and monoclonal antibodies, the protein was produced at a large scale and purified by ion-exchange chromatography using DEAE-sepharose. An HSV-1 type-specific diagnostic kit was designed and developed and the specificity and sensitivity of this kit were demonstrated to be 89.5% and 100%, respectively, as compared with a commercially available kit. A significant correlation was shown between the developed kit and the commercial kit.
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Wang, Qiang; Liu, Feiyang; Wang, Beilei; Zou, Fengming; Qi, Ziping; Chen, Cheng; Yu, Kailin; Hu, Chen; Qi, Shuang; Wang, Wenchao; Hu, Zhenquan; Liu, Juan; Wang, Wei; Wang, Li; Liang, Qianmao; Zhang, Shanchun; Ren, Tao; Liu, Qingsong; Liu, Jing
2017-01-12
The discovery of a novel potent type II ABL/c-KIT dual kinase inhibitor compound 34 (CHMFL-ABL/KIT-155), which utilized a hydrogen bond formed by NH on the kinase backbone and carbonyl oxygen of 34 as a unique hinge binding, is described. 34 potently inhibited purified ABL (IC 50 : 46 nM) and c-KIT kinase (IC 50 : 75 nM) in the biochemical assays and displayed high selectivity (S Score (1) = 0.03) at the concentration of 1 μM among 468 kinases/mutants in KINOMEscan assay. It exhibited strong antiproliferative activities against BCR-ABL/c-KIT driven CML/GISTs cancer cell lines through blockage of the BCR-ABL/c-KIT mediated signaling pathways, arresting cell cycle progression and induction of apoptosis. 34 possessed a good oral PK property and effectively suppressed the tumor progression in the K562 (CML) and GIST-T1 (GISTs) cells mediated xenograft mouse model. The distinct hinge-binding mode of 34 provided a novel pharmacophore for expanding the chemical structure diversity for the type II kinase inhibitors discovery.
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Loss of c-Kit function impairs arteriogenesis in a mouse model of hindlimb ischemia.
Hernandez, Diana R; Artiles, Adriana; Duque, Juan C; Martinez, Laisel; Pinto, Mariana T; Webster, Keith A; Velazquez, Omaida C; Vazquez-Padron, Roberto I; Lassance-Soares, Roberta M
2018-04-01
Arteriogenesis is a process whereby collateral vessels remodel usually in response to increased blood flow and/or wall stress. Remodeling of collaterals can function as a natural bypass to alleviate ischemia during arterial occlusion. Here we used a genetic approach to investigate possible roles of tyrosine receptor c-Kit in arteriogenesis. Mutant mice with loss of c-Kit function (Kit W/W-v ), and controls were subjected to hindlimb ischemia. Blood flow recovery was evaluated pre-, post-, and weekly after ischemia. Foot ischemic damage and function were assessed between days 1 to 14 post-ischemia while collaterals remodeling were measured 28 days post-ischemia. Both groups of mice also were subjected to wild type bone marrow cells transplantation 3 weeks before hindlimb ischemia to evaluate possible contributions of defective bone marrow c-Kit expression on vascular recovery. Kit W/W-v mice displayed impaired blood flow recovery, greater ischemic damage and foot dysfunction after ischemia compared to controls. Kit W/W-v mice also demonstrated impaired collateral remodeling consistent with flow recovery findings. Because arteriogenesis is a biological process that involves bone marrow-derived cells, we investigated which source of c-Kit signaling (bone marrow or vascular) plays a major role in arteriogenesis. Kit W/W-v mice transplanted with bone marrow wild type cells exhibited similar phenotype of impaired blood flow recovery, greater tissue ischemic damage and foot dysfunction as nontransplanted Kit W/W-v mice. This study provides evidence that c-Kit signaling is required during arteriogenesis. Also, it strongly suggests a vascular role for c-Kit signaling because rescue of systemic c-Kit activity by bone marrow transplantation did not augment the functional recovery of Kit W/W-v mouse hindlimbs. Copyright © 2017 Elsevier Inc. All rights reserved.
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The Classroom-Friendly ABO Blood Types Kit: Blood Agglutination Simulation
ERIC Educational Resources Information Center
Arnold, Savittree Rochanasmita; Kruatong, Tussatrin; Dahsah, Chanyah; Suwanjinda, Duongdearn
2012-01-01
The classroom-friendly ABO blood type kit was developed by combining advantages of modelling and a simulation laboratory to teach the topics of ABO blood types and blood transfusion. Teachers can easily simulate the agglutination reaction on a blood type testing plate in the classroom, and show the students how this reaction occurs by using the…
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Kalfusová, Alena; Kodet, Roman
2017-01-01
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Most of them arise due to activating mutations in KIT (75 - 85 %) or PDGFRA (less than 10 %) genes. Identification of the activating mutations in KIT and PDGFRA genes, which code for receptor tyrosine kinases (RTKs), has improved the outcome of targeted therapy of metastatic, unresectable or recurrent GISTs. Primary and/or secondary resistance represents a significant problem in the targeted therapy by Imatinib mesylate (IM) in patients with GIST. An important mechanism of the secondary resistance is the evolvement of secondary mutations. Except for primary and secondary resistance, there is another problem of disease progression - a failure of tumor cells eradication even in the long term therapy of tyrosine kinase inhibitors. GISTs without mutations in KIT/PDGFRA genes constitute 10 - 15% GISTs in adults, and a majority (85 %) of pediatric GISTs. KIT/PDGFRA wild-type GISTs represent a heterogeneous group of tumors with several molecular-genetics and/or morphologic differences. KIT/PDGFRA wild-type GISTs are different in their molecular features, for example in mutations in the BRAF, KRAS, NF1 genes or defects of succinate dehydrogenase (SDH) subunits. KIT/PDGFRA wild-type GISTs are generally less sensitive to targeted therapy by tyrosine kinase inhibitors in comparison with KIT/PDGFRA mutated GISTs. Inhibitors of BRAF, PI3K (mTOR) or inhibitors of IGF1R and VEGFR receptors provide alternative therapeutic strategies.
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Newton. MicroSIFT Courseware Evaluation.
ERIC Educational Resources Information Center
Northwest Regional Educational Lab., Portland, OR.
THE FOLLOWING IS THE FULL TEXT OF THIS DOCUMENT (Except for the Evaluation Summary Table): VERSION: Member's Apple Demonstration Kit. PRODUCER: Conduit, P.O. Box 388, Iowa City, Iowa 52244. EVALUATION COMPLETED: June 1982 by the staff and constituents of the Portland Public Schools, Multnomah ESD, Portland, Oregon. COST: $35.00. ABILITY LEVEL:…
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Furitsu, T; Tsujimura, T; Tono, T; Ikeda, H; Kitayama, H; Koshimizu, U; Sugahara, H; Butterfield, J H; Ashman, L K; Kanayama, Y
1993-01-01
The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells. Images PMID:7691885
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75 FR 75934 - Airworthiness Directives; Apical Industries Inc. (Apical) Emergency Float Kits
Federal Register 2010, 2011, 2012, 2013, 2014
2010-12-07
...-1190; Directorate Identifier 2010-SW-038-AD] RIN 2120-AA64 Airworthiness Directives; Apical Industries Inc. (Apical) Emergency Float Kits AGENCY: Federal Aviation Administration, DOT. ACTION: Notice of... the Apical emergency float kits installed on certain model helicopters under supplemental type...
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76 FR 77375 - Airworthiness Directives; Apical Industries, Inc., (Apical) Emergency Float Kits
Federal Register 2010, 2011, 2012, 2013, 2014
2011-12-13
... Airworthiness Directives; Apical Industries, Inc., (Apical) Emergency Float Kits AGENCY: Federal Aviation... the Apical emergency float kits installed on certain model helicopters under supplemental type... the service information identified in this AD from Apical Industries, Inc., 2608 Temple Heights Drive...
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Deng, Jingjing; Yang, Shu; Yuan, Qing; Chen, Yuzhong; Li, Dandan; Sun, Haimei; Tan, Xinghua; Zhang, Fuchun; Zhou, Deshan
2017-01-01
Acupuncture is a therapy effective in treating postoperative ileus (POI); its underlying mechanisms remain unclear. MicroRNAs (miRNAs) participate in inflammation and injury to the interstitial cells of Cajal (ICCs), both of which are considered to be contributors to POI. C-kit, encoding KIT, a specific marker of ICCs, is predicted to be targeted by miR-19a, an inflammation-related miRNA. Therefore, we investigated a possible link between inflammation, miR-19a, and ICCs in POI, as well as the mechanism by which these factors are affected by acupuncture. The effects of acupuncture on POI were assessed in patients after colorectal resection and in colocolic anastomosis mice. Immunofluorescence staining demonstrated that KIT[Formula: see text]/ano1[Formula: see text] ICCs dramatically decreased around the colonic incision in mice, which was negatively correlated with the pronounced increase in macrophage. However, this decrease was not due to apoptosis. IL-6R was expressed in ICCs, and IL-6 level was significantly increased, as measured by ELISA, in accompaniment with high miR-19a expression. The increase in IL-6 and miR-19a levels was negatively correlated with the decrease in KIT[Formula: see text]/ano1[Formula: see text] ICCs. A luciferase reporter assay demonstrated that miR-19a directly targeted C-kit, indicating that miR-19a caused ICC damage. Interestingly, acupuncture inhibited macrophage activation, IL-6 release, and miR-19a upregulation, while promoting KIT and ano1 restoration in ICCs. High serum miR-19a level in patients after colorectal resection was also reduced by acupuncture. Conclusively, the IL-6 released by macrophages during gastrointestinal surgery upregulated miR-19a, which downregulated KIT in ICCs and finally resulted in POI. Acupuncture can interfere with the "IL-6-miR-19a-KIT" axis, suggesting that it may be a therapeutic mechanism that works against POI.
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Lysozyme adsorption onto mesoporous materials: effect of pore geometry and stability of adsorbents.
Vinu, Ajayan; Miyahara, Masahiko; Hossain, Kazi Zakir; Takahashi, Motoi; Balasubramanian, Veerappan Vaithilingam; Mori, Toshiyuki; Ariga, Katsuhiko
2007-03-01
In this paper, adsorption of lysozyme onto two kinds of mesoporous adsorbents (KIT-5 and AISBA-15) has been investigated and the results on the effects of pore geometry and stability of the adsorbents are also discussed. The KIT-5 mesoporous silica materials possess cage-type pore geometry while the AISBA-15 adsorbent has mesopores of cylindrical type with rather large diameter (9.7 nm). Adsorption of lysozyme onto AISBA-15 aluminosilicate obeys a Langmuir isotherm, resulting in pore occupation of 25 to 30%. In contrast, the KIT-5 adsorbents showed very small adsorption capacities for the lysozyme adsorption, typically falling in 6 to 13% of pore occupation. The cage-type KIT-5 adsorbents have narrow channel (4 to 6 nm) where penetration of the lysozyme (3 x 3 x 4.5 nm) might be restricted. The KIT-5 adsorbent tends to collapse after long-time immersion in water, as indicated by XRD patterns, while the AISBA-15 adsorbent retains its regular structure even after immersion in basic water for 4 days. These facts confirm superiority of the AISBA-15 as an adsorbent as compared with the KIT-5 mesoporous silicates. This research strikingly demonstrates the selection of mesoporous materials is crucial to achieve efficient immobilization of biomaterials in aqueous environment.
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ERIC Educational Resources Information Center
Canadian Health Libraries Association.
Nine Canadian health libraries participated in a pilot test of the Benchmarking Tool Kit between January and April, 1998. Although the Tool Kit was designed specifically for health libraries, the content and approach are useful to other types of libraries as well. Used to its full potential, benchmarking can provide a common measuring stick to…
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Differential Use of Elementary Science Kits
ERIC Educational Resources Information Center
Jones, Gail; Robertson, Laura; Gardner, Grant E.; Dotger, Sharon; Blanchard, Margaret R.
2012-01-01
The use of kits in elementary science classes is a growing trend in some countries. Kits provide materials and inquiry lessons in a ready-to-teach format for teachers to use in their science instruction. This study examined elementary teachers' instructional strategies, classroom practices, and assessment types in relation to the frequency of…
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Liu, Fei; Zhu, Hua; Yu, Jiangyuan; Han, Xuedi; Xie, Qinghua; Liu, Teli; Xia, Chuanqin; Li, Nan; Yang, Zhi
2017-06-01
Somatostatin receptors are overexpressed in neuroendocrine tumors, whose endogenous ligands are somatostatin. DOTA-TATE is an analogue of somatostatin, which shows high binding affinity to somatostatin receptors. We aim to evaluate the 68 Ga/ 177 Lu-labeling DOTA-TATE kit in neuroendocrine tumor model for molecular imaging and to try human-positron emission tomography/computed tomography imaging of 68 Ga-DOTA-TATE in neuroendocrine tumor patients. DOTA-TATE kits were formulated and radiolabeled with 68 Ga/ 177 Lu for 68 Ga/ 177 Lu-DOTA-TATE (M-DOTA-TATE). In vitro and in vivo stability of 177 Lu-DOTA-TATE were performed. Nude mice bearing human tumors were injected with 68 Ga-DOTA-TATE or 177 Lu-DOTA-TATE for micro-positron emission tomography and micro-single-photon emission computed tomography/computed tomography imaging separately, and clinical positron emission tomography/computed tomography images of 68 Ga-DOTA-TATE were obtained at 1 h post-intravenous injection from patients with neuroendocrine tumors. Micro-positron emission tomography and micro-single-photon emission computed tomography/computed tomography imaging of 68 Ga-DOTA-TATE and 177 Lu-DOTA-TATE both showed clear tumor uptake which could be blocked by excess DOTA-TATE. In addition, 68 Ga-DOTA-TATE-positron emission tomography/computed tomography imaging in neuroendocrine tumor patients could show primary and metastatic lesions. 68 Ga-DOTA-TATE and 177 Lu-DOTA-TATE could accumulate in tumors in animal models, paving the way for better clinical peptide receptor radionuclide therapy for neuroendocrine tumor patients in Asian population.
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Satzger, Imke; Küttler, Uta; Völker, Bernward; Schenck, Florian; Kapp, Alexander; Gutzmer, Ralf
2010-01-01
Previously an increased frequency of KIT aberrations in mucosal melanomas was reported, whereas c-KIT in most types of cutaneous melanomas does not appear to be of pathogenetic importance. Imatinib has become the standard of care in other cancers with KIT mutations such as gastrointestinal stromal tumors. Recently 12 cases of metastatic melanoma and KIT-activating mutations have been published to be successfully treated with c-KIT blockers such as imatinib, sunitinib, dasatinib or sorafenib. We report here on one of our patients with KIT-activating mutation in metastatic anal mucosal melanoma, who showed a response to imatinib therapy and summarize the available literature regarding this new therapeutic option. Copyright 2009 S. Karger AG, Basel.
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Zheng, Si-Li; Li, Zhi-Yong; Zhang, Zheng; Wang, Dong-Sheng; Xu, Jian; Miao, Chao-Yu
2018-04-01
Metrnl is a newly discovered secreted protein with neurotrophic activity and metabolic effect, while in earlier studies its circulating level in human was not explored. We evaluated two commercial enzyme-linked immunosorbent assay kits (DY7867-05, R&D Systems and SK00478-02, Aviscera Bioscience) for the detection of human circulating Metrnl. The DY7867-05 kit showed superiority over the SK00478-02 kit since it generated better curve fitting degree, smaller variation among tests, higher inter-assay reproducibility and better specificity, and could effectively detect human Metrnl in six types of blood samples. Subsequent analysis was performed using the DY7867-05 kit. Sample storage conditions were investigated. No gender difference in circulating Metrnl levels was found, while people with newly diagnosed type 2 diabetes mellitus (T2DM) had significantly lower Metrnl levels compared to the healthy controls.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Standley, W.G.; Berry, W.H.; O'Farrell, T.P.
1992-09-01
Sources and rates of mortality of a San Joaquin kit fox population (Vulpes velox macrotis) were investigated at Camp Roberts Army National Guard Training Site, California, from November 1988 through September 1991. National Guard-authorized activities, including military training, caused the death of three of the 94 (3%) kit foxes radiocollared, and do not appear to jeopardize the continued existence of the population. Predation by larger carnivores, primarily coyotes (Canis latrans), caused the death of 75% of the 32 radiocollared kit foxes recovered dead for which a cause of death could be determined; vehicle impacts, disease (rabies), poisoning, and shooting weremore » each responsible for the deaths of 6.3%. Adult annual mortality rate was 0.47 and the juvenile mortality rate was 0.80, and both rates are similar to rates reported for kit foxes in other locations. There was no significant difference between male and female mortality rates in either age class. The proportions of dead kit foxes recovered in different habitat types were similar to the availability of the habitat types within the distribution of kit fox on the installation.« less
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Bank, Steffen; Nexø, Bjørn Andersen; Andersen, Vibeke; Vogel, Ulla; Andersen, Paal Skytt
2013-06-01
The recovery of biological samples for genetic epidemiological studies can be cumbersome. Blood clots are routinely collected for serological examinations. However, the extraction of DNA from blood clots can be difficult and often results in low yields. The aim was to compare the efficiency of commercial purification kits for extracting DNA from long-term frozen clotted blood. Serum tubes with clotted blood were stored at -20°C for 1 to 2.5 years before DNA extraction. DNA was extracted from 10 blood clot samples using PureGene (Qiagen) with and without glycogen, the QIAamp DNA Micro kit (Qiagen), and the Nucleospin 96 Blood kit (Macherey-Nagel). Furthermore, blood clots from 1055 inflammatory bowel disease patients were purified using the Maxwell 16 Blood purification kit (Promega). The DNA was extracted according to the manufacturers` instructions and real-time PCR and the A(260)/A(280) ratio were used to evaluate the quality of the extracted DNA. The highest DNA yield was obtained by the Maxwell 16 Blood purification kit (Promega) with a median of 4.90 μg (range 0.8-25 μg) pr 300 μL total blood. PureGene with glycogen (Qiagen) had the second highest yield with a median of 0.65 μg (range 0.5-2.6 μg) pr 300 μL total blood. The yield obtained by the different commercial kits varied considerably. Our work demonstrates that high-quality and -quantity DNA can be extracted with the Maxwell 16 Blood purification kit (Promega) from cryopreserved blood clots, even after prolonged storage. The recovered DNA served as a reliable PCR template for single-nucleotide polymorphism assays.
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Yun, SeongJu; Kim, Won Kyu; Kwon, Yujin; Jang, Mi; Bauer, Sebastian; Kim, Hoguen
2018-05-15
Gain-of-function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have been shown to be dysregulated in GISTs and impact KIT expression. Little is known though on KIT-independent targets of KIT-regulating mRNAs. We sought to investigate how miR-494 inhibits GIST proliferation and to identify novel target gene. We used microarray-based gene expression analyses to identify pathways and target genes affected by miR-494. The expressional relationship between survivin and miR-494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, promoter assays and chromatin immunoprecipitation (ChiP) were performed to clarify the roles of survivin in GIST progression. Gene expression microarray analysis revealed that miR-494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin (BIRC5) was a key target of miR-494, and its expression showed an inverse correlation with miR-494 expression in 35 GIST tissues (Pearson's correlation coefficient, r = -0.418, p = 0.012). Downregulation of survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced survivin overexpression relieved miR-494-mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of survivin. Survivin also regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GISTs revealed that survivin expression was significantly correlated with overall survival of GIST patients (p = 0.004). Our findings indicated that miR-494 synergistically suppressed GISTs by concomitantly targeting survivin and KIT. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.
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Fujiwara, Shigeki; Tachihara, Keiichi; Mori, Satoshi; Ouchi, Kentaro; Yokoe, Chizuko; Imaizumi, Uno; Morimoto, Yoshinari; Miki, Yoichiro; Toyoguchi, Izumi; Yoshida, Kazu-Ichi; Yokoyama, Takeshi
2016-12-01
Blood pressure transducer kits are equipped with two types of Planecta™ ports-the flat-type Planecta™ port (FTP) and the Planecta™ port with a three-way stopcock (PTS). We reported that FTP application decreased the natural frequency of the kits. However, Planecta™ is an invaluable tool as it prevents infection, ensures technical simplicity, and excludes air. Hence, an ideal Planecta™ port that does not decrease the frequency characteristics is required. As a first step in this direction, we aimed to assess the influence of PTSs on the natural frequency of blood transducer kits. A DTXplus transducer kit (DT4812J; Argon Medical Devices, TX, USA) was used along with ≥1 PTSs (JMS, Hiroshima, Japan), and the frequency characteristics were assessed. The natural frequency and damping coefficient of each kit were obtained by using frequency characteristics analysis software, and these parameters were evaluated by plotting them on Gardner's chart. Regardless of whether one or two PTSs were inserted, the natural frequency of the kits only slightly decreased (from 42.5 to 41.1 Hz, when 2 PTSs were used). Thus, the frequency characteristics of the kits with PTSs were adequate for pressure monitoring. The insertion of ≥2 FTPs in pressure transducer kits should be avoided, as they markedly decrease the natural frequency and lead to underdamping. However, the effect of PTS insertion in pressure transducer kits on the frequency characteristics is minimal. Thus, we found that the use of PTS markedly improved the frequency characteristics as compared to the use of FTP.
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Purohit, Rituraj
2014-01-01
KIT receptor is the prime target in gastrointestinal stromal tumor (GISTs) therapy. Second generation inhibitor, Sunitinib, binds to an inactivated conformation of KIT receptor and stabilizes it in order to prevent tumor formation. Here, we investigated the dynamic behavior of wild type and mutant D816H KIT receptor, and emphasized the extended A-loop (EAL) region (805-850) by conducting molecular dynamics simulation (∼100 ns). We analyzed different properties such as root mean square cutoff or deviation, root mean square fluctuation, radius of gyration, solvent-accessible surface area, hydrogen bonding network analysis, and essential dynamics. Apart from this, clustering and cross-correlation matrix approach was used to explore the conformational space of the wild type and mutant EAL region of KIT receptor. Molecular dynamics analysis indicated that mutation (D816H) was able to alter intramolecular hydrogen bonding pattern and affected the structural flexibility of EAL region. Moreover, flexible secondary elements, specially, coil and turns were dominated in EAL region of mutant KIT receptor during simulation. This phenomenon increased the movement of EAL region which in turn helped in shifting the equilibrium towards the active kinase conformation. Our atomic investigation of mutant KIT receptor which emphasized on EAL region provided a better insight into the understanding of Sunitinib resistance mechanism of KIT receptor and would help to discover new therapeutics for KIT-based resistant tumor cells in GIST therapy.
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19 CFR 122.133 - Stores list required on arrival.
Code of Federal Regulations, 2014 CFR
2014-04-01
... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.133 Stores list required on... liquor kit on board before an aircraft lands. The incoming stores list shall state for each type of... beverages in the kit. (b) Disposition of stores list copies. One copy of the incoming stores list shall be...
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19 CFR 122.133 - Stores list required on arrival.
Code of Federal Regulations, 2012 CFR
2012-04-01
... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.133 Stores list required on... liquor kit on board before an aircraft lands. The incoming stores list shall state for each type of... beverages in the kit. (b) Disposition of stores list copies. One copy of the incoming stores list shall be...
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METHOD FOR MICRORNA ISOLATION FROM CLINICAL SERUM SAMPLES
Li, Yu; Kowdley, Kris V.
2012-01-01
MicroRNAs are a group of intracellular non-coding RNA molecules that have been implicated in a variety of human diseases. Due to their high stability in blood, microRNAs released into circulation could be potentially utilized as non-invasive biomarkers for diagnosis or prognosis. Current microRNA isolation protocols are specifically designed for solid tissues and are impractical for biomarker development utilizing small-volume serum samples on a large scale. Thus, a protocol for microRNA isolation from serum is needed to accommodate these conditions in biomarker development. To establish such a protocol, we developed a simplified approach to normalize sample input by using single synthetic spike-in microRNA. We evaluated three commonly used commercial microRNA isolation kits for the best performance by comparing RNA quality and yield. The manufacturer’s protocol was further modified to improve the microRNA yield from 200 μL of human serum. MicroRNAs isolated from a large set of clinical serum samples were tested on the miRCURY LNA real-time PCR panel and confirmed to be suitable for high-throughput microRNA profiling. In conclusion, we have established a proven method for microRNA isolation from clinical serum samples suitable for microRNA biomarker development. PMID:22982505
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Kampa-Schittenhelm, Kerstin Maria; Frey, Julia; Haeusser, Lara A; Illing, Barbara; Pavlovsky, Ashly A; Blumenstock, Gunnar; Schittenhelm, Marcus Matthias
2017-10-10
Activating D816 mutations of the class III receptor tyrosine kinase KIT are associated with the majority of patients with systemic mastocytosis (SM), but also core binding factor (CBF) AML, making KIT mutations attractive therapeutic targets for the treatment of these cancers. Crenolanib is a potent and selective inhibitor of wild-type as well as mutant isoforms of the class III receptor tyrosine kinases FLT3 and PDGFRα/β. Notably, crenolanib inhibits constitutively active mutant-FLT3 isoforms resulting from amino acid substitutions of aspartic acid at codon 835, which is homologous to codon 816 in the KIT gene - suggesting sensitivity against mutant-KIT D816 isoforms as well. Here we demonstrate that crenolanib targets KIT D816 in SM and CBF AML models: crenolanib inhibits cellular proliferation and initiates apoptosis of mastocytosis cell lines expressing these mutations. Target-specificity was confirmed using an isogenic cell model. In addition, we demonstrate that KIT D816 mutations are targetable with clinically achievable doses of crenolanib. Further, a rationale to combine cladribine (2-CDA), the therapeutic standard in SM, with crenolanib is provided. In conclusion, we demonstrate that crenolanib is an inhibitor of mutant-KIT D816 isoforms at clinically achievable concentrations, and thus may be a potential treatment for SM and CBF AML as a monotherapy or in combination approaches.
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Kit W-sh Mutation Prevents Cancellous Bone Loss during Calcium Deprivation.
Lotinun, Sutada; Suwanwela, Jaijam; Poolthong, Suchit; Baron, Roland
2018-01-01
Calcium is essential for normal bone growth and development. Inadequate calcium intake increases the risk of osteoporosis and fractures. Kit ligand/c-Kit signaling plays an important role in regulating bone homeostasis. Mice with c-Kit mutations are osteopenic. The present study aimed to investigate whether impairment of or reduction in c-Kit signaling affects bone turnover during calcium deprivation. Three-week-old male WBB6F1/J-Kit W /Kit W-v /J (W/W v ) mice with c-Kit point mutation, Kit W-sh /HNihrJaeBsmJ (W sh /W sh ) mice with an inversion mutation in the regulatory elements upstream of the c-Kit promoter region, and their wild-type controls (WT) were fed either a normal (0.6% calcium) or a low calcium diet (0.02% calcium) for 3 weeks. μCT analysis indicated that both mutants fed normal calcium diet had significantly decreased cortical thickness and cancellous bone volume compared to WT. The low calcium diet resulted in a comparable reduction in cortical bone volume and cortical thickness in the W/W v and W sh /W sh mice, and their corresponding controls. As expected, the low calcium diet induced cancellous bone loss in the W/W v mice. In contrast, W sh /W sh cancellous bone did not respond to this diet. This c-Kit mutation prevented cancellous bone loss by antagonizing the low calcium diet-induced increase in osteoblast and osteoclast numbers in the W sh /W sh mice. Gene expression profiling showed that calcium deficiency increased Osx, Ocn, Alp, type I collagen, c-Fms, M-CSF, and RANKL/OPG mRNA expression in controls; however, the W sh mutation suppressed these effects. Our findings indicate that although calcium restriction increased bone turnover, leading to osteopenia, the decreased c-Kit expression levels in the W sh /W sh mice prevented the low calcium diet-induced increase in cancellous bone turnover and bone loss but not the cortical bone loss.
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Direct Y-STR amplification of body fluids deposited on commonly found crime scene substrates.
Dargay, Amanda; Roy, Reena
2016-04-01
Body fluids detected on commonly found crime scene substrates require extraction, purification and quantitation of DNA prior to amplification and generation of short tandem repeat (STR) DNA profiles. In this research Y-STR profiles were generated via direct amplification of blood and saliva deposited on 12 different substrates. These included cigarette butts, straws, grass, leaves, woodchips and seven different types of fabric. After depositing either 0.1 μL of blood or 0.5 μL of saliva, each substrate containing the dry body fluid stain was punched using a Harris 1.2 mm micro-punch. Each of these punched substrates, a total of 720 samples, containing minute amount of blood or saliva was either amplified directly without any pre-treatment, or was treated with one of the four washing reagents or buffer. In each of these five experimental groups the substrates containing the body fluid remained in the amplification reagent during the thermal cycling process. Each sample was amplified with the three direct Y-STR amplification kits; AmpFℓSTR(®) Yfiler(®) Direct, Yfiler(®) Plus Amplification Kits and the PowerPlex(®) Y23 System. Complete and concordant Y-STR profiles were successfully obtained from most of these 12 challenging crime scene objects when the stains were analyzed by at least one of the five experimental groups. The reagents and buffer were interchangeable among the three amplification kits, however, pre-treatment with these solutions did not appear to enhance the quality or the number of the full profiles generated with direct amplification. This study demonstrates that blood and saliva deposited on these simulated crime scene objects can be amplified directly. Copyright © 2016 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
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Obata, Yuuki; Toyoshima, Shota; Wakamatsu, Ei; Suzuki, Shunichi; Ogawa, Shuhei; Esumi, Hiroyasu; Abe, Ryo
2014-01-01
Kit is a receptor-type tyrosine kinase found on the plasma membrane. It can transform mast cells through activating mutations. Here, we show that a mutant Kit from neoplastic mast cells from mice, Kit(D814Y), is permanently active and allows cells to proliferate autonomously. It does so by activating two signalling pathways from different intracellular compartments. Mutant Kit from the cell surface accumulates on endolysosomes through clathrin-mediated endocytosis, which requires Kit’s kinase activity. Kit(D814Y) is constitutively associated with phosphatidylinositol 3-kinase, but the complex activates Akt only on the cytoplasmic surface of endolysosomes. It resists destruction because it is under-ubiquitinated. Kit(D814Y) also appears in the endoplasmic reticulum soon after biosynthesis, and there, can activate STAT5 aberrantly. These mechanisms of oncogenic signalling are also seen in rat and human mast cell leukemia cells. Thus, oncogenic Kit signalling occurs from different intracellular compartments, and the mutation acts by altering Kit trafficking as well as activation. PMID:25493654
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Yantiss, Rhonda K; Rosenberg, Andrew E; Sarran, Lisa; Besmer, Peter; Antonescu, Cristina R
2005-04-01
Multiple gastrointestinal stromal tumors typically occur in familial form associated with KIT receptor tyrosine kinase or platelet-derived growth factor receptor-alpha (PDGFRA) germline mutations, but may also develop in the setting of type 1 neurofibromatosis. The molecular abnormalities of gastrointestinal stromal tumors arising in neurofibromatosis have not been extensively studied. We identified three patients with type 1 neuro-fibromatosis and multiple small intestinal stromal tumors. Immunostains for CD117, CD34, desmin, actins, S-100 protein, and keratins were performed on all of the tumors. DNA was extracted from representative paraffin blocks from separate tumor nodules in each case and subjected to a nested polymerase chain reaction, using primers for KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18, followed by direct sequencing. The mean patient age was 56 years (range: 37-86 years, male/female ratio: 2/1). One patient had three tumors, one had five, and one had greater than 10 tumor nodules, all of which demonstrated histologic features characteristic of gastrointestinal stromal tumors and stained strongly for CD117 and CD34. One patient died of disease at 35 months, one was disease free at 12 months and one was lost to follow-up. DNA extracts from 10 gastrointestinal stromal tumors (three from each of two patients and four from one patient) were subjected to polymerase chain reactions and assessed for mutations. All of the tumors were wild type for KIT exons 9, 13, and 17 and PDGFRA exons 12 and 18. Three tumors from one patient had identical point mutations in KIT exon 11, whereas the other tumors were wild type at this locus. We conclude that, although most patients with type 1 neurofibromatosis and gastrointestinal stromal tumors do not have KIT or PDGFRA mutations, KIT germline mutations might be implicated in the pathogenesis of gastrointestinal stromal tumors in some patients.
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Cell type-specific deficiency of c-kit gene expression in mutant mice of mi/mi genotype.
Isozaki, K.; Tsujimura, T.; Nomura, S.; Morii, E.; Koshimizu, U.; Nishimune, Y.; Kitamura, Y.
1994-01-01
The mi locus of mice encodes a novel member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called mi factor). In addition to microphthalmus, osteopetrosis, and lack of melanocytes, mice of mi/mi genotype are deficient in mast cells. Since the c-kit receptor tyrosine kinase plays an important role in the development of mast cells, and since the c-kit expression by cultured mast cells from mi/mi mice is deficient in both mRNA and protein levels, the mast cell deficiency of mi/mi mice has been attributed at least in part to the deficient expression of c-kit. However, it remained to be examined whether the c-kit expression was also deficient in tissues of mi/mi mice. In the present study, we examined the c-kit expression by mi/mi skin mast cells using in situ hybridization and immunohistochemistry. Moreover, we examined the c-kit expression by various cells other than mast cells in tissues of mi/mi mice. We found that the c-kit expression was deficient in mast cells but not in erythroid precursors, testicular germ cells, and neurons of mi/mi mice. This suggested that the regulation of the c-kit transcription by the mi factor was dependent on cell types. Mice of mi/mi genotype appeared to be a useful model to analyze the function of transcription factors in the whole-animal level. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7524330
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A change in structural integrity of c-Kit mutant D816V causes constitutive signaling.
Raghav, Pawan Kumar; Singh, Ajay Kumar; Gangenahalli, Gurudutta
2018-03-01
Several signaling pathways, ligands, and genes that regulate proliferative and self-renewal properties of the Hematopoietic Stem Cells (HSCs) have been studied meticulously. One of the signaling pathways that play a crucial role in the process of hematopoiesis is the Stem Cell Factor (SCF) mediated c-Kit pathway. The c-Kit is a Receptor Tyrosine Kinase (RTK), which is expressed in the cells including HSCs. It undergoes dimerization upon binding with its cognate ligand SCF. As a result, phosphorylation of the Juxtamembrane (JM) domain of c-Kit takes place at Tyr568 and Tyr570 residues. These phosphorylated residues become the docking sites for protein tyrosine phosphatases (PTPs) namely SHP-1 and SHP-2, which in turn cause dephosphorylation and negative regulation of the downstream signaling responsible for the cell proliferation. Interestingly, it has been reported that the mutation of c-Kit at D816V makes it independent of SCF stimulation and SHP-1/SHP-2 inhibition, thereby, causing its constitutive activation. The present study was commenced to elucidate the structural behavior of this mutation in the JM and A-loop region of c-Kit using Molecular Dynamics (MD) simulations of the wild-type and mutant c-Kit in unphosphorylated and phosphorylated states. The energy difference computed between the wild type and mutant (D816V) c-Kit, and protein-protein docking and complex analysis revealed the impact of this single residue mutation on the integrity dynamics of c-Kit that makes it independent of SHP-1/SHP-2 negative regulation. Copyright © 2018 Elsevier B.V. All rights reserved.
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Protein kinase C-δ-mediated recycling of active KIT in colon cancer.
Park, Misun; Kim, Won Kyu; Song, Meiying; Park, Minhee; Kim, Hyunki; Nam, Hye Jin; Baek, Sung Hee; Kim, Hoguen
2013-09-15
Abnormal signaling through receptor tyrosine kinase (RTK) moieties is important in tumorigenesis and drug targeting of colorectal cancers. Wild-type KIT (WT-KIT), a RTK that is activated upon binding with stem cell factor (SCF), is highly expressed in some colon cancers; however, little is known about the functional role of SCF-dependent KIT activation in colon cancer pathogenesis. We aimed to elucidate the conditions and roles of WT-KIT activation in colon cancer tumorigenesis. Colorectal cancers with KIT expression were characterized by immunoblotting and immunohistochemistry. The biologic alterations after KIT-SCF binding were analyzed with or without protein kinase C (PKC) activation. We found that WT-KIT was expressed in a subset of colon cancer cell lines and was activated by SCF, leading to activation of downstream AKT and extracellular signal-regulated kinase (ERK) signaling pathways. We also showed that KIT expression gradually decreased, after prolonged SCF stimulation, due to lysosomal degradation. Degradation of WT-KIT after SCF binding was significantly rescued when PKC was activated. We also showed the involvement of activated PKC-δ in the recycling of WT-KIT. We further showed that a subset of colorectal cancers exhibit expressions of both WT-KIT and activated PKC-δ and that expression of KIT is correlated with poor patient survival (P = 0.004). Continuous downstream signal activation after KIT-SCF binding is accomplished through PKC-δ-mediated recycling of KIT. This sustained KIT activation may contribute to tumor progression in a subset of colon cancers with KIT expression and might provide the rationale for a therapeutic approach targeting KIT. ©2013 AACR.
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Aisner, Seena C.; Dahlberg, Suzanne; Hameed, Meera R.; Ettinger, David S.; Schiller, Joan H.; Johnson, David H.; Aisner, Joseph; Loehrer, Patrick J.
2011-01-01
Background Advanced or recurrent nonresectable thymic epithelial tumors show only a modest response to standard chemotherapy. A recent study using octreotide and prednisone in thymic tumors, Eastern Cooperative Oncology Group study E1C97, was conducted to verify the activity of octreotide for thymic tumors. The aim of this study was to determine whether epidermal growth factor receptor (EGFR) immunoreactivity correlated with outcomes and to identify new biologic markers for potential targeted therapy. Three markers, EGFR, C-kit, and Her2/neu, were selected for evaluation in patients with advanced thymic epithelial tumors treated on E1C97. Methods Of the 42 patients entered onto E1C97, 34 patients (World Health Organization [WHO] categories: type A = 1, type AB = 1, type B1 = 10, type B2 = 11 type B3 = 8, and type C = 3) had sufficient tissue available for immunohistologic study. Each tumor was assessed to have 0, 1+, 2+, or 3+ immunore-activity in the cytoplasm or membranes of the neoplastic cells for Her2/neu and EGFR and for the presence or absence of C-kit immunoreactivity. Results EGFR immunoreactivity of 2+ or 3+ was associated with more aggressive thymic tumors (WHO types B2 and B3). However, strong EGFR immunoreactivity was not consistently seen with thymic carcinoma. The presence of EGFR within cells was associated with a significantly improved progression-free survival (PFS) and a trend for overall survival (OS). Twelve patients demonstrated C-kit immunoreactivity; the lack of C-kit immunoreactivity was significantly associated with superior PFS but not OS. Her2/neu immunoreactivity was uniformly negative for all tumors evaluated. There was no association between response and biomarker status. Conclusions High EGFR immunoreactivity is seen in more aggressive thymic neoplasms as classified according to the 2004 WHO, but regardless of classification, the presence of EGFR in tumor cells (1+, 2+, and 3+) is associated with improved performance free survival (PFS) and a trend for better OS. In contrast, the absence of C-kit immunoreactivity was associated with improved PFS. These data suggest that EGFR and C-kit may be prognostic, and further studies of these markers in subcategories of thymic malignancies is warranted. PMID:20421818
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Dayton, Melody; Koskinen, Mikko T; Tom, Bradley K; Mattila, Anna-Maria; Johnston, Eric; Halverson, Joy; Fantin, Dennis; DeNise, Sue; Budowle, Bruce; Smith, David Glenn; Kanthaswamy, Sree
2009-01-01
Aim To develop a reagent kit that enables multiplex polymerase chain reaction (PCR) amplification of 18 short tandem repeats (STR) and the canine sex-determining Zinc Finger marker. Methods Validation studies to determine the robustness and reliability in forensic DNA typing of this multiplex assay included sensitivity testing, reproducibility studies, intra- and inter-locus color balance studies, annealing temperature and cycle number studies, peak height ratio determination, characterization of artifacts such as stutter percentages and dye blobs, mixture analyses, species-specificity, case type samples analyses and population studies. Results The kit robustly amplified domesticated dog samples and consistently generated full 19-locus profiles from as little as 125 pg of dog DNA. In addition, wolf DNA samples could be analyzed with the kit. Conclusion The kit, which produces robust, reliable, and reproducible results, will be made available for the forensic research community after modifications based on this study’s evaluation to comply with the quality standards expected for forensic casework. PMID:19480022
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Pochop, Jaroslav; Kačániová, Miroslava; Hleba, Lukáš; Lopasovský, L'ubomír; Bobková, Alica; Zeleňáková, Lucia; Stričík, Michal
2012-01-01
The aim of this study was to follow contamination of ready-to-eat food with Listeria monocytogenes by using the Step One real time polymerase chain reaction (PCR). We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and MicroSEQ® Listeria monocytogenes Detection Kit for the real-time PCR performance. In 30 samples of ready-to-eat milk and meat products without incubation we detected strains of Listeria monocytogenes in five samples (swabs). Internal positive control (IPC) was positive in all samples. Our results indicated that the real-time PCR assay developed in this study could sensitively detect Listeria monocytogenes in ready-to-eat food without incubation.
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Antonescu, Cristina R; Sommer, Gunhild; Sarran, Lisa; Tschernyavsky, Sylvia J; Riedel, Elyn; Woodruff, James M; Robson, Mark; Maki, Robert; Brennan, Murray F; Ladanyi, Marc; DeMatteo, Ronald P; Besmer, Peter
2003-08-15
Activating mutations of the KIT juxtamembrane region are the most common genetic events in gastrointestinal stromal tumors (GISTs) and have been noted as independent prognostic factors. The impact of KIT mutation in other regions, such as the extracellular or kinase domains, is not well-defined and fewer than 30 cases have been published to date. One hundred twenty GISTs, confirmed by KIT immunoreactivity, were evaluated for the presence of KIT exon 9, 11, 13, and 17 mutations. The relation between the presence/type of KIT mutation and clinicopathological factors was analyzed using Fisher's exact test and log-rank test. Forty-four % of the tumors were located in the stomach, 47% in the small bowel, 6% in the rectum, and 3% in the retroperitoneum. Overall, KIT mutations were detected in 78% of patients as follows: 67% in exon 11, 11% in exon 9, and none in exon 13 or 17. The types of KIT exon 11 mutations were heterogeneous and clustered in the classic "hot spot" at the 5' end of exon 11. Seven % of cases showed internal tandem duplications (ITD) at the 3' end of exon 11, in a region that we designate as a second hot spot for KIT mutations. Interestingly, these cases were associated with: female predominance, stomach location, occurrence in older patients, and favorable outcome. There were significant associations between exon 9 mutations and large tumor size (P < 0.001) and extragastric location (P = 0.02). Ten of these 13 patients with more than 1-year follow-up have developed recurrent disease. Most KIT-expressing GISTs show KIT mutations that are preferentially located within the classic hot spot of exon 11. In addition, we found an association between a second hot spot at the 3'end of exon 11, characterized by ITDs, and a subgroup of clinically indolent gastric GISTs in older females. KIT exon 9 mutations seem to define a distinct subset of GISTs, located predominantly in the small bowel and associated with an unfavorable clinical course.
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Student chapters: effective dissemination networks for informal optics and photonics education
NASA Astrophysics Data System (ADS)
Fabian, Dirk; Vermeulen, Nathalie; Van Overmeire, Sara
2009-06-01
Professional societies sponsor student chapters in order to foster scholarship and training in photonics at the college and graduate level, but they are also an excellent resource for disseminating photonics knowledge to pre-college students and teachers. Starting in 2006, we tracked the involvement of SPIE student chapter volunteers in informal pre-college education settings. Chapter students reached 2800, 4900 and 11800 pre-college students respectively from 2006-2008 with some form of informal instruction in optics and photonics. As a case study, the EduKit, a self-contained instruction module featuring refractive and diffractive micro-optics developed by the European Network of Excellence on Micro-Optics (NEMO), was disseminated through student chapters in Argentina, Belgium, Canada, China, Colombia, India, Latvia, Mexico, Peru, Russia, Singapore, South Africa, and the United States. We tracked the movement of this material through the network, up to the student-teacher feedback stage. The student chapter network provided rapid dissemination of the material, translation of the material into the local language, and leveraged existing chapter contacts in schools to provide an audience. We describe the student chapter network and its impact on the development of the EduKit teaching module.
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78 FR 46927 - Procurement List; Proposed Deletions
Federal Register 2010, 2011, 2012, 2013, 2014
2013-08-02
...: 7520-01-453-1968--4 PC STAMPS KIT, ``ACCOUNTANT SET'' NSN: 7520-01-453-1969--4 PC STAMPS KIT, ``MAIL... COORDINATOR, WASHINGTON, DC Service Type/Location: Microfilming Tax Forms Service, Internal Revenue Service...
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Mahameed, Mohamed; Tirosh, Boaz
2017-11-01
An optimized biomanufacturing process in mammalian cells is contingent on the ability of the producing cells to reach high viable cell densities. In addition, at the peak of growth, cells need to continue producing the biological entity at a consistent quality. Thus, engineering cells with robust growth performance and resilience to variable stress conditions is highly desirable. The tyrosine kinase receptor, KIT, plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity, and resistance to kinase inhibitors. Importantly, this onco-KIT mutant when introduced into various cell types is arrested in the endoplasmic reticulum in a constitutively active form. Here, we investigated the effect of oncogenic D816V KIT on the performance of CHO-K1 cells under conventional tissue culture growth settings and when adapted, to shaking conditions. The onco-KIT promoted global protein synthesis, elevated the expression of a secretable transgene, enhanced proliferation, and improved the overall titers of a model glycoprotein. Moreover, the expression of the onco-KIT endowed the cells with a remarkable resistance to various stress conditions. Our data suggest that the introduction of onco-KIT can serve as a strategy for improving glycoprotein biomanufacturing. Biotechnol. Bioeng. 2017;114: 2560-2570. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
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Biranjia-Hurdoyal, Susheela D; Seetulsingh-Goorah, Sharmila P
2016-01-01
The aim was to determine the performances of four Helicobacter pylori serological detection kits in different target groups, using Amplified IDEIA™ Hp StAR™ as gold standard. Kits studied were Rapid Immunochromatoghraphic Hexagon, Helicoblot 2.1, an EIA IgG kit and EIA IgA kit. Stool and blood samples were collected from 162 apparently healthy participants (control) and 60 Type 2 diabetes mellitus (T2DM) patients. The performances of the four serological detection kits were found to be affected by gender, age, health status and ethnicity of the participants. In the control group, the Helicoblot 2.1 kit had the best performance (AUC = 0.85; p<0.05, accuracy = 86.4%), followed by EIA IgG (AUC = 0.75; p<0.05, accuracy = 75.2%). The Rapid Hexagon and EIA IgA kits had relatively poor performances. In the T2DM subgroup, the kits H2.1 and EIA IgG had best performances, with accuracies of 96.5% and 93.1% respectively. The performance of EIA IgG improved with adjustment of its cut-off value. The performances of the detection kits were affected by various factors which should be taken into consideration.
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Comparison of two techniques for removing fiber posts.
Gesi, A; Magnolfi, S; Goracci, C; Ferrari, M
2003-09-01
The purpose of this study was to evaluate the time needed to remove several types of fiber posts using two different bur kits. Estimates refer to the time needed to pass the fiber post until arriving at the gutta-percha. Sixty extracted anterior teeth were treated endodontically. A post space with a standard depth of 10 mm was prepared in each root canal. The sample was randomly divided into 3 groups of 20 specimens each. Three different types of posts were cemented: group 1, Conic 6% tapered fiber posts (Ghimas); group 2, FRC Poster fiber posts (Ivoclar-Vivadent); and group 3, Composipost carbon fiber posts (RTD). To remove the post, for half of each group's specimens the burs from the RTD fiber posts removal kit were used (subgroup A). From the other half of the teeth in each group (subgroup B), posts were removed by using a diamond bur and a Largo bur. Composipost carbon fiber posts (group 3) took significantly less time to remove than the other two types of posts (p < 0.05). For the bur kits, the procedure involving the use of a diamond and a Largo bur (subgroup B) was significantly faster (p < 0.05). The interaction between the type of post and the type of bur kit used was not significant (p > 0.05).
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T & I--Electric Motors. Kit No. 621. Instructor's Manual and Student Learning Activity Guide.
ERIC Educational Resources Information Center
Bomar, William
This instructor's manual and student learning activity guide comprise a kit for trade and industrial education (T & I) activities on electric motors. Purpose stated for the activities is to teach the student the four basic types of electric motors, the advantages and disadvantages of each, the types of jobs each can perform, and how to disassemble…
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Desneux, Jérémy; Pourcher, Anne-Marie
2014-01-01
Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil®, PowerFecal®, NucleoSpin® Soil kits and QIAamp® DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin® Soil kit (NS kit), and to a lesser extent the PowerFecal® kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. PMID:24838631
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Rupar, Kaja; Moharram, Sausan A; Kazi, Julhash U; Rönnstrand, Lars
2018-04-23
KIT is a receptor tyrosine kinase (RTK) involved in several cellular processes such as regulation of proliferation, survival and differentiation of early hematopoietic cells, germ cells and melanocytes. Activation of KIT results in phosphorylation of tyrosine residues in the receptor, and recruitment of proteins that mediate downstream signaling and also modulate receptor signaling. Here we show that the SRC-like adaptor protein 2 (SLAP2) binds to wild-type KIT in a ligand-dependent manner and is furthermore found constitutively associated with the oncogenic mutant KIT-D816V. Peptide fishing analysis mapped pY568 and pY570 as potential SLAP2 association sites in KIT, which overlaps with the SRC binding sites in KIT. Expression of SLAP2 in cells expressing the transforming mutant KIT-D816V led to reduced cell viability and reduced colony formation. SLAP2 also partially blocked phosphorylation of several signal transduction molecules downstream of KIT such as AKT, ERK, p38 and STAT3. Finally, SLAP2 expression enhanced ubiquitination of KIT and its subsequent degradation. Taken together, our data demonstrate that SLAP2 negatively modulates KIT-D816V-mediated transformation by enhancing degradation of the receptor.
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Roh, Eun-Youn; Song, Eun-Young; Chang, Jee-Young; Yoon, Jong-Hyun; Shin, Sue
2016-08-01
The performance of a new intermediate-resolution method using a PCR-Luminex platform and LABType® SSO A, B DRB1 kits as an HLA typing method for the cord blood (CB) registry of the Korean population was investigated. A total of 1,413 cord blood units (CBUs) were enrolled - 1,382 from Koreans and 31 from non-Koreans or mixed-ancestry individuals. HLA-A, -B, and -DRB1 typing was performed using the LABType® SSO typing kits. HLA typing with the DNA method and 2-digit results are mandatory for the public CB bank in Korea according to the "CB Act." The proportions of ambiguous results in the 2-digit assignment were 14.6% (206/1,413) and 14.8% (205/ 1,382) among the total subjects and the Korean donors, respectively. In the 2-digit resolution, 3 different HLA-A types (69 CBUs), 31 HLA-B types (124 CBUs), and 3 HLA-DRB1 types (13 CBUs) showed ambiguous results. The 'most probable type' to the ambiguous results based on the reported Korean HLA allele frequencies were able to be assigned. The most probable results were 100% consistent with the confirmed types as determined by the HD kits (DRB1) and additional PCR-SBT or PCR-SSP tests (A and B). Luminex technology is more automated and less labor intensive than the conventional SSO typing method, and the results are less affected by differences between inspectors. Although it is not satisfactory as a sole confirmation test and cannot be used as a replacement for the PCR-SBT test, the combination of Luminex technology with LABType® SSO kits and population frequency data provides a proper typing platform that can be used as a qualifying test for CB registries.
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GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells
Barroeta Seijas, Amairelys Belen; Simonetti, Sonia; Vitale, Sara; Runci, Daniele; Quinci, Angela Caterina; Soriani, Alessandra; Criscuoli, Mattia; Filippi, Irene; Naldini, Antonella; Sacchetti, Federico Maria; Tarantino, Umberto; Oliva, Francesco; Piccirilli, Eleonora; Santoni, Angela; Di Rosa, Francesca
2017-01-01
Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity. PMID:28261209
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Characterization of Human Salivary Extracellular RNA by Next-generation Sequencing.
Li, Feng; Kaczor-Urbanowicz, Karolina Elżbieta; Sun, Jie; Majem, Blanca; Lo, Hsien-Chun; Kim, Yong; Koyano, Kikuye; Liu Rao, Shannon; Young Kang, So; Mi Kim, Su; Kim, Kyoung-Mee; Kim, Sung; Chia, David; Elashoff, David; Grogan, Tristan R; Xiao, Xinshu; Wong, David T W
2018-04-23
It was recently discovered that abundant and stable extracellular RNA (exRNA) species exist in bodily fluids. Saliva is an emerging biofluid for biomarker development for noninvasive detection and screening of local and systemic diseases. Use of RNA-Sequencing (RNA-Seq) to profile exRNA is rapidly growing; however, no single preparation and analysis protocol can be used for all biofluids. Specifically, RNA-Seq of saliva is particularly challenging owing to high abundance of bacterial contents and low abundance of salivary exRNA. Given the laborious procedures needed for RNA-Seq library construction, sequencing, data storage, and data analysis, saliva-specific and optimized protocols are essential. We compared different RNA isolation methods and library construction kits for long and small RNA sequencing. The role of ribosomal RNA (rRNA) depletion also was evaluated. The miRNeasy Micro Kit (Qiagen) showed the highest total RNA yield (70.8 ng/mL cell-free saliva) and best small RNA recovery, and the NEBNext library preparation kits resulted in the highest number of detected human genes [5649-6813 at 1 reads per kilobase RNA per million mapped (RPKM)] and small RNAs [482-696 microRNAs (miRNAs) and 190-214 other small RNAs]. The proportion of human RNA-Seq reads was much higher in rRNA-depleted saliva samples (41%) than in samples without rRNA depletion (14%). In addition, the transfer RNA (tRNA)-derived RNA fragments (tRFs), a novel class of small RNAs, were highly abundant in human saliva, specifically tRF-4 (4%) and tRF-5 (15.25%). Our results may help in selection of the best adapted methods of RNA isolation and small and long RNA library constructions for salivary exRNA studies. © 2018 American Association for Clinical Chemistry.
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Small RNA profiling of low biomass samples: identification and removal of contaminants.
Heintz-Buschart, Anna; Yusuf, Dilmurat; Kaysen, Anne; Etheridge, Alton; Fritz, Joëlle V; May, Patrick; de Beaufort, Carine; Upadhyaya, Bimal B; Ghosal, Anubrata; Galas, David J; Wilmes, Paul
2018-05-14
Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. DNA contamination has been previously reported, yet contamination with RNA is usually considered to be very unlikely due to its inherent instability. Small RNAs (sRNAs) identified in tissues and bodily fluids, such as blood plasma, have implications for physiology and pathology, and therefore the potential to act as disease biomarkers. Thus, the possibility for RNA contaminants demands careful evaluation. Herein, we report on the presence of small RNA (sRNA) contaminants in widely used microRNA extraction kits and propose an approach for their depletion. We sequenced sRNAs extracted from human plasma samples and detected important levels of non-human (exogenous) sequences whose source could be traced to the microRNA extraction columns through a careful qPCR-based analysis of several laboratory reagents. Furthermore, we also detected the presence of artefactual sequences related to these contaminants in a range of published datasets, thereby arguing in particular for a re-evaluation of reports suggesting the presence of exogenous RNAs of microbial and dietary origin in blood plasma. To avoid artefacts in future experiments, we also devise several protocols for the removal of contaminant RNAs, define minimal amounts of starting material for artefact-free analyses, and confirm the reduction of contaminant levels for identification of bona fide sequences using 'ultra-clean' extraction kits. This is the first report on the presence of RNA molecules as contaminants in RNA extraction kits. The described protocols should be applied in the future to avoid confounding sRNA studies.
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Loss of c-KIT expression in thyroid cancer cells.
Franceschi, Sara; Lessi, Francesca; Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria
2017-01-01
Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.
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Loss of c-KIT expression in thyroid cancer cells
Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria
2017-01-01
Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression. PMID:28301608
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Conditional Deletion of Kit in Melanocytes: White Spotting Phenotype Is Cell Autonomous.
Aoki, Hitomi; Tomita, Hiroyuki; Hara, Akira; Kunisada, Takahiro
2015-07-01
It is well established that cell-intrinsic signaling through the receptor tyrosine kinase KIT is critical for the development of neural crest-derived melanocytes. Nevertheless, it is not entirely clear whether Kit acts exclusively in a melanocyte-autonomous manner or in addition indirectly through other cell types. To address this question in vivo, we generated a targeted allele of Kit that allowed for CRE recombinase-mediated deletion of the transmembrane domain of KIT. Mice carrying one copy of the targeted allele and expressing CRE under the melanoblast/melanocyte-specific tyrosinase promoter exhibited a white spotting phenotype that was even more extensive compared with that found in mice heterozygous for a Kit-null allele. This phenotype is unlikely the result of sequestration of KIT ligand by neighboring cells or by potentially secreted forms of KIT because the spotting phenotype could not be rescued by overexpression of KITL. Likewise, overexpression of endothelin-3 or hepatocyte growth factor was unable to rescue melanocytes in these mice. Although the severity of the observed phenotype remains to be explained, the findings indicate that melanocyte-selective impairment of Kit is sufficient to interfere with normal melanocyte development.
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Comparison of five methods for extraction of Legionella pneumophila from respiratory specimens.
Wilson, Deborah; Yen-Lieberman, Belinda; Reischl, Udo; Warshawsky, Ilka; Procop, Gary W
2004-12-01
The efficiencies of five commercially available nucleic acid extraction methods were evaluated for the recovery of a standardized inoculum of Legionella pneumophila in respiratory specimens (sputum and bronchoalveolar lavage [BAL] specimens). The concentrations of Legionella DNA recovered from sputa with the automated MagNA Pure (526,200 CFU/ml) and NucliSens (171,800 CFU/ml) extractors were greater than those recovered with the manual methods (i.e., Roche High Pure kit [133,900 CFU/ml], QIAamp DNA Mini kit [46,380 CFU/ml], and ViralXpress kit [13,635 CFU/ml]). The rank order was the same for extracts from BAL specimens, except that for this specimen type the QIAamp DNA Mini kit recovered more than the Roche High Pure kit.
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DNA extraction from benthic Cyanobacteria: comparative assessment and optimization.
Gaget, V; Keulen, A; Lau, M; Monis, P; Brookes, J D
2017-01-01
Benthic Cyanobacteria produce toxic and odorous compounds similar to their planktonic counterparts, challenging the quality of drinking water supplies. The biofilm that benthic algae and other micro-organisms produce is a complex and protective matrix. Monitoring to determine the abundance and identification of Cyanobacteria, therefore, relies on molecular techniques, with the choice of DNA isolation technique critical. This study investigated which DNA extraction method is optimal for DNA recovery in order to guarantee the best DNA yield for PCR-based analysis of benthic Cyanobacteria. The conventional phenol-chloroform extraction method was compared with five commercial kits, with the addition of chemical and physical cell-lysis steps also trialled. The efficacy of the various methods was evaluated by measuring the quantity and quality of DNA by UV spectrophotometry and by quantitative PCR (qPCR) using Cyanobacteria-specific primers. The yield and quality of DNA retrieved with the commercial kits was significantly higher than that of DNA obtained with the phenol-chloroform protocol. Kits including a physical cell-lysis step, such as the MO BIO Power Soil and Biofilm kits, were the most efficient for DNA isolation from benthic Cyanobacteria. These commercial kits allow greater recovery and the elimination of dangerous chemicals for DNA extraction, making them the method of choice for the isolation of DNA from benthic mats. They also facilitate the extraction of DNA from benthic Cyanobacteria, which can help to improve the characterization of Cyanobacteria in environmental studies using qPCRs or population composition analysis using next-generation sequencing. © 2016 The Society for Applied Microbiology.
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c-Kit modifies the inflammatory status of smooth muscle cells
Song, Lei; Martinez, Laisel; Zigmond, Zachary M.; Hernandez, Diana R.; Lassance-Soares, Roberta M.; Selman, Guillermo
2017-01-01
Background c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. Methods High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (KitW/W–v) and control (Kit+/+) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. Results The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Discussion Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation. PMID:28626608
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c-Kit modifies the inflammatory status of smooth muscle cells.
Song, Lei; Martinez, Laisel; Zigmond, Zachary M; Hernandez, Diana R; Lassance-Soares, Roberta M; Selman, Guillermo; Vazquez-Padron, Roberto I
2017-01-01
c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (Kit W/W-v ) and control (Kit +/+ ) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation.
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MEMS phase former kit for high-resolution wavefront control
NASA Astrophysics Data System (ADS)
Gehner, Andreas; Wildenhain, Michael; Neumann, Hannes; Elgner, Andreas; Schenk, Harald
2005-08-01
The MEMS Phase Former Kit developed by the Fraunhofer IPMS is a complete Spatial Light Modulator system based on a piston-type Micro Mirror Array (MMA) for the use in high-resolution, high-speed optical phase control. It has been designed for an easy system integration into an user-specific environment to offer a platform for first practical investigations to open up new applications in Adaptive Optics. The key component is a fine segmented 240 x 200 array of 40 μm piston-type mirror elements capable of 400 nm analog deflection for a 2pi phase modulation in the visible. Each mirror can be addressed and deflected independently by means of an integrated CMOS backplane address circuitry at an 8bit height resolution. Full user programmability and control is provided by a newly developed comfortable driver software for Windows XP based PCs supporting both a Graphical User Interface (GUI) for stand-alone operation with pre-defined data patterns as well as an open ActiveX programming interface for a closed-loop operation with real-time data from an external source. An IEEE1394a FireWire interface is used for high-speed data communication with an electronic driving board performing the actual MMA programming and control allowing for an overall frame rate of up to 500 Hz. Successful proof-of-concept demonstrations already have been given for eye aberration correction in ophthalmology, for error compensation of leightweight primary mirrors of future space telescopes and for ultra-short laser pulse shaping. Besides a presentation of the basic device concept and system architecture the paper will give an overview of the obtained results from these applications.
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KIT Mutations Are Common in Testicular Seminomas
Kemmer, Kathleen; Corless, Christopher L.; Fletcher, Jonathan A.; McGreevey, Laura; Haley, Andrea; Griffith, Diana; Cummings, Oscar W.; Wait, Cecily; Town, Ajia; Heinrich, Michael C.
2004-01-01
Expression of KIT tyrosine kinase is critical for normal germ cell development and is observed in the majority of seminomas. Activating mutations in KIT are common in gastrointestinal stromal tumors and mastocytosis. In this study we examined the frequency and spectrum of KIT mutations in 54 testicular seminomas, 1 ovarian dysgerminoma and 37 non-seminomatous germ cell tumors (NSGCT). Fourteen seminomas (25.9%) contained exon 17 point mutations including D816V (6 cases), D816H (3 cases), Y823D (2 cases), and single examples of Y823C, N822K, and T801I. No KIT mutations were found in the ovarian dysgerminoma or the NSGCTs. In transient transfection assays, mutant isoforms D816V, D816H, Y823D, and N822K were constitutively phosphorylated in the absence of the natural ligand for KIT, stem cell factor (SCF). In contrast, activation of T801I and wild-type KIT required SCF. Mutants N822K and Y823D were inhibited by imatinib mesylate (Gleevec, previously STI571) whereas D816V and D816H were both resistant to imatinib mesylate. Biochemical evidence of KIT activation, as assessed by KIT phosphorylation and KIT association with phosphatidylinositol (PI) 3-kinase in tumor cell lysates, was largely confined to seminomas with a genomic KIT mutation. These findings suggest that activating KIT mutations may contribute to tumorigenesis in a subset of seminomas, but are not involved in NSGCT. PMID:14695343
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A national survey of home-based care kits for palliative HIV/AIDS care in South Africa.
Mabude, Z A; Beksinska, M E; Ramkissoon, A; Wood, S; Folsom, M
2008-09-01
The objective of this study was to assess home-based care (HBC) kits and programs in South Africa to evaluate the feasibility of scaling up kit production and distribution. South African HBC organizations received structured questionnaires; key informant interviews and a literature review were completed to assess systems for production, distribution and supply of HBC kits. Meetings with stakeholders were held in two Provinces to share and analyze the study findings. The study team distributed questionnaires to 466 organizations and conducted interviews with representatives from 45 organizations, the Provincial Department of Health (DoH) and manufacturers of kits. All identifiable HBC organizations in South Africa were included in the survey. As a result 215 HBC organizations returned questionnaires; including non-governmental organizations (56%), community-based organizations (32%) and organizations affiliated with government health departments. Two types of kits were available: a home kit and a professional kit. The demand for HBC kits exceeded availability, kit contents and availability varied considerably and the supply chain was irregular. Kit production and distribution systems were fragmented. Replenishment of kit items was problematic. End-users are mostly caregivers who have not received adequate training on their use. The study shows that substantial work has been done by HBC organizations in South Africa to respond to the need for palliative care supplies within resource constraints. The growing demand for kits exceeds the supply. There is a need to improve the supply chain management of HBC kits, strengthen referral systems and links between community-based organizations and government departments, expand training opportunities for care givers, and develop monitoring and evaluation systems.
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Hattori, Mai; Ishikawa, Osamu; Oikawa, Daisuke; Amano, Hiroo; Yasuda, Masahito; Kaira, Kyoichi; Ishida-Yamamoto, Akemi; Nakano, Hajime; Sawamura, Daisuke; Terawaki, Shin-Ichi; Wakamatsu, Kaori; Tokunaga, Fuminori; Shimizu, Akira
2018-03-21
Piebaldism is a pigmentary disorder characterized by a white forelock and depigmented patches. Although the loss-of-function mutations in the KIT gene underlie the disease, the intracellular dynamics of the mutant KIT are largely unknown. We herein report a Japanese family with piebaldism in which the affected members showed a mild phenotype. The objective of this study is to investigate the functions and intracellular dynamics of the mutant KIT protein. We performed genetic analyses of the KIT gene using peripheral blood cells. We analyzed the intracellular localization of the mutant KIT protein in HEK293T cells transfected with wild-type (Wt) and/or mutant KIT genes. Immunoprecipitation analyses, immunoblotting and immunofluorescence studies were performed using antibodies against KIT and downstream signaling proteins. Glycosidase digestion analysis was performed to clarify the intracellular localization of KIT protein. A genetic analysis revealed a novel heterozygous mutation c.645_650delTGTGTC which results in the in-frame deletion of Val 216 and Ser 217 in the extracellular domain of KIT. Immunoprecipitation analyses confirmed that the wild and mutant KIT formed a heterodimer after treatment with stem cell factor (SCF); however, the phosphorylation of the downstream signaling factors was decreased. In an immunofluorescence study, the mutant KIT accumulated predominantly in the endoplasmic reticulum (ER) and was sparsely expressed on the cell surface. A glycosidase digestion study revealed that the mutant KIT is predominantly localized in the ER. These data reveal an aberrant function and intracellular localization of mutant KIT protein in piebaldism. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
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Emergency medical kit for commercial airlines: an update.
Thibeault, Claude
2002-06-01
As expected, the issue of medical kits for commercial airlines continues to attract attention, especially in light of the recent United States regulation on the subject. As promised in its first recommendation in 1998, the Air Transport Medicine (ATM) Committee has continued to monitor medical kit usage as well as pharmaceutical scientific developments and wishes to propose an update to its 1998 recommendation. Lists of contents are provided for emergency medical kits of two types: 1) those without defibrillator/monitor or monitor; and 2) those with defibrillator/monitor or monitor alone. Follow up and updates on this issue will be an ongoing task of the ATM Committee.
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Michel, Anastasija; Schüler, Andrea; Friedrich, Pamela; Döner, Fatma; Bopp, Tobias; Radsak, Markus; Hoffmann, Markus; Relle, Manfred; Distler, Ute; Kuharev, Jörg; Tenzer, Stefan; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Schild, Hansjörg; Schmitt, Edgar; Becker, Marc; Stassen, Michael
2013-06-01
Mast cell-deficient Kit(W-sh) "sash" mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that Kit(W-sh) causes aberrant extramedullary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also highly expanded in the spleen of sash mice. These cells are able to suppress T cell responses in vitro and phenotypically and functionally resemble myeloid-derived suppressor cells (MDSC). MDSC typically accumulate in tumor-bearing hosts and are able to dampen immune responses. Consequently, transfer of MDSC from naive sash mice into line 1 alveolar cell carcinoma tumor-bearing wild-type littermates leads to enhanced tumor progression. However, although it can also be observed in sash mice, accelerated growth of transplanted line 1 alveolar cell carcinoma tumors is a mast cell-independent phenomenon. Thus, the Kit(W-sh) mutation broadly affects key steps in myelopoiesis that may have an impact on mast cell research.
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KIT pathway alterations in mucosal melanomas of the vulva and other sites.
Omholt, Katarina; Grafström, Eva; Kanter-Lewensohn, Lena; Hansson, Johan; Ragnarsson-Olding, Boel K
2011-06-15
A significant proportion of mucosal melanomas contain alterations in KIT. The aim of this study was to characterize the pattern of KIT, NRAS, and BRAF mutations in mucosal melanomas at specific sites and to assess activation of the KIT downstream RAF/MEK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways in mucosal melanoma specimens. Seventy-one primary mucosal melanomas from various sites were studied. Mutation analysis was done by DNA sequencing. Expression of KIT, phosphorylated (p)-ERK, and p-AKT was evaluated by immunohistochemistry. KIT mutations were detected in 35% (8 of 23) of vulvar, 9% (2 of 22) of anorectal, 7% (1 of 14) of nasal cavity, and 20% (1 of 5) of penile melanomas. No KIT mutations were found in 7 vaginal melanomas. The difference in KIT mutation frequency between vulvar and nonvulvar cases was statistically significant (P = 0.014). The overall frequencies of NRAS and BRAF mutations were 10% and 6%, respectively. Notably, vaginal melanomas showed a NRAS mutation rate of 43%. KIT gene amplification (≥4 copies), as assessed by quantitative real-time PCR, was observed in 19% of cases. KIT expression was associated with KIT mutation status (P < 0.001) and was more common in vulvar than nonvulvar tumors (P = 0.016). Expression of p-ERK and p-AKT was observed in 42% and 59% of tumors, respectively, and occurred irrespective of KIT/NRAS/BRAF mutation status. NRAS mutation was associated with worse overall survival in univariate analysis. Results show that KIT mutations are more common in vulvar melanomas than other types of mucosal melanomas and that both the RAF/MEK/ERK and PI3K/AKT pathways are activated in mucosal melanoma specimens. ©2011 AACR.
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Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples.
Holmes, Amy S; Houston, Rachel; Elwick, Kyleen; Gangitano, David; Hughes-Stamm, Sheree
2018-05-01
DNA quantification is a vital step in forensic DNA analysis to determine the optimal input amount for DNA typing. A quantitative real-time polymerase chain reaction (qPCR) assay that can predict DNA degradation or inhibitors present in the sample prior to DNA amplification could aid forensic laboratories in creating a more streamlined and efficient workflow. This study compares the results from four commercial qPCR kits: (1) Investigator® Quantiplex® Pro Kit, (2) Quantifiler® Trio DNA Quantification Kit, (3) PowerQuant® System, and (4) InnoQuant® HY with high molecular weight DNA, low template samples, degraded samples, and DNA spiked with various inhibitors.The results of this study indicate that all kits were comparable in accurately predicting quantities of high quality DNA down to the sub-picogram level. However, the InnoQuant(R) HY kit showed the highest precision across the DNA concentration range tested in this study. In addition, all kits performed similarly with low concentrations of forensically relevant PCR inhibitors. However, in general, the Investigator® Quantiplex® Pro Kit was the most tolerant kit to inhibitors and provided the most accurate quantification results with higher concentrations of inhibitors (except with salt). PowerQuant® and InnoQuant® HY were the most sensitive to inhibitors, but they did indicate significant levels of PCR inhibition. When quantifying degraded samples, each kit provided different degradation indices (DI), with Investigator® Quantiplex® Pro indicating the largest DI and Quantifiler® Trio indicating the smallest DI. When the qPCR kits were paired with their respective STR kit to genotype highly degraded samples, the Investigator® 24plex QS and GlobalFiler® kits generated more complete profiles when the small target concentrations were used for calculating input amount.
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NASA Astrophysics Data System (ADS)
Holasek, Rick; Nakanishi, Keith; Ziph-Schatzberg, Leah; Santman, Jeff; Woodman, Patrick; Zacaroli, Richard; Wiggins, Richard
2017-04-01
Hyperspectral imaging (HSI) has been used for over two decades in laboratory research, academic, environmental and defense applications. In more recent time, HSI has started to be adopted for commercial applications in machine vision, conservation, resource exploration, and precision agriculture, to name just a few of the economically viable uses for the technology. Corning Incorporated (Corning) has been developing and manufacturing HSI sensors, sensor systems, and sensor optical engines, as well as HSI sensor components such as gratings and slits for over a decade and a half. This depth of experience and technological breadth has allowed Corning to design and develop unique HSI spectrometers with an unprecedented combination of high performance, low cost and low Size, Weight, and Power (SWaP). These sensors and sensor systems are offered with wavelength coverage ranges from the visible to the Long Wave Infrared (LWIR). The extremely low SWaP of Corning's HSI sensors and sensor systems enables their deployment using limited payload platforms such as small unmanned aerial vehicles (UAVs). This paper discusses use of the Corning patented monolithic design Offner spectrometer, the microHSI™, to build a highly compact 400-1000 nm HSI sensor in combination with a small Inertial Navigation System (INS) and micro-computer to make a complete turn-key airborne remote sensing payload. This Selectable Hyperspectral Airborne Remote sensing Kit (SHARK) has industry leading SWaP (1.5 lbs) at a disruptively low price due, in large part, to Corning's ability to manufacture the monolithic spectrometer out of polymers (i.e. plastic) and therefore reduce manufacturing costs considerably. The other factor in lowering costs is Corning's well established in house manufacturing capability in optical components and sensors that further enable cost-effective fabrication. The competitive SWaP and low cost of the microHSI™ sensor is approaching, and in some cases less than the price point of Multi Spectral Imaging (MSI) sensors. Specific designs of the Corning microHSI™ SHARK visNIR turn-key system are presented along with salient performance characteristics. Initial focus market areas include precision agriculture and historic and recent microHSI™ SHARK prototype test results are presented.
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Evaluation of commercial kits for dual extraction of DNA and RNA from human body fluids.
Schweighardt, Andrew J; Tate, Courtney M; Scott, Kristina A; Harper, Kathryn A; Robertson, James M
2015-01-01
STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body-fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR-Duet(™) kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand-alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.
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Synthesis and biological evaluation of di-aryl urea derivatives as c-Kit inhibitors.
Ravez, Séverine; Arsenlis, Stéphane; Barczyk, Amélie; Dupont, Anthony; Frédérick, Raphaël; Hesse, Stéphanie; Kirsch, Gilbert; Depreux, Patrick; Goossens, Laurence
2015-11-15
Inhibition of receptor tyrosine kinases (RTKs) continued to be a successful approach for the treatment of many types of human cancers and many potent small molecules kinase inhibitors have been discovered the last decade. In the present study, we describe the synthesis of thienopyrimidine derivatives and their pharmacological evaluation against nine kinases (EGFR, PDGFR-ß, c-Kit, c-Met, Src, Raf, VEGFR-1, -2 and -3). Most of the synthesized compounds showed from moderate to potent activities against c-Kit with IC50 values in the nanomolar range. Among them, 4-anilino(urea)thienopyrimidine analogs showed selectivity and potent c-Kit inhibition with IC50 values less than 6 nM. Docking simulation was performed for the most promising compound 9 into the c-Kit active site to determine the potential binding mode. This study reveal that the 4-anilino(urea)thienopyrimidine is an interesting scaffold to design novel potent and selective c-Kit inhibitors which may make promising candidates for cancers where c-Kit receptors are overexpressed. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Vojkovska, H; Kubikova, I; Kralik, P
2015-03-01
Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false-negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice. In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model micro-organism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real-time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoil(™) Microbial DNA Isolation Kit, is suitable for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. Coupled with the triplex real-time qPCR assay, this DNA isolation kit is applicable to the samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. Several recent outbreaks of Listeria monocytogenes have been associated with the consumption of fruits and vegetables. Real-time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real-time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoil(™) Microbial DNA Isolation Kit can be used for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. This method is applicable to samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. © 2014 The Society for Applied Microbiology.
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Yun, SeongJu; Kim, Won Kyu; Kwon, Yujin; Jang, Mi; Bauer, Sebastian
2018-01-01
Gain‐of‐function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have been shown to be dysregulated in GISTs and impact KIT expression. Little is known though on KIT‐independent targets of KIT‐regulating mRNAs. We sought to investigate how miR‐494 inhibits GIST proliferation and to identify novel target gene. We used microarray‐based gene expression analyses to identify pathways and target genes affected by miR‐494. The expressional relationship between survivin and miR‐494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, promoter assays and chromatin immunoprecipitation (ChiP) were performed to clarify the roles of survivin in GIST progression. Gene expression microarray analysis revealed that miR‐494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin (BIRC5) was a key target of miR‐494, and its expression showed an inverse correlation with miR‐494 expression in 35 GIST tissues (Pearson's correlation coefficient, r = −0.418, p = 0.012). Downregulation of survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced survivin overexpression relieved miR‐494‐mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of survivin. Survivin also regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GISTs revealed that survivin expression was significantly correlated with overall survival of GIST patients (p = 0.004). Our findings indicated that miR‐494 synergistically suppressed GISTs by concomitantly targeting survivin and KIT. PMID:29277888
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Land Use as a Driver of Patterns of Rodenticide Exposure in Modeled Kit Fox Populations
Nogeire, Theresa M.; Lawler, Joshua J.; Schumaker, Nathan H.; Cypher, Brian L.; Phillips, Scott E.
2015-01-01
Although rodenticides are increasingly regulated, they nonetheless cause poisonings in many non-target wildlife species. Second-generation anticoagulant rodenticide use is common in agricultural and residential landscapes. Here, we use an individual-based population model to assess potential population-wide effects of rodenticide exposures on the endangered San Joaquin kit fox (Vulpes macrotis mutica). We estimate likelihood of rodenticide exposure across the species range for each land cover type based on a database of reported pesticide use and literature. Using a spatially-explicit population model, we find that 36% of modeled kit foxes are likely exposed, resulting in a 7-18% decline in the range-wide modeled kit fox population that can be linked to rodenticide use. Exposures of kit foxes in low-density developed areas accounted for 70% of the population-wide exposures to rodenticides. We conclude that exposures of non-target kit foxes could be greatly mitigated by reducing the use of second-generation anticoagulant rodenticides in low-density developed areas near vulnerable populations. PMID:26244655
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Electronic Reference Service. SPEC Kit 251.
ERIC Educational Resources Information Center
Goetsch, Lori, Comp.
1999-01-01
The goals of this SPEC Kit were to report on the extent to which ARL (Association of Research Libraries) libraries provide electronic reference services and to offer a snapshot of the types of users reached, questions received, policies established, data-gathering techniques utilized, and innovations implemented. The first section of the kit…
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78 FR 67022 - Airworthiness Directives; Bell Helicopter Textron, Inc., Helicopters
Federal Register 2010, 2011, 2012, 2013, 2014
2013-11-08
... Apical Industries, Inc. (Apical) emergency float kit installed under Supplemental Type Certificate (STC... service information identified in this AD, contact Apical Industries, Inc., 2608 Temple Heights Drive..., 206L-4, and 407 helicopters with an Apical emergency float kit installed under STC number SR01535LA...
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The stem cell growth factor receptor KIT is not expressed on interstitial cells in bladder.
Gevaert, Thomas; Ridder, Dirk De; Vanstreels, Els; Daelemans, Dirk; Everaerts, Wouter; Aa, Frank Van Der; Pintelon, Isabel; Timmermans, Jean-Pierre; Roskams, Tania; Steiner, Clara; Neuhaus, Jochen
2017-06-01
The mast/stem cell growth factor receptor KIT has long been assumed to be a specific marker for interstitial cells of Cajal (ICC) in the bladder, with possible druggable perspectives. However, several authors have challenged the presence of KIT + ICC in recent years. The aim of this study was therefore to attempt to clarify the conflicting reports on KIT expression in the bladder of human beings, rat, mouse and guinea pig and to elucidate the possible role of antibody-related issues and interspecies differences in this matter. Fresh samples were obtained from human, rat, mouse and guinea pig cystectomies and processed for single/double immunohistochemistry/immunofluorescence. Specific antibodies against KIT, mast cell tryptase (MCT), anoctamin-1 (ANO1) and vimentin were used to characterize the cell types expressing KIT. Gut (jejunum) tissue was used as an external antibody control. Our results revealed KIT expression on mast cells but not on ICC in human, rat, mouse and guinea pig bladder. Parallel immunohistochemistry showed KIT expression on ICC in human, rat, mouse and guinea pig gut, which confirmed the selectivity of the KIT antibody clones. In conclusion, we have shown that KIT + cells in human, rat, mouse and guinea pig bladder are mast cells and not ICC. The present report is important as it opposes the idea that KIT + ICC are present in bladder. In this perspective, functional concepts of KIT + ICC being involved in sensory and/or motor aspects of bladder physiology should be revised. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
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Comparison of lesional skin c-KIT mutations with clinical phenotype in patients with mastocytosis.
Chan, I J; Tharp, M D
2018-06-01
Activating c-KIT mutations cause abnormal mast cell growth and appear to play a role in mastocytosis. However, the correlation of c-KIT mutations with disease phenotypes is poorly characterized. To evaluate the correlation of c-KIT mutations with clinical presentations and laboratory findings. Total cellular RNA was isolated from the skin lesions of 43 adults and 7 children with mastocytosis, and PCR amplicons of cDNA were sequenced for c-KIT mutations. The most common activating mutation, KIT-D816V, was identified in 72% of adults and 57% of children. Additional activating mutations, namely, V560G and the internal tandem duplications (ITDs) 502-503dupAY, were detected in 12% of adults and 8% of children. V560G occurred more commonly in our patients than previously reported, and it appeared to be associated with more advanced disease. Otherwise, the presence or absence of activating mutations did not correlate with skin lesion morphology, disease extent or total serum tryptase levels. Four adults had expression only of wild-type KIT, while two others had expression of a truncated KIT lacking tyrosine kinase activity; yet these patients were clinically indistinguishable from those patients with activating c-KIT mutations. Activating c-KIT mutations exist in a significant portion of patients with mastocytosis, but not all patients showed expression of these mutations. Except for V560G, the presence or absence of activating c-KIT mutations did not predict the extent of disease. These observations suggest that although activating c-KIT mutations are associated with mast cell growth, other genes probably play a role in the cause of mastocytosis. © 2018 British Association of Dermatologists.
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Immunochemical analytical methods for the determination of peanut proteins in foods.
Whitaker, Thomas B; Williams, Kristina M; Trucksess, Mary W; Slate, Andrew B
2005-01-01
Peanut proteins can cause allergenic reactions that can result in respiratory and circulatory effects in the body sometimes leading to shock and death. The determination of peanut proteins in foods by analytical methods can reduce the risk of serious reactions in the highly sensitized individual by allowing for the detection of these proteins in a food at various stages of the manufacturing process. The method performance of 4 commercially available enzyme-linked immunosorbent assay (ELISA) kits was evaluated for the detection of peanut proteins in milk chocolate, ice cream, cookies, and breakfast cereals: ELISA-TEK Peanut Protein Assay, now known as "Bio-Kit" for peanut proteins, from ELISA Technologies Inc.; Veratox for Peanut Allergens from Neogen Corp.; RIDASCREEN Peanut Kit from R-Biopharm GmbH; and ProLisa from Canadian Food Technology Ltd. The 4 test kits were evaluated for accuracy (recovery) and precision using known concentrations of peanut or peanut proteins in the 4 food matrixes. Two different techniques, incurred and spiked, were used to prepare samples with 4 known concentrations of peanut protein. Defatted peanut flour was added in the incurred samples, and water-soluble peanut proteins were added in the spiked samples. The incurred levels were 0.0, 10, 20, and 100 microg whole peanut per g food; the spiked levels were 0.0, 5, 10, and 20 microg peanut protein per g food. Performance varied by test kit, protein concentration, and food matrix. The Veratox kit had the best accuracy or lowest percent difference between measured and incurred levels of 15.7% when averaged across all incurred levels and food matrixes. Recoveries associated with the Veratox kit varied from 93 to 115% for all food matrixes except cookies. Recoveries for all kits were about 50% for cookies. The analytical precision, as measured by the variance, increased with an increase in protein concentration. However, the coefficient of variation (CV) was stable across the 4 incurred protein levels and was 7.0% when averaged across the 4 food matrixes and analytical kits. The R-Biopharm test kit had the best precision or a CV of 4.2% when averaged across all incurred levels and food matrixes. Because measured protein values varied by test kit and food matrix, a method was developed to normalize or transform measured protein concentrations to an adjusted protein value that was equal to the known protein concentration. The normalization method adjusts measured protein values to equal the true protein value regardless of the type test kit or type food matrix.
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Nannini, Margherita; Ravegnini, Gloria; Angelini, Sabrina; Astolfi, Annalisa; Biasco, Guido; Pantaleo, Maria A
2015-01-01
MicroRNAs are a class of short noncoding RNAs, that play a relevant role in multiple biological processes, such as differentiation, proliferation and apoptosis. Gastrointestinal stromal tumors (GIST) are considered as a paradigm of molecular biology in solid tumors worldwide, and after the discovery of specific alterations in the KIT and PDGFRA genes, they have emerged from anonymity to become a model for targeted therapy. Epigenetics have an emerging and relevant role in different steps of GIST biology such as tumorigenesis, disease progression, prognosis and drug resistance. The aim of the present review was to summarize the current evidence about the role of microRNAs in GIST, including their potential application as well as their limits.
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Kalinsky, Kevin; Lee, Sandra; Rubin, Krista M; Lawrence, Donald P; Iafrarte, Anthony J; Borger, Darell R; Margolin, Kim A; Leitao, Mario M; Tarhini, Ahmad A; Koon, Henry B; Pecora, Andrew L; Jaslowski, Anthony J; Cohen, Gary I; Kuzel, Timothy M; Lao, Christopher D; Kirkwood, John M
2017-07-15
KIT-directed tyrosine kinase inhibitors such as imatinib have demonstrated benefits in KIT-mutant (KIT+) mucosal, acral, vulvovaginal, and chronically sun-damaged (CSD) melanoma. Dasatinib has superior preclinical activity in comparison with other tyrosine kinase inhibitors against cells with the most common KIT mutation, exon 11 L576P . The ECOG-ACRIN E2607 trial assessed dasatinib in patients with these melanoma subtypes. Patients received 70 mg of oral dasatinib twice daily. The primary objective for this 2-stage phase 2 trial was response rate. Stage I was open to KIT+ and wild-type KIT (KIT-) mucosal, acral, and CSD melanoma (n = 57). Stage II accrued only KIT+ tumors (n = 30). To enrich the trial for KIT+ tumors, vulvovaginal melanoma was added, and CSD melanoma was removed from eligibility. Secondary objectives included progression-free survival (PFS), overall survival (OS), and safety. From May 2009 to December 2010, the first stage enrolled 57 patients. Among the evaluable patients, 3 of 51 (5.9%) achieved a partial response: all were KIT-. Stage II closed early because of slow accrual (November 2011 to December 2015). In stage II, 4 of 22 evaluable patients (18.2%) had a partial response; the median duration was 4.2 months. The median PFS was 2.1 months (n = 73; 95% confidence interval [CI], 1.5-2.9 months). The median OS was 7.5 months (95% CI, 6.0-11.9 months). In exploratory analyses, no differences were seen in PFS or OS with the KIT status or subtype. Dasatinib was discontinued because of adverse events in 9 of 75 patients (12%). The dasatinib response rate among KIT+ melanoma patients was low. In view of its clinical activity, it is recommended that imatinib remain the KIT tyrosine kinase inhibitor of choice for unresectable KIT+ melanoma. Cancer 2017;123:2688-97. © 2017 American Cancer Society. © 2017 American Cancer Society.
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Flores, Shahida; Sun, Jie; King, Jonathan; Budowle, Bruce
2014-05-01
The GlobalFiler™ Express PCR Amplification Kit uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit is specifically designed for processing reference DNA samples in a high throughput manner. Validation studies were conducted to assess the performance and define the limitations of this direct amplification kit for typing blood and buccal reference DNA samples on various punchable collection media. Studies included thermal cycling sensitivity, reproducibility, precision, sensitivity of detection, minimum detection threshold, system contamination, stochastic threshold and concordance. Results showed that optimal amplification and injection parameters for a 1.2mm punch from blood and buccal samples were 27 and 28 cycles, respectively, combined with a 12s injection on an ABI 3500xL Genetic Analyzer. Minimum detection thresholds were set at 100 and 120RFUs for 27 and 28 cycles, respectively, and it was suggested that data from positive amplification controls provided a better threshold representation. Stochastic thresholds were set at 250 and 400RFUs for 27 and 28 cycles, respectively, as stochastic effects increased with cycle number. The minimum amount of input DNA resulting in a full profile was 0.5ng, however, the optimum range determined was 2.5-10ng. Profile quality from the GlobalFiler™ Express Kit and the previously validated AmpFlSTR(®) Identifiler(®) Direct Kit was comparable. The validation data support that reliable DNA typing results from reference DNA samples can be obtained using the GlobalFiler™ Express PCR Amplification Kit. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
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Validation of ALK/ROS1 Dual Break Apart FISH Probe probe in non-small-cell lung cancer.
Lim, Sun Min; Chang, Hyun; Cha, Yoon Jin; Liang, Shile; Tai, Yan Chin; Li, Gu; Pestova, Ekaterina; Policht, Frank; Perez, Thomas; Soo, Ross A; Park, Won Young; Kim, Hye Ryun; Shim, Hyo Sup; Cho, Byoung Chul
2017-09-01
ALK and ROS1 gene rearrangements are distinct molecular subsets of non-small-cell lung cancer (NSCLC), and they are strong predictive biomarkers of response to ALK/ROS1 inhibitors, such as crizotinib. Thus, it is clinically important to develop an effective screening strategy to detect patients who will benefit from such treatment. In this study, we aimed to validate analytical performance of Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) in NSCLC. Study population composed of three patient cohorts with histologically confirmed lung adenocarcinoma (patients with ALK rearrangement, patients with ROS1 rearrangement and patients with wild-type ALK and ROS1). Specimens consisted of 12 ALK-positive, 8 ROS1-positive and 21 ALK/ROS1-wild type formalin-fixed paraffin-embedded samples obtained from surgical resection or excisional biopsy. ALK rearrangement was previously assessed by Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Abbot Park, IL, USA) and ROS1 rearrangement was previously assessed by ZytoLight ® SPEC ROS1 Break Apart Probe (ZytoVision, GmbH). All specimens were re-evaluated by Vysis ALK/ROS1 Dual Break Apart Probe Kit. FISH images were scanned on BioView AllegroPlus system and interpreted via BioView SoloWeb remotely. For a total of 41 patient samples, the concordance of the results by Vysis ALK/ROS1 Dual Break Apart Probe Kit was evaluated and compared to the known ALK and ROS1 rearrangement status of the specimen. Of the 12 ALK-positive cases, hybridization with Vysis ALK/ROS1 Dual Break Apart Probe Kit was successful in 10 cases (success rate 10/12, 83%) and of these 10 cases, all showed ALK rearrangement (100% concordance with the results of Vysis ALK Break Apart FISH Probe Kit). Two of the ALK+ cases were excluded due to weak ROS1 signals that could not be enumerated. Of the 8 ROS1-positive cases, 6 cases were successfully evaluated using Vysis ALK/ROS1 Dual Break Apart Probe Kit. The success rate was 75% (6/8), and of these 6 cases, all showed ROS1 rearrangement, giving a 100% concordance with ZytoLight ® SPEC ROS1 Break Apart Probe. Two of the cases were excluded due to weak ROS1 gold signal or high background. In the cohort of 21 wild-type cases, the success rate using Vysis ALK/ROS1 Dual Break Apart FISH Probe Kit was 85% (18/21) and the concordance with ALK and ROS1 probe kit was 100% (18/18). Vysis ALK/ROS1 Dual Break Apart Probe Kit (RUO) can detect ALK and ROS1 rearrangement simultaneously in NSCLC. Copyright © 2017 Elsevier B.V. All rights reserved.
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Fox, J C; Jordan, H E; Kocan, K M; George, T J; Mullins, S T; Barnett, C E; Glenn, B L; Cowell, R L
1986-03-01
Current methods and commercial test systems for the diagnosis of parasitic infections in both animals and humans are reviewed. Lists of test kits and their manufacturers are provided along with ordering information: the only commercially available test kits are for the diagnosis of toxoplasmosis in humans or animals and dirofilariasis (heartworm) in dogs. A partial list of diagnostic laboratories and the parasite tests they perform is also provided. Complete lists of diagnostic tests that could be obtained in the private sector are not available but would be useful. Two microfluorometric solid-phase assay systems are reviewed, and adaptations to custom assays for several kinds of parasites are briefly described. User problems in performing tests and interpreting results are stressed with emphasis placed on diagnosis of dirofilariasis in dogs. False-positive serology in dogs without heartworms and negative antibody responses in micro-filariae-positive animals are discussed with respect to proper interpretation of results.
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Killey, R; Mynors, C; Pearce, R; Nell, A; Prentis, A; Day, M J
2018-01-01
To determine the utility of an in-practice test kit to detect protective serum antibody against canine distemper virus, canine adenovirus and canine parvovirus type 2 in a sample of the UK dog population. Serum samples from 486 dogs, last vaccinated between less than 1 month and 124 months previously, were tested with the VacciCheck™ test kit for protective antibodies against distemper, adenovirus and parvovirus type 2. A high proportion of the dogs tested (93·6%) had protective antibody against all three of the core vaccine antigens: 95·7% of the dogs were seropositive against canine distemper virus, 97·3% against canine adenovirus and 98·5% against canine parvovirus type 2. The small number of dogs that were seronegative for one or more of the antigens (n = 31) may have had waning of previous serum antibody or may have been rare genetic non-responders to that specific antigen. UK veterinarians can be reassured that triennial revaccination of adult dogs with core vaccines provides long-lived protective immunity. In-practice serological test kits are a valuable tool for informing decision-making about canine core revaccination. © 2017 British Small Animal Veterinary Association.
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Performance and welfare of rabbit does in various caging systems.
Mikó, A; Matics, Zs; Gerencsér, Zs; Odermatt, M; Radnai, I; Nagy, I; Szendrő, K; Szendrő, Zs
2014-07-01
The objective of the study was to compare production and welfare of rabbit does and their kits housed in various types of cages. Female rabbits were randomly allocated to four groups with the following cage types: CN: common wire-mesh flat-deck cage, without footrest; CF: cage similar to the CN but with plastic footrest; ECWP: enlarged cage with wire-mesh platform; and ECPP: extra enlarged cage with plastic-mesh platform. All does were inseminated on the same day, 11 days after kindlings. Reproductive performance was evaluated during the first five consecutive kindlings. Severity of sore hocks was scored at each insemination. Location preference of the does and the platform usage of their kits were evaluated. Kindling rate, litter size (total born, born alive, alive at 21 and 35 days) and kit mortality were not significantly influenced by the cage types. The litter weight at 21 days was higher in ECWP and ECPP cages than in the CF group (3516, 3576 and 3291 g, respectively; P2.5 cm) and 3 to 4 (3=callus opened, cracks present; 4=wounds) were 58%, 60%, 78% and 48%, and 0%, 5%, 0% and 48% in groups ECPP, ECWP, CF and CN, respectively. Higher number of daily nest visits was observed for CF does than for ECWP does (12.5 v. 5.9; P2/day) was higher in the CF group than in the ECWP group (12.1 v. 3.2%; P<0.01). Within large cages, the does were observed on the platform more frequently in the ECPP cages compared with the ECWP cages (56.9% v. 31.7%; P<0.001). Similarly, 2.7% and 0.2% of kits at 21 days of age, and 33.2% and 5.2% of kits at 28 days of age, were found on the platforms of ECPP and ECWP cages, respectively. In conclusion, cages larger than the conventional ones improved kits' weaning weight, plastic footrests and plastic-mesh platforms in conventional and/or large cages reduced sore hocks' problems, plastic-mesh platforms were more used by both does and kits compared with the wire-mesh platforms.
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Zhao, Meng-Chuan; Li, Gui-Xia; Zhang, Dan; Zhou, Hang-Yu; Wang, Hao; Yang, Shuo; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun
2017-06-01
Respiratory Pathogen 13 Detection Kit (13× kit) is able to simultaneously detect 11 respiratory viruses, Mycoplasma pneumoniae (MP) and Chlamydia in a single reaction. Using 572 Nasopharyngeal aspirates collected from hospitalized children, the clinical performance of 13× kit for detecting 11 respiratory viruses was evaluated in comparison with a routinely used 2-tube multiplex reverse transcription PCR assay (2-tube assay) at provincial Centers for Disease Control and Prevention in China. The clinical performance of 13× kit for detecting MP and Chlamydia was evaluated by commercial real-time quantitative PCR (qPCR) kits or sequencing. For tested viruses, the assay concordance was 95.98% and the kappa coefficient was 0.89. All the MP and Chlamydia positive samples detected by 13× kit were confirmed as true positives. The utilization of the 13× kit in clinical settings will be helpful for doctors to assess clinical outcome according to virus type or multiple infections, and to limit the use of antibiotics. Copyright © 2017 Elsevier Inc. All rights reserved.
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Two brothers' alleged paternity for a child: who is the father?
Dogan, Muhammed; Kara, Umut; Emre, Ramazan; Fung, Wing Kam; Canturk, Kemal Murat
2015-06-01
In paternity cases where individuals are close relatives, it may be necessary to evaluate mother's DNA profile (trio test) and to increase the number of polymorphic STR loci that are analyzed. In our case, two alleged fathers who are brothers and the child (duo case) were analyzed based on 20 STR loci; however, no exclusions could be achieved. Then trio test (with mother) was performed using the Identifiler Plus kit (Applied Biosystems) and no exclusions could be achieved again. Analysis performed with the ESS Plex Plus kit (Qiagen), the paternity of one of the two alleged fathers was rejected only on 2 STR loci. We made the calculations of power of exclusion values to interpret our results more properly. The probability of exclusion (PE) is calculated as 0.9776546 in 15 loci of Identifiler Plus kit without mother. The PE is calculated as 0.9942803, if 5 additional loci from ESS Plex Plus kit are typed. The PE becomes 0.9961048 for the Identifiler Plus kit in trio analysis. If both Identifiler Plus and ESS Plex Plus kits are used for testing, the PE is calculated as 0.999431654, which indicates that the combined kits are highly discriminating.
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ERIC Educational Resources Information Center
Eslinger, Leslie Silk
Recognizing the long-lasting impact of young childrens learning through themes as well as the amount of teacher time spent in preparing for this type of teaching, this kit is designed to help teachers avoid the shortcomings of theme-based teaching, while capitalizing on the benefits of this approach. The book is presented in two sections. The…
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Park, Cheol Keun; Kim, Won Kyu; Kim, Hoguen
2017-10-01
KIT overexpression is frequently observed in adenoid cystic carcinomas (AdCCs), chromophobe renal cell carcinomas (ChRCCs), and gastrointestinal stromal tumours (GISTs). Persistent KIT activation has been reported to be mediated by protein kinase C (PKC)-δ in a subset of colon cancers with wild-type KIT overexpression, and by PKC-θ in GISTs with mutant KIT overexpression. To elucidate the clinical implications of PKC-δ and PKC-θ expression in KIT-expressing tumours, we investigated the expression of KIT, PKC-δ and PKC-θ in AdCCs and ChRCCs in comparison with GISTs. KIT expression, PKC-δ expression and PKC-θ expression were analysed in whole sections from 41 AdCCs, 40 ChRCCs and 56 GISTs by immunohistochemistry. Membranous expression of KIT was found in 34 AdCCs and all ChRCCs, whereas cytoplasmic expression of KIT was found in 46 GISTs. In AdCCs, PKC-δ expression was associated with histological grade (P = 0.049), lymphovascular invasion (P = 0.004), perineural invasion (P = 0.002), and KIT positivity (P = 0.002). PKC-δ positivity was associated with shorter relapse-free survival (RFS) (P = 0.017) and a tendency for there to be shorter overall survival (OS) (P = 0.090) in patients with AdCCs. No clinicopathological associations were observed between PKC-δ and KIT expression in ChRCCs. In GISTs, PKC-θ expression was associated with higher mitotic count (P = 0.011) and high grade according to the modified National Institutes of Health criteria (P < 0.001). PKC-θ positivity was associated with shorter RFS (P = 0.016) and a tendency for there to be shorter OS (P = 0.051) in patients with GISTs. PKC-δ expression is associated with KIT expression and the prognosis of patients with AdCCs, suggesting that PKC-δ may be a potential therapeutic target for AdCCs. © 2017 The Authors. Histopathology published by John Wiley & Sons Ltd.
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Foster, R; Byrnes, E; Meldrum, C; Griffith, R; Ross, G; Upjohn, E; Braue, A; Scott, R; Varigos, G; Ferrao, P; Ashman, L K
2008-11-01
The receptor tyrosine kinase c-KIT plays a key role in normal mast cell development. Point mutations in c-KIT have been associated with sporadic or familial mastocytosis. Two unrelated pairs of apparently identical twins affected by cutaneous mastocytosis attending the Mastocytosis Clinic at the Royal Children's Hospital, Melbourne, provided an opportunity to assess the possible contribution of c-KIT germline mutations or polymorphisms in this disease. Tissue biopsy, blood and/or buccal swab specimens were collected from 10 children with mastocytosis. To detect germline mutations/polymorphisms in c-KIT, we studied all coding exons by denaturing high pressure liquid chromatography. Exons showing mismatches were examined by direct sequencing. The influence of the substitution identified was further examined by expressing the variant form of c-KIT in factor-dependent FDC-P1 cells. In both pairs of twins, a heterozygous ATG to CTG transition in codon 541 was observed, resulting in the substitution of a methionine residue in the transmembrane domain by leucine (M541L). In each case, one parent was also heterozygous for this allele. Expression of M541L KIT in FDC-P1 cells enabled them to grow in human KIT ligand (stem cell factor, SCF) but did not confer factor independence. Compared with cells expressing wild-type KIT at a similar level, M541L KIT-expressing cells displayed enhanced growth at low levels of SCF, and heightened sensitivity to the KIT inhibitor, imatinib mesylate. The data suggest that the single nucleotide polymorphism resulting in the substitution M541L may predispose to paediatric mastocytosis.
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Tyrosine Kinase Inhibitors Induce Down-Regulation of c-Kit by Targeting the ATP Pocket
Descarpentries, Clotilde; Frisan, Emilie; Adam, Kevin; Verdier, Frederique; Floquet, Célia; Dubreuil, Patrice; Lacombe, Catherine; Fontenay, Michaela; Mayeux, Patrick; Kosmider, Olivier
2013-01-01
The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket. PMID:23637779
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Comparison of DNA extraction protocols for microbial communities from soil treated with biochar
Leite, D.C.A.; Balieiro, F.C.; Pires, C.A.; Madari, B.E.; Rosado, A.S.; Coutinho, H.L.C.; Peixoto, R.S.
2014-01-01
Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep™ (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples. PMID:24948928
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Comparison of DNA extraction protocols for microbial communities from soil treated with biochar.
Leite, D C A; Balieiro, F C; Pires, C A; Madari, B E; Rosado, A S; Coutinho, H L C; Peixoto, R S
2014-01-01
Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep™ (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples.
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Rhodes, Johanna; Beale, Mathew A; Fisher, Matthew C
2014-01-01
The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use.
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Arbefeville, S; Harris, A; Ferrieri, P
2017-09-01
Fungal infections cause considerable morbidity and mortality in immunocompromised patients. Rapid and accurate identification of fungi is essential to guide accurately targeted antifungal therapy. With the advent of molecular methods, clinical laboratories can use new technologies to supplement traditional phenotypic identification of fungi. The aims of the study were to evaluate the sole commercially available MicroSEQ® D2 LSU rDNA Fungal Identification Kit compared to the in-house developed internal transcribed spacer (ITS) regions assay in identifying moulds, using two well-known online public databases to analyze sequenced data. 85 common and uncommon clinically relevant fungi isolated from clinical specimens were sequenced for the D2 region of the large subunit (LSU) of ribosomal RNA (rRNA) gene with the MicroSEQ® Kit and the ITS regions with the in house developed assay. The generated sequenced data were analyzed with the online GenBank and MycoBank public databases. The D2 region of the LSU rRNA gene identified 89.4% or 92.9% of the 85 isolates to the genus level and the full ITS region (f-ITS) 96.5% or 100%, using GenBank or MycoBank, respectively, when compared to the consensus ID. When comparing species-level designations to the consensus ID, D2 region of the LSU rRNA gene aligned with 44.7% (38/85) or 52.9% (45/85) of these isolates in GenBank or MycoBank, respectively. By comparison, f-ITS possessed greater specificity, followed by ITS1, then ITS2 regions using GenBank or MycoBank. Using GenBank or MycoBank, D2 region of the LSU rRNA gene outperformed phenotypic based ID at the genus level. Comparing rates of ID between D2 region of the LSU rRNA gene and the ITS regions in GenBank or MycoBank at the species level against the consensus ID, f-ITS and ITS2 exceeded performance of the D2 region of the LSU rRNA gene, but ITS1 had similar performance to the D2 region of the LSU rRNA gene using MycoBank. Our results indicated that the MicroSEQ® D2 LSU rDNA Fungal Identification Kit was equivalent to the in-house developed ITS regions assay to identify fungi at the genus level. The MycoBank database gave a better curated database and thus allowed a better genus and species identification for both D2 region of the LSU rRNA gene and ITS regions. Copyright © 2017 Elsevier B.V. All rights reserved.
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Klomtong, P; Chaweewan, K; Phasuk, Y; Duangjinda, M
2015-10-19
Mutations in melanocortin 1 receptor (MC1R) gene and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) gene have been shown to affect coat color patterns in pigs. Additional functional marker genes, such as insulin like growth factor-2 (IGF2) and orphan nuclear receptor, germ cell nuclear factor (NR6A1), have been described for variations in factors such as fat deposition, litter size, and vertebra number in pigs. In this study, we investigated 129 pigs representing 4 breeds: Thai indigenous, classified into black (similar to Raad or Ka done pig) and black and white (similar to the Hailum and Kwai pig) coat color types; wild boar; Duroc; and Chinese Meishan. Mutations of MC1R, KIT, IGF2, and NR6A1 were detected using polymerase chain reaction-restriction fragment length polymorphism. The genotypes variation in MC1R and KIT genes could be used to differentiate four groups of coat color: solid black, black and white, red, and wild type. For IGF2, the GG genotype was present in wild boar only; for NR6A1 the TT genotype was found only in Duroc pigs. We identified novel 14-bp deletions in KIT that were associated with black and white coat color in Thai indigenous pigs. Insights into variations in genes presented in this study will be useful in future developmental breeding programs for the Thai native pig.
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Tamada, Hiromi; Kiyama, Hiroshi
2015-01-01
Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice. PMID:26727725
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Tamada, Hiromi; Kiyama, Hiroshi
2015-01-01
Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/W(v) mice carrying W and W(v) mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/W(v) mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/W(v) mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/W(v) mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/W(v) mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/W(v) mutant mice.
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Tamada, Hiromi; Kiyama, Hiroshi
2015-01-01
Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon.The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers,but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice.
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Arnau-Bonachera, A; Savietto, D; Pascual, J J
2017-12-11
The specialization process associated with genetic selection could be associated with functional disorders, affecting the reproductive success of females (fitness). We hypothesized that by modulating energy acquisition and allocation of females we could balance productivity and reproductive success. To test this hypothesis, we used 203 rabbit females belonging to three genetic types: H (n=66) maternal line specialized in prolificacy, LP (n=67) generalist maternal line, R (n=70) paternal line specialized in growth rate. We fed each genetic type with two diets specifically designed to promote milk yield (AF) or body reserves recovery (CS). We controlled females between their first and fifth reproductive cycles, recording traits related with productivity and fitness of females. H females fed CS had on average 11.2±0.43 kits with an individual weight of 54±1.2 g at birth and 525±11 g at weaning. Their conception rate when multiparous was 44% and their survival rate at the end of the experiment 30%. When they were fed AF, the individual weight of kits was 3.8 g heavier (P<0.05) at birth and 38 g heavier at weaning (P<0.05), the conception rate when multiparous increased 23 percentage points (P<0.05) and the survival rate at the end of the experiment 25 percentage points (P<0.05). LP females fed CS had on average 10.8±0.43 kits with an individual weight of 52±1.2 g at birth and 578±11 g at weaning. Their conception rate when multiparous was 79% and their survival rate at the end of the experiment 75%. When they were fed AF, it only increased individual weight of kits at weaning (+39 g; P<0.05). R females fed CS had on average 8.4±0.43 kits with an individual weight of 60±1.2 g at birth and 568±11 g at weaning. Their conception rate when multiparous was 60% and their survival rate at the end of the experiment 37%. When they were fed AF, they presented 1.4 kits less at birth (P<0.05) but heavier at birth (+4.9 g; P<0.05) and at weaning (+37 g; P<0.05). Therefore, we observed that genetic types prioritized different fitness components and that diets could affected them. In this sense, seems that more specialized genetic types, were more sensitive to diets than the more generalist type.
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Prediction of autosomal STR typing success in ancient and Second World War bone samples.
Zupanič Pajnič, Irena; Zupanc, Tomaž; Balažic, Jože; Geršak, Živa Miriam; Stojković, Oliver; Skadrić, Ivan; Črešnar, Matija
2017-03-01
Human-specific quantitative PCR (qPCR) has been developed for forensic use in the last 10 years and is the preferred DNA quantification technique since it is very accurate, sensitive, objective, time-effective and automatable. The amount of information that can be gleaned from a single quantification reaction using commercially available quantification kits has increased from the quantity of nuclear DNA to the amount of male DNA, presence of inhibitors and, most recently, to the degree of DNA degradation. In skeletal remains samples from disaster victims, missing persons and war conflict victims, the DNA is usually degraded. Therefore the new commercial qPCR kits able to assess the degree of degradation are potentially able to predict the success of downstream short tandem repeat (STR) typing. The goal of this study was to verify the quantification step using the PowerQuant kit with regard to its suitability as a screening method for autosomal STR typing success on ancient and Second World War (WWII) skeletal remains. We analysed 60 skeletons excavated from five archaeological sites and four WWII mass graves from Slovenia. The bones were cleaned, surface contamination was removed and the bones ground to a powder. Genomic DNA was obtained from 0.5g of bone powder after total demineralization. The DNA was purified using a Biorobot EZ1 device. Following PowerQuant quantification, DNA samples were subjected to autosomal STR amplification using the NGM kit. Up to 2.51ng DNA/g of powder were extracted. No inhibition was detected in any of bones analysed. 82% of the WWII bones gave full profiles while 73% of the ancient bones gave profiles not suitable for interpretation. Four bone extracts yielded no detectable amplification or zero quantification results and no profiles were obtained from any of them. Full or useful partial profiles were produced only from bone extracts where short autosomal (Auto) and long degradation (Deg) PowerQuant targets were detected. It is concluded that STR typing of old bones after quantification with the PowerQuant should be performed only when both Auto and Deg targets are detected simultaneously with no respect to [Auto]/[Deg] ratio. Prediction of STR typing success could be made according to successful amplification of Deg fragment. The PowerQuant kit is capable of identifying bone DNA samples that will not yield useful STR profiles using the NGM kit, and it can be used as a predictor of autosomal STR typing success of bone extracts obtained from ancient and WWII skeletal remains. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
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Picard-Meyer, Evelyne; Peytavin de Garam, Carine; Schereffer, Jean Luc; Marchal, Clotilde; Robardet, Emmanuelle; Cliquet, Florence
2015-01-01
This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R (2) values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.
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Picard-Meyer, Evelyne; Peytavin de Garam, Carine; Schereffer, Jean Luc; Marchal, Clotilde; Robardet, Emmanuelle; Cliquet, Florence
2015-01-01
This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R 2 values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes. PMID:25785274
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Preparation and Presentation of the Library Budget. SPEC Kit 32.
ERIC Educational Resources Information Center
Association of Research Libraries, Washington, DC. Office of Management Studies.
This kit on the preparation and presentation of the library budget in Association of Research Libraries (ARL) institutions contains a concise summary of the results of a 1977 member survey on budget preparation and eight related primary source documents. The summary of the Systems and Procedures Exchange Center (SPEC) survey focuses on types of…
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Comparison of Three Different Commercial Kits for the Human Papilloma Virus Genotyping.
Lim, Yong Kwan; Choi, Jee-Hye; Park, Serah; Kweon, Oh Joo; Park, Ae Ja
2016-11-01
High-risk type human papilloma virus (HPV) is the most important cause of cervical cancer. Recently, real-time polymerase chain reaction and reverse blot hybridization assay-based HPV DNA genotyping kits are developed. So, we compared the performances of different three HPV genotyping kits using different analytical principles and methods. Two hundred positive and 100 negative cervical swab specimens were used. DNA was extracted and all samples were tested by the MolecuTech REBA HPV-ID, Anyplex II HPV28 Detection, and HPVDNAChip. Direct sequencing was performed as a reference method for confirming high-risk HPV genotypes 16, 18, 45, 52, and 58. Although high-level agreement results were observed in negative samples, three kits showed decreased interassay agreement as screening setting in positive samples. Comparing the genotyping results, three assays showed acceptable sensitivity and specificity for the detection of HPV 16 and 18. Otherwise, various sensitivities showed in the detection of HPV 45, 52, and 58. The three assays had dissimilar performance of HPV screening capacity and exhibited moderate level of concordance in HPV genotyping. These discrepant results were unavoidable due to difference in type-specific analytical sensitivity and lack of standardization; therefore, we suggested that the efforts to standardization of HPV genotyping kits and adjusting analytical sensitivity would be important for the best clinical performance. © 2016 Wiley Periodicals, Inc.
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Kang, Keren; Wu, Peidian; Li, Wenmei; Tang, Shixing; Wang, Jihua; Luo, Xiaochun; Xie, Mingquan
2015-01-01
To develop a rapid, sensitive and specific assay for quantification of serum heart-type fatty acid binding protein (H-FABP) based on immunofluorescence of specific monoclonal antibodies. We generated novel H-FABP-directed monoclonal antibodies by cloning of spleen cells of mice immunized with H-FABP. Epitopes were mapped and antigen affinity was assessed by surface plasmon resonance (SPR). The H-FABP specific monoclonal antibodies were coupled to fluorescent beads and sprayed onto a nitrocellulose membrane facilitating quantification of H-FABP by immunofluorescence. Reagent cross-reactivity, interference resistance, accuracy and sensitivity were examined. A total of 103 clinical samples were used to compare the sensitivity and specificity of the new assay to a commercially available Randox kit. This new assay could be finished within 15 min, with sensitivity reaching 1 ng/ml. In a trial of 103 clinical serum samples, the new testing kit results were highly correlated with those from the Randox kit (R(2) = 0.9707). Using the Randox kit as the reference kit, the sensitivity of the new assay was 98.25%, and specificity was 100%. An immunofluorescence-based H-FABP assay employing novel monoclonal antibodies could rapidly, specifically and sensitively detect H-FABP in serum samples, providing an effective method for rapid clinical assessment of H-FABP index in the clinic.
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Maliken, Bryan D; Kanisicak, Onur; Karch, Jason; Khalil, Hadi; Fu, Xing; Boyer, Justin G; Prasad, Vikram; Zheng, Yi; Molkentin, Jeffery D
2018-04-17
Background -While c-Kit + adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells (CPCs). Methods - Kit allele-dependent lineage tracing and fusion analysis was performed in mice following simultaneous Gata4 and Gata6 cell-type specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit + cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2 CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. Results -Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2 CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion due to defective endothelial cell differentiation in the absence of Gata4 Conclusions -Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit + cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit + endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and capillary network integrity.
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Feng, Tang; Jian-Xia, Tang; Feng, Lu; Sui, Xu; Ya-Ping, Gu; De-Sheng, Tong; Guo-Ding, Zhu; Hai-Yong, Hua; Hua-Yun, Zhou; Jun, Cao
2016-03-21
To evaluate the Wondfo Rapid Diagnostic Kit (Pf-LDH/Pan -pLDH) for detecting Plasmodium ovale and analyze the influence of parasitaemia, concentration and polymorphism of pLDH on the performances. A total of 100 blood samples from P. ovale patients confirmed by PCR were detected with the Wondfo Rapid Diagnostic Kit according to the manufacturers'instructions. The parasitaemia was determined by the microscopic examination. The concentration of pLDH was measured by ELISA tests. The LDH gene of P. ovale was amplified by PCR and sequenced. The influence of these three factors on the positive rate was analyzed. The overall positive rate of Wondfo Rapid Diagnostic Kit was 70.0% (70/100). The positive rate was 27.3% for the samples with parasitaemia ≤ 500 parasites/μl and reached 75.0%-75.4% when parasitaemia > 500 parasites/μl. The positive rate was 6.7% for samples with a low pLDH concentration ( A values ≤ 0.100) and reached 95.1%-100% at a high pLDH concentration ( A values > 0.100). The results of sequence analysis indicated that all the samples could be divided into 2 types, P. o. curtisi and P. o. wallikeri . The gene homology of LDH between 2 types was 97%. There were 24 single nucleotide polymorphism (s) (SNPs) between 2 types, while only 3 SNPs were non-synonymous mutations. The homology of LDH amino acid sequences between 2 types was 99%; only 3 amino acids were different. The positive rates for P. o. curtisi and P. o. wallikeri were 73.1% (38/52) and 66.7% (32/48) respectively; there was no statistically significant difference ( P > 0.05). The Wondfo Rapid Diagnostic Kit (Pf-LDH/Pan-pLDH) performs better than most of the similar products for the detection of P. ovale , and the positive rates are closely related to the parasitaemia and concentration of pLDH, while no related to the polymorphism of pLDH gene.
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Kissova, Miroslava; Maga, Giovanni; Crespan, Emmanuele
2016-10-01
The tyrosine kinase Kit, a receptor for Stem Cell Factor, is involved, among others, in processes associated to cell survival, proliferation and migration. Upon physiological conditions, the activity of Kit is tightly regulated. However, primary mutations that lead to its constitutive activation are the causal oncogenic driver of gastrointestinal stromal tumours (GISTs). GISTs are known to be refractory to conventional therapies but the introduction of Imatinib, a selective inhibitor of tyrosine kinases Abl and Kit, significantly ameliorated the treatment options of GISTs patients. However, the acquisition of secondary mutations renders Kit resistant towards all available drugs. Mutation involving gatekeeper residues (such as V654a and T670I) influence both the structure and the catalytic activity of the enzyme. Therefore, detailed knowledge of the enzymatic properties of the mutant forms, in comparison with the wild type enzyme, is an important pre-requisite for the rational development of specific inhibitors. In this paper we report a thorough kinetic analysis of the reaction catalyzed by the Kit kinase and its gatekeeper mutated form T670I. Our results revealed the different mechanisms of action of these two enzymes and may open a new avenue for the future design of specific Kit inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Keating, Glenda; Bliwise, Donald L; Saini, Prabhjyot; Rye, David B; Trotti, Lynn Marie
2017-09-01
The hypothalamic peptide hypocretin 1 (orexin A) may be assayed in cerebrospinal fluid to diagnose narcolepsy type 1. This testing is not commercially available, and factors contributing to assay variability have not previously been comprehensively explored. In the present study, cerebrospinal fluid hypocretin concentrations were determined in duplicate in 155 patient samples, across a range of sleep disorders. Intra-assay variability of these measures was analyzed. Inter-assay correlation between samples tested at Emory and at Stanford was high (r = 0.79, p < 0.0001). Intra-assay correlation between samples tested in duplicate in our center was also high (r = 0.88, p < 0.0001); intra-assay variability, expressed as the difference between values as a percentage of the higher value, was low at 9.4% (SD = 7.9%). Although both time the sample spent in the freezer (r = 0.16, p = 0.04) and age of the kit used for assay (t = 3.64, p = 0.0004) were significant predictors of intra-kit variability in univariate analyses, only age of kit was significant in multivariate linear regression (F = 4.93, p = 0.03). Age of radioimmunoassay kit affects intra-kit variability of measured hypocretin values, such that kits closer to expiration exhibit significantly more variability.
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Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.
Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi
2017-12-21
High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.
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Maddens, Stéphane; Charruyer, Alexandra; Plo, Isabelle; Dubreuil, Patrice; Berger, Stuart; Salles, Bernard; Laurent, Guy; Jaffrézou, Jean-Pierre
2002-08-15
Previous studies demonstrated that Kit activation confers radioprotection. However, the mechanism by which Kit signaling interferes with cellular response to ionizing radiation (IR) has not been firmly established. Based on the role of the sphingomyelin (SM) cycle apoptotic pathway in IR-induced apoptosis, we hypothesized that one of the Kit signaling components might inhibit IR-induced ceramide production or ceramide-induced apoptosis. Results show that, in both Ba/F3 and 32D murine cell lines transfected with wild-type c-kit, stem cell factor (SCF) stimulation resulted in a significant reduction of IR-induced apoptosis and cytotoxicity, whereas DNA repair remained unaffected. Moreover, SCF stimulation inhibited IR-induced neutral sphingomyelinase (N-SMase) stimulation and ceramide production. The SCF inhibitory effect on SM cycle was not influenced by wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor. The SCF protective effect was maintained in 32D-KitYF719 cells in which the PI3K/Akt signaling pathway is abolished due to mutation in Kit docking site for PI3K. In contrast, phospholipase C gamma (PLC gamma) inhibition by U73122 totally restored IR-induced N-SMase stimulation, ceramide production, and apoptosis in Kit-activated cells. Moreover, SCF did not protect 32D-KitYF728 cells (lacking a functional docking site for PLC gamma 1), from IR-induced SM cycle. Finally, SCF-induced radioprotection of human CD34(+) bone marrow cells was also inhibited by U73122. Altogether, these results suggest that SCF radioprotection is due to PLC gamma 1-dependent negative regulation of IR-induced N-SMase stimulation. Beyond the scope of Kit-expressing cells, it suggests that PLC gamma 1 status could greatly influence the post-DNA damage cellular response to IR, and perhaps, to other genotoxic agents.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yang, Lei; Wu, Zhong; Yin, Gang
2014-12-12
Highlights: • SCF receptor c-Kit is functionally expressed in primary and transformed osteoblasts. • SCF protects primary and transformed osteoblasts from H{sub 2}O{sub 2}. • SCF activation of c-Kit in osteoblasts, required for its cyto-protective effects. • c-Kit mediates SCF-induced Akt activation in cultured osteoblasts. • Akt activation is required for SCF-regulated cyto-protective effects in osteoblasts. - Abstract: Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but littlemore » is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H{sub 2}O{sub 2}). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H{sub 2}O{sub 2}. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H{sub 2}O{sub 2} was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H{sub 2}O{sub 2} activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H{sub 2}O{sub 2}-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.« less
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KIT amplification and gene mutations in acral/mucosal melanoma in Korea.
Yun, Jina; Lee, Jeeyun; Jang, Jiryeon; Lee, Eui Jin; Jang, Kee Taek; Kim, Jung Han; Kim, Kyoung-Mee
2011-06-01
Mucosal and acral melanomas have demonstrated different genetic alterations and biological behavior compared with more common cutaneous melanomas. It was recently reported that gain-of-function KIT mutations and/or copy number increases are more common in mucosal and acral melanomas. Thus, we studied the frequency and pattern of KIT aberrations in mucosal and acral melanomas in Korea. We analyzed 97 patients who were pathologically confirmed with mucosal or acral melanoma between 1997 and 2010 at Samsung Medical Center. Of the 97 melanoma patients, 92 were screened for mutations in KIT exons 11, 13, 17, and 18, BRAF and NRAS genes. KIT copy number was assessed by quantitative, real-time PCR. Of the 97 patients, 55 (56.7%) were mucosal, 40 (41.2%) were acral melanoma, and two were of unknown primary origin. Among seven cases with KIT mutation, five (60.0%) occurred in exon 11, one (20.0%) in exon 17, and one (20.0%) in exon 13. Point mutations were the most common, resulting in substitutions in exon 11 (K558R, T574A, L576P, and V559A), exon 13 (N655K), and exon 17 (N822K). A novel Thr574Ala (c.1720A>G) KIT mutation, which has not been reported in melanoma or other tumor types, was identified in one genital melanoma case. Of the 97 mucosal or acral melanoma specimens, 49 were tested for KIT gene copy number changes using quantitative PCR. Increased KIT copy number was identified in 15 patients: seven (40%) of 20 acral melanomas and eight (31%) of 26 mucosal melanomas. Our study implicates that a significant proportion of acral and mucosal melanomas have KIT mutations in Asian population. © 2011 The Authors. APMIS © 2011 APMIS.
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Steiner, René; Beier, Ulrike S; Heiss-Kisielewsky, Irene; Engelmeier, Robert; Dumfahrt, Herbert; Dhima, Matilda
2015-06-01
During the insertion appointment, the practitioner is often faced with the need to adjust ceramic surfaces to fit a restoration to the adjacent or opposing dentition and soft tissues. The purpose of this study was to assess the ceramic surface smoothness achieved with various commercially available ceramic polishing kits on different commonly used ceramic systems. The reliability of the cost of a polishing kit as an indicator of improved surface smoothness was assessed. A total of 350 ceramic surfaces representing 5 commonly available ceramic systems (IPS Empress Esthetic, IPS e.max Press, Cergo Kiss, Vita PM 9, Imagine PressX) were treated with 5 types of ceramic polishing systems (Cerapreshine, 94006C, Ceramiste, Optrafine, Zenostar) by following the manufacturers' guidelines. The surface roughness was measured with a profilometer (Taylor Hobson; Precision Taylor Hobson Ltd). The effects of ceramic systems and polishing kits of interest on surface roughness were analyzed by 2-way ANOVA, paired t test, and Bonferroni corrected significance level. The ceramic systems and polishing kits statistically affected surface roughness (P<.001).The polishing kit Zenostar on IPS e.max Press created the smoothest ceramic surface. No correlation could be established between the high cost of the polishing kit and low surface roughness. None of the commonly used ceramic polishing kits could create a surface smoother than that of glazed ceramic (P<.001). The inclusion of a diamond polishing paste step is recommended to improve surface smoothness (P<.001). The cost of ceramic polishing kits is not recommended as a reliable indicator of better performance of ceramic polishing kits (P>.30). Copyright © 2015 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.
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Olekšáková, Tereza; Žurovcová, Martina; Klimešová, Vanda; Barták, Miroslav; Šuláková, Hana
2018-04-01
Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.
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Kaminski, Alexander; Ma, Nan; Donndorf, Peter; Lindenblatt, Nicole; Feldmeier, Gregor; Ong, Lee-Lee; Furlani, Dario; Skrabal, Christian A; Liebold, Andreas; Vollmar, Brigitte; Steinhoff, Gustav
2008-01-01
In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1alpha)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1alpha enhanced c-kit+-cell migration. In vivo, SDF-1alpha alone triggered endothelial rolling-not firm adherence-of c-kit+ cells in WT mice. While TNF-alpha alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1alpha+TNF-alpha, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1alpha mediates firm adhesion c-kit+ cells only in the presence of TNF-alpha stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.
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The pivotal role of fibrocytes and mast cells in mediating fibrotic reactions to biomaterials
Thevenot, Paul T.; Baker, David W.; Weng, Hong; Sun, Man-Wu; Tang, Liping
2011-01-01
Almost all biomaterial implants are surrounded by a fibrotic capsule, although the mechanism of biomaterial-mediated fibrotic reactions is mostly unclear. To search for the types of cells responsible for triggering the tissue responses, we used poly-L glycolic acid polymers capable of releasing various reagents. We first identified that CD45+ /Collagen 1+ fibrocytes are recruited and resided within the fibrotic capsule at the implant interface. Interestingly, we found that the recruitment of fibrocytes and the extent of fibrotic tissue formation (collagen type I production) were substantially enhanced and reduced by the localized release of compound 48/80 and cromolyn, respectively. Since it is well established that compound 48/80 and cromolyn alter mast cell reactions, we hypothesized that mast cells are responsible for triggering fibrocyte recruitment and subsequent fibrotic capsule formation surrounding biomaterial implants. To directly test this hypothesis, similar studies were carried out using mast cell deficient mice, WBB6F1/J-KitW/KitW-v/, and their congenic controls. Indeed, mast cell deficient mice prompted substantially less fibrocyte and myofibroblast responses in comparison to C-57 wild type mice controls. Most interestingly, subcutaneous mast cell reconstitution of WBB6F1/J-KitW/KitW-v/J mice almost completely restored the fibrocyte response in comparison to the C-57 wild type response. These results indicate that the initial biomaterial interaction resulting in the stimulation of mast cells and degranulation byproducts not only stimulates the inflammatory cascade but significantly alters the downstream fibrocyte response and degree of fibrosis. PMID:21864899
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Korshun, Ludmila; Vudmaska, Mariya; Moysa, Larissa; Kovtonjuk, Galina; Mikhalap, Svetlana; Ganova, Larissa; Spivak, Nikolay
2013-12-01
In order to the detection of type-specific IgG to herpes simplex virus type 2 (HSV-2) in human serum or plasma the recombinant analog of HSV-2 glycoprotein G (gG2) was created. To construct an expression vector the DNA fragment with a sequence identical to immunodominant regions of HSV-2 gG2 was cloned into modified vector pET28a containing of the glutation-S-transferase sequence (pET28-GST). Escherichia coli BL21 (DE3) were transformed with the recombinant plasmid. The target protein was expressed mainly in soluble form. Chromatographic purification of soluble GST-gG2 protein was performed taking into account the features of its primary structure that are 6His-tag and GST-tag. To determine the affinity constant of the specific IgG to GST-gG2 we used the method proposed by Friguet et al. (1985). The affinity constants were within the range of 10(7)-10(8)M(-1) proving their high-affinity. The purified recombinant HSV-2 antigen was used to design a diagnostic ELISA kit, which was evaluated with referent controls and standard panels of sera containing and/or not containing anti-HSV-2 IgG. Comparative evaluation of this kit and the commercially available "HSV-Type 2 IgG-ELISA" (NovaTec, Dietzenbach, Germany) kit was performed. There was no significant difference (P>0.05). It allows to use developed ELISA kit for clinical diagnosis of HSV-2 infection. Copyright © 2013 Elsevier B.V. All rights reserved.
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Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane
2012-09-01
Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.
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Dempsey, Steven J; Gese, Eric M; Kluever, Bryan M; Lonsinger, Robert C; Waits, Lisette P
2015-01-01
Development and evaluation of noninvasive methods for monitoring species distribution and abundance is a growing area of ecological research. While noninvasive methods have the advantage of reduced risk of negative factors associated with capture, comparisons to methods using more traditional invasive sampling is lacking. Historically kit foxes (Vulpes macrotis) occupied the desert and semi-arid regions of southwestern North America. Once the most abundant carnivore in the Great Basin Desert of Utah, the species is now considered rare. In recent decades, attempts have been made to model the environmental variables influencing kit fox distribution. Using noninvasive scat deposition surveys for determination of kit fox presence, we modeled resource selection functions to predict kit fox distribution using three popular techniques (Maxent, fixed-effects, and mixed-effects generalized linear models) and compared these with similar models developed from invasive sampling (telemetry locations from radio-collared foxes). Resource selection functions were developed using a combination of landscape variables including elevation, slope, aspect, vegetation height, and soil type. All models were tested against subsequent scat collections as a method of model validation. We demonstrate the importance of comparing multiple model types for development of resource selection functions used to predict a species distribution, and evaluating the importance of environmental variables on species distribution. All models we examined showed a large effect of elevation on kit fox presence, followed by slope and vegetation height. However, the invasive sampling method (i.e., radio-telemetry) appeared to be better at determining resource selection, and therefore may be more robust in predicting kit fox distribution. In contrast, the distribution maps created from the noninvasive sampling (i.e., scat transects) were significantly different than the invasive method, thus scat transects may be appropriate when used in an occupancy framework to predict species distribution. We concluded that while scat deposition transects may be useful for monitoring kit fox abundance and possibly occupancy, they do not appear to be appropriate for determining resource selection. On our study area, scat transects were biased to roadways, while data collected using radio-telemetry was dictated by movements of the kit foxes themselves. We recommend that future studies applying noninvasive scat sampling should consider a more robust random sampling design across the landscape (e.g., random transects or more complete road coverage) that would then provide a more accurate and unbiased depiction of resource selection useful to predict kit fox distribution.
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2010-01-01
Background Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST. Results Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation. Conclusions As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It remains to be clarified whether an autocrine or paracrine mechanism by overexpression of receptor tyrosine kinase ligands is responsible for the tumorigenesis of wt-GIST. PMID:21171987
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Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment.
Arai, Yasuyuki; Choi, Uimook; Corsino, Cristina I; Koontz, Sherry M; Tajima, Masaki; Sweeney, Colin L; Black, Mary A; Feldman, Steven A; Dinauer, Mary C; Malech, Harry L
2018-05-02
We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (<1%-13.1%). This resulted in significant depletion of the BM c-kit + population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue. Published by Elsevier Inc.
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Manzone, Marco; Balsari, Paolo; Marucco, Paolo; Tamagnone, Mario
2017-03-01
All maize drills produce a fine dust due to the seed coating abrasions that occur inside the seeding element. The air stream generated by the fan of pneumatic drills - necessary to create a depression in the sowing element of the machine and to guarantee correct seed deposition - can blow away the solid particles detached from the seeds. In order to reduce this phenomenon, a coated maize seeds company (Syngenta®) has set up an ad hoc dual-pipe deflector kit that easily fits different pneumatic drills (also old drills). In this study, the efficiency of this kit and the influence of different drill types on the kit's performance in reducing environmental pollution were evaluated using three different pneumatic seed drill models. The research showed that a dual-pipe deflector installed on a drill in standard configuration did not change the seeder performance, and by using this kit on pneumatic drills, irrespective of their design, it is possible to reduce by up to 69% the amount of dust drift in comparison with the conventional machine set-up. The dual-pipe deflector, under the conditions employed in the present experiments, showed good performance with all types of maize pneumatic drill used. Irrespective of the seeder model on which it is mounted, it is able to obtain similar results, indicating its high operational versatility. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.
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miR-148b-3p functions as a tumor suppressor in GISTs by directly targeting KIT.
Wang, Yu; Li, Jun; Kuang, Dong; Wang, Xiaoyan; Zhu, Yuanli; Xu, Sanpeng; Chen, Yaobing; Cheng, Henghui; Zhao, Qiu; Duan, Yaqi; Wang, Guoping
2018-04-16
Gain-of-function mutations and overexpression of KIT are characteristic features of gastrointestinal stromal tumor (GIST). Dysregulation in miRNA expression may lead to KIT overexpression and tumorigenesis. miRNA microarray analysis and real-time PCR were used to determine the miRNA expression profiles in a cohort of 69 clinical samples including 50 CD117 IHC+ /KIT mutation GISTs and 19 CD117 IHC- /wild-type GISTs. GO enrichment and KEGG pathway analyses were performed to reveal the predicted targets of the dysregulated miRNAs. Of the dysregulated miRNAs whose expression was inversely correlated with that of KIT miRNAs were predicted by bioinformatics analysis and confirmed by luciferase reporter assay. Cell counting kit-8 (CCK-8) and flow cytometry were used to measure the cell proliferation, cycle arrest and apoptosis. Wound healing and transwell assays were used to evaluate migration and invasion. A xenograft BALB/c nude mouse model was applied to investigate the tumorigenesis in vivo. Western blot and qRT-PCR were used to investigate the protein and mRNA levels of KIT and its downstream effectors including ERK, AKT and STAT3. Of the six miRNAs whose expression was inversely correlated with that of KIT, we found that miR-148b-3p was significantly downregulated in the CD117 IHC+ /KIT mutation GIST cohort. This miRNA was subsequently found to inhibit proliferation, migration and invasion of GIST882 cells. Mechanistically, miR-148b-3p was shown to regulate KIT expression through directly binding to the 3'-UTR of the KIT mRNA. Restoration of miR-148b-3p expression in GIST882 cells led to reduced expression of KIT and the downstream effectors proteins ERK, AKT and STAT3. However, overexpression of KIT reversed the inhibitory effect of miR-148b-3p on cell proliferation, migration and invasion. Furthermore, we found that reduced miR-148b-3p expression correlated with poor overall survival (OS) and disease-free survival (DFS) in GIST patients. miR-148b-3p functions as an important regulator of KIT expression and a potential prognostic biomarker for GISTs.
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Hysteroscopic sterilization: 10-year retrospective analysis of worldwide pregnancy reports.
Munro, M G; Nichols, J E; Levy, B; Vleugels, M P H; Veersema, S
2014-01-01
To identify factors that might contribute to pregnancies reported after hysteroscopic sterilization worldwide. Retrospective review of commercial data compiled from the MAUDE database, medical literature, and manufacturer reports received during commercial distribution of hysteroscopic sterilization micro-inserts from 2001 through 2010 (Canadian Taskforce classification III descriptive study). From 2001 through 2010, 497 305 hysteroscopic sterilization kits were distributed worldwide, and 748 pregnancies were reported, i.e., 0.15% of the estimated user population based on the number of distributed kits. The data were sufficient to enable analysis of 508 pregnancies for potential contributing factors and showed most to be associated with patient or physician noncompliance (n = 264) or misinterpreted confirmation tests (n = 212). Conceptions deemed to have occurred within 2 weeks of the procedure and therefore too early for detection were identified in 32 cases. Although there are limitations to the dataset and the study design is retrospective, it represents the largest body of cumulative hysteroscopic sterilization data available to date. Of the 748 pregnancies reported, it is apparent that some might have been prevented with greater patient and clinician attention to interim contraceptive use and counseling and with more rigorous evaluation and informed interpretation of the procedure confirmation tests. Although the estimated pregnancy rate based on such a dataset is likely an underestimation, it does suggest that the evaluable field performance of hysteroscopic sterilization micro-inserts is consistent with the labeled age-adjusted effectiveness of 99.74% at 5 years. Copyright © 2014 AAGL. Published by Elsevier Inc. All rights reserved.
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SensorKit: An End-to-End Solution for Environmental Sensor Networking
NASA Astrophysics Data System (ADS)
Silva, F.; Graham, E.; Deschon, A.; Lam, Y.; Goldman, J.; Wroclawski, J.; Kaiser, W.; Benzel, T.
2008-12-01
Modern day sensor network technology has shown great promise to transform environmental data collection. However, despite the promise, these systems have remained the purview of the engineers and computer scientists who design them rather than a useful tool for the environmental scientists who need them. SensorKit is conceived of as a way to make wireless sensor networks accessible to The People: it is an advanced, powerful tool for sensor data collection that does not require advanced technological know-how. We are aiming to make wireless sensor networks for environmental science as simple as setting up a standard home computer network by providing simple, tested configurations of commercially-available hardware, free and easy-to-use software, and step-by-step tutorials. We designed and built SensorKit using a simplicity-through-sophistication approach, supplying users a powerful sensor to database end-to-end system with a simple and intuitive user interface. Our objective in building SensorKit was to make the prospect of using environmental sensor networks as simple as possible. We built SensorKit from off the shelf hardware components, using the Compact RIO platform from National Instruments for data acquisition due to its modular architecture and flexibility to support a large number of sensor types. In SensorKit, we support various types of analog, digital and networked sensors. Our modular software architecture allows us to abstract sensor details and provide users a common way to acquire data and to command different types of sensors. SensorKit is built on top of the Sensor Processing and Acquisition Network (SPAN), a modular framework for acquiring data in the field, moving it reliably to the scientist institution, and storing it in an easily-accessible database. SPAN allows real-time access to the data in the field by providing various options for long haul communication, such as cellular and satellite links. Our system also features reliable data storage and transmission, using a custody transfer mechanism that ensures data is retained until successful delivery to the scientist can be confirmed. The ability for the scientist to communicate in real-time with the sensor network in the field enables remote sensor reconfiguration and system health and status monitoring. We use a spiral approach of design, test, deploy and revise, and, by going to the field frequently and getting feedback from field scientists, we have been able to include additional functionality that is useful to the scientist while ensuring SensorKit remains intuitive to operate. Users can configure, control, and monitor SensorKit using a number of tools we have developed. An intuitive user interface running on a desktop or laptop allows scientists to setup the system, add and configure sensors, and specify when and how the data will be collected. We also have a mobile version of our interface that runs on a PDA and lets scientists calibrate sensors and "tune" the system while in the field, allowing for data validation before leaving the field and returning to the research lab. SensorKit also features SensorBase, an intuitive user interface built on top of a standard SQL database, which allows scientists to store and share their data with other researchers. SensorKit has been used for diverse scientific applications and deployed throughout the world: from studying mercury cycling in rice paddies in China, to ecological research in the neotropical rainforests of Costa Rica, to monitoring the contamination of salt lakes in Argentina.
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SLAM family member 8 is involved in oncogenic KIT-mediated signaling in human mastocytosis.
Sugimoto, Akihiko; Kataoka, Tatsuki R; Ueshima, Chiyuki; Takei, Yusuke; Kitamura, Kyohei; Hirata, Masahiro; Nomura, Takashi; Haga, Hironori
2018-03-02
The signaling lymphocytic activation molecule family member 8 (SLAMF8)/CD353 is a member of the CD2 family of proteins. Its ligand has not been identified. SLAMF8 is expressed by macrophages and suppresses cellular functions. No study has yet explored SLAMF8 expression or function in human mastocytosis, which features oncogenic KIT-mediated proliferation of human mast cells. SLAMF8 protein was expressed in human mastocytosis cells, immunohistochemically. SLAMF8 expression was also evident in the human mast cell lines, HMC1.2 (expressing oncogenic KIT) and LAD2 (expressing wild-type KIT) cells. SLAMF8-knockdown significantly reduced the KIT-mediated growth of HMC1.2 cells but not that of LAD2 cells. SLAMF8-knockdown HMC1.2 cells exhibited significant attenuation of SHP-2 activation and oncogenic KIT-mediated RAS-RAF-ERK signaling. An interaction between SLAMF8 and SHP-2 was confirmed in HMC1.2 cells and all pathological mastocytosis specimens examined (19 of 19 cases, 100%). Thus, SLAMF8 is involved in oncogenic KIT-mediated RAS-RAF-ERK signaling and the subsequent growth of human neoplastic mast cells mediated by SHP-2. SLAMF8 is a possible therapeutic target in human mastocytosis patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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A Frameshift Mutation in KIT is Associated with White Spotting in the Arabian Camel
Holl, Heather; Isaza, Ramiro; Mohamoud, Yasmin; Ahmed, Ayeda; Almathen, Faisal; Youcef, Cherifi; Gaouar, Semir; Antczak, Douglas F.; Brooks, Samantha
2017-01-01
While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five KITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation. PMID:28282952
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Integrated Molecular Characterization of Testicular Germ Cell Tumors.
Shen, Hui; Shih, Juliann; Hollern, Daniel P; Wang, Linghua; Bowlby, Reanne; Tickoo, Satish K; Thorsson, Vésteinn; Mungall, Andrew J; Newton, Yulia; Hegde, Apurva M; Armenia, Joshua; Sánchez-Vega, Francisco; Pluta, John; Pyle, Louise C; Mehra, Rohit; Reuter, Victor E; Godoy, Guilherme; Jones, Jeffrey; Shelley, Carl S; Feldman, Darren R; Vidal, Daniel O; Lessel, Davor; Kulis, Tomislav; Cárcano, Flavio M; Leraas, Kristen M; Lichtenberg, Tara M; Brooks, Denise; Cherniack, Andrew D; Cho, Juok; Heiman, David I; Kasaian, Katayoon; Liu, Minwei; Noble, Michael S; Xi, Liu; Zhang, Hailei; Zhou, Wanding; ZenKlusen, Jean C; Hutter, Carolyn M; Felau, Ina; Zhang, Jiashan; Schultz, Nikolaus; Getz, Gad; Meyerson, Matthew; Stuart, Joshua M; Akbani, Rehan; Wheeler, David A; Laird, Peter W; Nathanson, Katherine L; Cortessis, Victoria K; Hoadley, Katherine A
2018-06-12
We studied 137 primary testicular germ cell tumors (TGCTs) using high-dimensional assays of genomic, epigenomic, transcriptomic, and proteomic features. These tumors exhibited high aneuploidy and a paucity of somatic mutations. Somatic mutation of only three genes achieved significance-KIT, KRAS, and NRAS-exclusively in samples with seminoma components. Integrated analyses identified distinct molecular patterns that characterized the major recognized histologic subtypes of TGCT: seminoma, embryonal carcinoma, yolk sac tumor, and teratoma. Striking differences in global DNA methylation and microRNA expression between histology subtypes highlight a likely role of epigenomic processes in determining histologic fates in TGCTs. We also identified a subset of pure seminomas defined by KIT mutations, increased immune infiltration, globally demethylated DNA, and decreased KRAS copy number. We report potential biomarkers for risk stratification, such as miRNA specifically expressed in teratoma, and others with molecular diagnostic potential, such as CpH (CpA/CpC/CpT) methylation identifying embryonal carcinomas. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
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Colomar, Mercedes; Cafferata, Maria Luisa; Aleman, Alicia; Tomasso, Giselle; Betran, Ana Pilar
2017-03-31
Antenatal care reduces maternal and perinatal mortality and morbidity through the detection and treatment of some conditions, but its coverage is less than optimal within certain populations. Supply kits for maternal health were designed to overcome barriers present when providing care during pregnancy and childbirth particularly to women from underserved population.We conducted a mixed-methods systematic review on the use of supply kits. This manuscript presents the findings from qualitative studies that reported barriers, facilitators, and user's recommendation in the adoption and implementation of any type of kit designed to be used during pregnancy or childbirth.This review included eight studies, and seven were implemented in developing countries. Most studies assessed the implementation of clean delivery kits to be used during labour and delivery, and contributed to gain insights into factors that may hinder or foster the use of kits.Clean delivery kits were conceived to cope with barriers related mainly to access. The most important barrier identified were those related to the socio-cultural and the lack of knowledge dimension such as who held the decision-making authority in the household, as well as popular beliefs behind the idea that birth preparation could bring bad luck, may prevent clients from adhering to their use. In addition, financial constraints and limited understanding of the instructions of use were accessibility barriers found. On the other hand, once used, clean delivery kits for maternal health were accepted by women and health workers. Convenience, hygienic components, and avoidance of delays in receiving care were viewed as satisfactory features.Supply kits are mostly affordable and easily deployable. Increasing awareness among the population about the offered kits and providing information on their benefits emerges as a critical step to foster use in settings where kits are available. Implementation of this strategy requires low complexity resources and could make the use of kits an accepted alternative to increase the use of evidence-based interventions and thus improve quality of care during pregnancy, childbirth and neonatal period mainly at the community level in low income countries and remote areas with low access.
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... Infants Online Diabetes Support Team TypeOneNation Community Forum Health Insurance Guide Bag of Hope® T1D Connections Program T1D Care Kit Toolkits For Healthcare Providers (PEAK) Get Involved ...
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Direct fluorescence polarization assay for the detection of glycopeptide antibiotics.
Yu, Linliang; Zhong, Meng; Wei, Yinan
2010-08-15
Glycopeptide antibiotics are widely used in the treatment of infections caused by Gram-positive bacteria. They inhibit the biosynthesis of the bacterial cell wall through binding to the D-alanyl-D-alanine (D-Ala-D-Ala) terminal peptide of the peptidoglycan precursor. Taking advantage of this highly specific interaction, we developed a direct fluorescence polarization based method for the detection of glycopeptide antibiotics. Briefly, we labeled the acetylated tripeptide Ac-L-Lys-D-Ala-D-Ala-OH with a fluorophore to create a peptide probe. Using three glycopeptide antibiotics, vancomycin, teicoplanin, and telavancin, as model compounds, we demonstrated that the fluorescence polarization of the peptide probe increased upon binding to antibiotics in a concentration dependent manner. The dissociation constants (K(d)) between the peptide probes and the antibiotics were consistent with those reported between free d-Ala-d-Ala and the antibiotics in the literature. The assay is highly reproducible and selective toward glycopeptide antibiotics. Its detection limit and work concentration range are 0.5 microM and 0.5-4 microM for vancomycin, 0.25 microM and 0.25-2 microM for teicoplanin, and 1 microM and 1-8 microM for telavancin. Furthermore, we compared our assay in parallel with a commercial fluorescence polarization immunoassay (FPIA) kit in detecting teicoplanin spiked in human blood samples. The accuracy and precision of the two methods are comparable. We expect our assay to be useful in both research and clinical laboratories.
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A distinct type of cell in myocardium: interstitial Cajal-like cells (ICLCs)
Kostin, S; Popescu, L M
2009-01-01
Abstract The existence of a novel type of interstitial cells in the heart, interstitial Cajal-like cells (ICLCs), had been described for the first time in 2005. Their identification was mainly based on ultrastructural criteria: very long (tens up to hundreds of micrometres) and moniliform prolongations, which are extremely thin (less than 0.2 μm), below the resolving power of light microscopy. Myocardial ICLCs were also identified by methylene-blue vital staining, silver impregnation, and immunoreactivity for CD 34, vimentin, CD117/c-kit, etc. Although a series of studies provided evidence for the existence of ICLCs in human atria and rat ventricles, further investigations in other laboratories, using additional techniques, are required to substantiate the consistency of these findings. Here we provide further evidence for the existence of ICLCs in human and mammalian hearts (by transmission and scanning electron microscopy, as well as confocal laser scanning microscopy). Noteworthy, we confirm that ICLCs communicate with neighbouring cells via shedding (micro)vesicles. Although these so-called ICLCs represent a distinct type of cells, different from classical interstitial cells of Cajal, or fibroblasts, their role(s) in myocardium remain(s) to be established. Several hypotheses are proposed: (i) adult stromal (mesenchymal) stem cells, which might participate in cardiac repair/remodelling; (ii) intercellular signalling (e.g. via shedding microvesicles); (iii) chemo-mechanical transducers and (iv) players in pacemaking and/or arrhytmogenesis, and so on. PMID:19183408
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A silent allele in the locus D5S818 contained within the PowerPlex®21 PCR Amplification Kit.
Chen, Ling; Tai, Yunchun; Qiu, Pingming; Du, Weian; Liu, Chao
2015-11-01
Three paternity tests cases were found with a single locus mismatch at the locus D5S818 with PowerPlex®21 PCR Amplification Kit (Promega). Forward and reverse primers were redesigned to type the samples again and to evaluate if there were alleles dropped out. The results showed the existence of a silent allele 12 in all the three families, due to a point mutation that changed cytosine to adenine at 90 nucleotides upstream from the 5' end of the AGAT repeat sequences in all the six individuals. A single locus mismatch due to a silent allele may occur in any locus using any kit. Therefore, we recommend using multiple kits to confirm the results in paternity testing cases with mismatches, especially when there is a single locus mismatch with homozygote involved. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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Das, Tapas; Banerjee, Sharmila; Shinto, Ajit; Kamaleshwaran, K K; Sarma, H D
2014-01-01
Patient dose of (177)Lu-DOTA-TATE, used for providing radiotherapeutic treatment to the patients suffering from cancers of neuroendocrine origin, could be prepared at the hospital radiopharmacy either 'in-situ' or by using freezedried kits. The objective of the present work is to formulate and evaluate a single vial freeze-dried DOTA-TATE kit, which is capable of producing up to 7.4 GBq (200 mCi) dose of (177)Lu-DOTA-TATE and to compare the two methodologies presently used for the preparation of the agent. Freeze-dried DOTA-TATE kits, comprising a lyophilized mixture of DOTA-TATE, gentisic acid and ammonium acetate, were prepared and used for the formulation of patient doses of (177)Lu-DOTA-TATE. The kits were subjected to detailed radiochemical evaluation and the shelf-life of the kits was determined. The pharmacokinetic behavior of the agent was studied in normal Wistar rats. These kits were utilized for treating the patients suffering from various types of neuroendocrine cancers. The freeze-dried kits were used for the preparation of up to 7.4 GBq (200 mCi) therapeutic doses of (177)Lu- DOTA-TATE with a radiochemical purity of >99% and were found to have sufficiently long shelf-life. Biological studies carried out in normal Wistar rats exhibited no significant accumulation of activity in any of the vital organs/tissue except in kidneys and non-accumulated activity showed major renal clearance. Clinical studies carried out in cancer patients exhibited accumulation of activity in the cancerous lesions and metastatic sites. The kit was useful for the convenient preparation of therapeutic dose of (177)Lu-DOTA-TATE, suitable for human administration. The use of kit is expected to reduce the batch failure and radiation exposure to the working personnel.
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A novel piezoelectric quartz micro-array immunosensor for detection of immunoglobulinE.
Yao, Chunyan; Chen, Qinghai; Chen, Ming; Zhang, Bo; Luo, Yang; Huang, Qing; Huang, Junfu; Fu, Weiling
2006-12-01
A novel multi-channel 2 x 5 model of piezoelectric (PZ) micro-array immunosensor has been developed for quantitative detection of human immunoglobulinE (IgE) in serum. Every crystal unit of the fabricated piezoelectric IgE micro-array immunosensor can oscillate without interfering each other. A multi-channel 2 x 5 model micro-array immunosensor as compared with the traditional one-channel immunosensor can provide eight times higher detection speeds for IgE assay. The anti-IgE antibody is deposited on the gold electrode's surface of 10 MHz AT-cut quartz crystals by SPA (staphylococcal protein A), and serves as an antibody recognizing layer. The highly ordered antibody monolayers ensure well-controlled surface structure and offer many advantages to the performance of the sensor. The uniform amount of antibody monolayer coated by the SPA is good, and non-specific reaction caused by other immunoglobulin in sample is found. The fabricated PZ immunosensor can be used for human IgE determination in the range of 5-300 IU/ml with high precision (CV is 4%). 50 human serum samples were detected by the micro-array immunosensor, and the results agreed well with those given by the commercially ELISA test kits. The correlation coefficient is 0.94 between ELISA and PZ immunosensor. After regeneration with NaOH the coated immunosensor can be reused 6 times without appreciable loss of activity.
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First aid kits for recreational dive boats, what should they contain?
Pye, Jacqueline; Greenhalgh, Trisha
2010-09-01
Well-equipped first-aid kits are necessary but not always provided on recreational dive boats. We aimed to review the types of illness and injury likely to be encountered on such boats and inform a content list for such kits. We conducted a 3-round Delphi study by email using a volunteer panel of 18 experts drawn from diving, dive medicine and nursing. In round 1, panellists shared examples of illnesses and injuries they had come across personally. These scenarios were circulated along with findings from a literature review, including existing recommendations. In rounds 2 and 3, the list of kit for dive boats in different settings was iteratively refined through online discussion and feedback. Passengers and crew on recreational dive boats may encounter a range of medical problems from minor injuries to serious accidents and non-dive-related illnesses. Recommended kit varied depending on context and setting (e.g. distance from land, qualifications and experience of crew). Consensus was quickly reached on key first-aid items but experts' views on emergency medicines differed. The study highlights the diversity of medical problems encountered on recreational dive boats. We offer preliminary guidance on the content of suitable first-aid kits and suggest areas for further research. Copyright © 2010 Elsevier Ltd. All rights reserved.
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NASA Astrophysics Data System (ADS)
Acciarri, R.; Adams, C.; An, R.; Anthony, J.; Asaadi, J.; Auger, M.; Bagby, L.; Balasubramanian, S.; Baller, B.; Barnes, C.; Barr, G.; Bass, M.; Bay, F.; Bishai, M.; Blake, A.; Bolton, T.; Camilleri, L.; Caratelli, D.; Carls, B.; Castillo Fernandez, R.; Cavanna, F.; Chen, H.; Church, E.; Cianci, D.; Cohen, E.; Collin, G. H.; Conrad, J. M.; Convery, M.; Crespo-Anadón, J. I.; Del Tutto, M.; Devitt, D.; Dytman, S.; Eberly, B.; Ereditato, A.; Escudero Sanchez, L.; Esquivel, J.; Fadeeva, A. A.; Fleming, B. T.; Foreman, W.; Furmanski, A. P.; Garcia-Gamez, D.; Garvey, G. T.; Genty, V.; Goeldi, D.; Gollapinni, S.; Graf, N.; Gramellini, E.; Greenlee, H.; Grosso, R.; Guenette, R.; Hackenburg, A.; Hamilton, P.; Hen, O.; Hewes, J.; Hill, C.; Ho, J.; Horton-Smith, G.; Hourlier, A.; Huang, E.-C.; James, C.; Jan de Vries, J.; Jen, C.-M.; Jiang, L.; Johnson, R. A.; Joshi, J.; Jostlein, H.; Kaleko, D.; Karagiorgi, G.; Ketchum, W.; Kirby, B.; Kirby, M.; Kobilarcik, T.; Kreslo, I.; Laube, A.; Li, Y.; Lister, A.; Littlejohn, B. R.; Lockwitz, S.; Lorca, D.; Louis, W. C.; Luethi, M.; Lundberg, B.; Luo, X.; Marchionni, A.; Mariani, C.; Marshall, J.; Martinez Caicedo, D. A.; Meddage, V.; Miceli, T.; Mills, G. B.; Moon, J.; Mooney, M.; Moore, C. D.; Mousseau, J.; Murrells, R.; Naples, D.; Nienaber, P.; Nowak, J.; Palamara, O.; Paolone, V.; Papavassiliou, V.; Pate, S. F.; Pavlovic, Z.; Piasetzky, E.; Porzio, D.; Pulliam, G.; Qian, X.; Raaf, J. L.; Rafique, A.; Rochester, L.; Rudolf von Rohr, C.; Russell, B.; Schmitz, D. W.; Schukraft, A.; Seligman, W.; Shaevitz, M. H.; Sinclair, J.; Smith, A.; Snider, E. L.; Soderberg, M.; Söldner-Rembold, S.; Soleti, S. R.; Spentzouris, P.; Spitz, J.; St. John, J.; Strauss, T.; Szelc, A. M.; Tagg, N.; Terao, K.; Thomson, M.; Toups, M.; Tsai, Y.-T.; Tufanli, S.; Usher, T.; Van De Pontseele, W.; Van de Water, R. G.; Viren, B.; Weber, M.; Wickremasinghe, D. A.; Wolbers, S.; Wongjirad, T.; Woodruff, K.; Yang, T.; Yates, L.; Zeller, G. P.; Zennamo, J.; Zhang, C.
2018-01-01
The development and operation of liquid-argon time-projection chambers for neutrino physics has created a need for new approaches to pattern recognition in order to fully exploit the imaging capabilities offered by this technology. Whereas the human brain can excel at identifying features in the recorded events, it is a significant challenge to develop an automated, algorithmic solution. The Pandora Software Development Kit provides functionality to aid the design and implementation of pattern-recognition algorithms. It promotes the use of a multi-algorithm approach to pattern recognition, in which individual algorithms each address a specific task in a particular topology. Many tens of algorithms then carefully build up a picture of the event and, together, provide a robust automated pattern-recognition solution. This paper describes details of the chain of over one hundred Pandora algorithms and tools used to reconstruct cosmic-ray muon and neutrino events in the MicroBooNE detector. Metrics that assess the current pattern-recognition performance are presented for simulated MicroBooNE events, using a selection of final-state event topologies.
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MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells.
Molnár, Viktor; Érsek, Barbara; Wiener, Zoltán; Tömböl, Zsófia; Szabó, Péter M; Igaz, Péter; Falus, András
2012-03-01
MicroRNAs provide an additional layer in the regulation of gene expression acting as repressors with several targets at the posttranscriptional level. This study describes microRNA expression patterns during differentiation and activation of mast cells. The expression levels of 567 different mouse miRNAs were compared by microarray between c-Kit+ committed progenitors, mucosal mast cells, resting and IgE-crosslinked BMMCs in vitro. The strongest upregulation of miR-132 upon IgE-mediated activation was validated in human cord blood-derived mast cells as well. HB-EGF growth factor also upregulated upon activation and was ranked high by more prediction algorithms. Co-transfection of miR-132 mimicking precursor and the 3'UTR of human Hbegf-containing luciferase vector proves that the predicted binding site is functional. In line with this, neutralization of miR-132 by anti-miR inhibitor leads to sustained production of HB-EGF protein in activated mast cells. Our data provide a novel example for negative regulation of a growth factor by an upregulated miRNA. © Springer Basel AG 2011
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Ogden, Samantha J; Horton, Jeffrey K; Stubbs, Simon L; Tatnell, Peter J
2015-01-01
The 1.2 mm Electric Coring Tool (e-Core™) was developed to increase the throughput of FTA(™) sample collection cards used during forensic workflows and is similar to a 1.2 mm Harris manual micro-punch for sampling dried blood spots. Direct short tandem repeat (STR) DNA profiling was used to compare samples taken by the e-Core tool with those taken by the manual micro-punch. The performance of the e-Core device was evaluated using a commercially available PowerPlex™ 18D STR System. In addition, an analysis was performed that investigated the potential carryover of DNA via the e-Core punch from one FTA disc to another. This contamination study was carried out using Applied Biosystems AmpflSTR™ Identifiler™ Direct PCR Amplification kits. The e-Core instrument does not contaminate FTA discs when a cleaning punch is used following excision of discs containing samples and generates STR profiles that are comparable to those generated by the manual micro-punch. © 2014 American Academy of Forensic Sciences.
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Diagnostic performance of the "MESACUP anti-Skin profile TEST".
Horváth, Orsolya N; Varga, Rita; Kaneda, Makoto; Schmidt, Enno; Ruzicka, Thomas; Sárdy, Miklós
2016-01-01
The "MESACUP anti-Skin profile TEST" is a new, commercially available ELISA kit to detect circulating IgG autoantibodies against desmoglein 1, desmoglein 3, BP180, BP230, and type VII collagen, both simultaneously and more rapidly than previous assays. The aim of this study was to evaluate the diagnostic accuracy of this kit for the diagnosis of pemphigus foliaceus, pemphigus vulgaris, bullous pemphigoid and epidermolysis bullosa acquisita. Dual-centre retrospective study in which 138 patients with autoimmune blistering diseases were compared to 40 controls Using the MESACUP anti-Skin profile TEST, both sensitivities and specificities for desmoglein 1, desmoglein 3, BP180, BP230, and type VII collagen autoantibodies were similar to those obtained using previous, specific ELISA systems and 88% of the results were concordant without any significant difference. The MESACUP anti-Skin profile TEST had a similar performance to previously produced ELISA systems. The novel kit can be used for rapid diagnosis of most common autoimmune blistering diseases and is especially suitable for identifying overlapping disorders.
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The Ultracool Typing Kit - An Open-Source, Qualitative Spectral Typing GUI for L Dwarfs
NASA Astrophysics Data System (ADS)
Schwab, Ellianna; Cruz, Kelle; Núñez, Alejandro; Burgasser, Adam J.; Rice, Emily; Reid, Neill; Faherty, Jacqueline K.; BDNYC
2018-01-01
The Ultracool Typing Kit (UTK) is an open-source graphical user interface for classifying the NIR spectral types of L dwarfs, including field and low-gravity dwarfs spanning L0-L9. The user is able to input an NIR spectrum and qualitatively compare the input spectrum to a full suite of spectral templates, including low-gravity beta and gamma templates. The user can choose to view the input spectrum as both a band-by-band comparison with the templates and a full bandwidth comparison with NIR spectral standards. Once an optimal qualitative comparison is selected, the user can save their spectral type selection both graphically and to a database. Using UTK to classify 78 previously typed L dwarfs, we show that a band-by-band classification method more accurately agrees with optical spectral typing systems than previous L dwarf NIR classification schemes. UTK is written in python, released on Zenodo with a BSD-3 clause license and publicly available on the BDNYC Github page.
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Li, Shaoheng; Yan, Yuhui; Jiao, Yanan; Gao, Zhong; Xia, Yang; Kong, Liang; Yao, Yingjia; Tao, Zhenyu; Song, Jie; Yan, Yaping; Zhang, Guangxian; Yang, Jingxian
2016-09-01
Accumulation of β-amyloid peptide (Aβ) in the brain plays an important role in the pathogenesis of Alzheimer's disease (AD). It has been reported that osthole exerts its neuroprotective effect on neuronal synapses, but its exact mechanism is obscure. Recently, microRNAs have been demonstrated to play a crucial role in inducing synaptotoxicity by Aβ, implying that targeting microRNAs could be a therapeutic approach of AD. In the present study, we investigated the neuroprotective effects of osthole on a cell model of AD by transducing APP695 Swedish mutant (APP695swe, APP) into mouse cortical neurons and human SH-SY5Y cells. In this study, the cell counting kit CCK-8, apoptosis assay, immunofluorescence analysis, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction, and Western blot assay were used. We found that osthole could enhance cell viability, prevent cell death, and reverse the reduction of synaptic proteins (synapsin-1, synaptophysin, and postsynaptic density-95) in APP-overexpressed cells, which was attributed to increases in microRNA-9 (miR-9) expression and subsequent decreases in CAMKK2 and p-AMPKα expressions. These results demonstrated that osthole plays a neuroprotective activity role in part through upregulating miR-9 in AD.
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Hagiwara, Ken'ichi; Iwamaru, Yoshifumi; Tabeta, Naoko; Yokoyama, Takashi; Tobiume, Minoru
2017-03-04
A classical type of bovine spongiform encephalopathy (C-BSE), recognized in 1987, had a large impact on public health due to its zoonotic link to variant Creutzfeldt-Jakob disease by the human consumption of dietary products contaminated with the C-BSE prion. Thus, a number of countries implemented BSE surveillance using rapid post-mortem test kits that were approved for detection of the C-BSE prion in the cattle brain. However, as atypical BSE (L- and H-BSE) cases emerged in subsequent years, the efficacy of the kits for the detection of atypical BSE prions became a matter of concern. In response to this, laboratories in the European Union and Canada evaluated the kits used in their countries. Here, we carried out an evaluation study of NippiBL®, a kit currently used for BSE screening in Japan. By applying the kit to cattle brains of field cases of C-BSE and L-BSE, and an experimental case of H-BSE, we showed its comparable sensitivities to C, L-, and H-BSE prions, and satisfactory performance required by the European Food Safety Authority. In addition to NippiBL®, two kits (TeSeE® and FRELISA®) formerly used in Japan were effective for detection of the L-BSE prion, although the two kits were unable to be tested for the H-BSE prion due to the discontinuation of domestic sales during this study. These results indicate that BSE screening in Japan is as effective as those in other countries, and it is unlikely that cases of atypical BSE have been overlooked.
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Shen, Cheng-Cheng; Chen, Bing; Gu, Jian-Teng; Ning, Jiao-Lin; Zeng, Jing; Yi, Bin; Lu, Kai-Zhi
2018-03-01
Recent studies have shown that pulmonary angiogenesis is an important pathological process in the development of hepatopulmonary syndrome (HPS), and growing evidence has indicated that Stromal cell-derived factor 1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) axis is involved in pulmonary vascular disease by mediating the accumulation of c-kit+ cells. This study aimed to test the effect of AMD3100, an antagonist of CXCR4, in HPS pulmonary angiogenesis. Common bile duct ligation (CBDL) rats were used as experimental HPS model and were treated with AMD3100 (1.25mg/kg/day, i.p.) or 0.9% saline for 3weeks. The sham rats underwent common bile duct exposure without ligation. The c-kit+ cells accounts and its angiogenic-related functions, prosurvival signals, pulmonary angiogenesis and arterial oxygenation were analysed in these groups. Our results showed that pulmonary SDF-1/CXCR4, Akt, Erk and VEGF/VEGFR2 were significantly activated in CBDL rats, and the numbers of circulating and pulmonary c-kit+ cells were increased in CBDL rats compared with control rats. Additionally, the angiogenic-related functions of c-kit+ cells and pulmonary microvessel counts were also elevated in CBDL rats. CXCR4 inhibition reduced pulmonary c-kit+ cells and microvessel counts and improved arterial oxygenation within 3weeks in CBDL rats. The pulmonary prosurvival signals and pro-angiogenic activity of c-kit+ cells were also down-regulated in AMD3100-treated rats. In conclusion, AMD3100 treatment attenuated pulmonary angiogenesis in CBDL rats and prevented the development of HPS via reductions in pulmonary c-kit+ cells and inhibition of the prosurvival signals. Our study provides new insights in HPS treatment. Copyright © 2017. Published by Elsevier B.V.
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Hang, Dong; Yuan, Hua; Liu, Li; Wang, Lihua; Miao, Limin; Zhu, Meng; Du, Jiangbo; Dai, Juncheng; Hu, Zhibin; Chen, Ning; Shen, Hongbing; Ma, Hongxia
2017-01-01
KITLG/KIT pathway plays a vital role in multiple types of human cancer including head and neck squamous cell carcinoma (HNSCC). Genetic variations in KITLG and KIT may affect the expression or function of these genes, thereby modifying cancer risk. In this study, we evaluated the association of KITLG and KIT polymorphisms with HNSCC risk among Chinese population. Twenty-two tagging SNPs in KITLG and KIT genes were genotyped in a case-control study with 576 HNSCC patients and 1552 healthy controls. Logistic regression analyses revealed that an upstream SNP rs6554198 [additive model: adjusted odds ratio (OR) = 0.85, 95% confidence interval (CI) = 0.74-0.97, P = 0.019] and two intron SNPs rs2237025 (additive model: adjusted OR = 0.82, 95%CI = 0.70-0.95, P = 0.007), and rs17084687 (additive model: adjusted OR = 0.85, 95%CI = 0.73-0.99, P = 0.042) of KIT were significantly associated with the decreased risk of HNSCC. Combined analysis of the three SNPs showed that subjects carrying the protective alleles had decreased risk of HNSCC in a dose-response manner (P trend = 0.001). Furthermore, interaction analyses revealed a significant multiplicative interaction between rs17084687 and drinking on HNSCC risk (P = 0.012). Luciferase activity assay indicated that the allele A of potentially functional rs6554198 led to significantly lower transcription activity of KIT compared to the risk allele G. Summarily, our findings suggested that SNPs in KIT gene may play a role in genetic susceptibility to HNSCC, which may improve our understanding of the pathogenic mechanisms of this disease. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
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A Frameshift Mutation in KIT is Associated with White Spotting in the Arabian Camel.
Holl, Heather; Isaza, Ramiro; Mohamoud, Yasmin; Ahmed, Ayeda; Almathen, Faisal; Youcef, Cherifi; Gaouar, Semir; Antczak, Douglas F; Brooks, Samantha
2017-03-09
While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five kITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation.
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2012-01-01
Background Vulvar carcinomas are rare tumors, and there is limited data regarding molecular alterations. To our knowledge there are no published studies on c-KIT and squamous cell carcinomas of the vulva (VSCC). Although there are a significant number of other tumor types which express c-KIT, there remains controversy as to its relationship to patient outcome. Thus, we wished to investigate such controversial findings to determine the prognostic importance of c-KIT by evaluating its protein and mRNA expression in VSCCs, correlating these findings with clinicopathological features and Human Papillomavirus (HPV) infection. Methods c-KIT expression was scored by immunohistochemistry (IHC) as positive or negative in 139 formalin-fixed paraffin-embedded (FFPE) cases of vulvar carcinomas arrayed in a tissue microarray (TMA) using the DAKO® A4502 rabbit polyclonal c-KIT antibody (diluted 1:100). c-KIT mRNA was evaluated by qRT-PCR in 34 frozen samples from AC Camargo Hospital Biobank (17 tumoral and 17 non-tumoral samples) using TaqMan probes–Applied Biosystems [Hs00174029_m1]. HPV genotyping was assessed in 103 samples using Linear Array® HPV Genotyping Test kit (Roche Molecular Diagnostics, Basel, Switzerland). All results obtained were correlated with clinical and pathological data of the patients. Results c-KIT protein was positive by immunohistochemistry in 70.5% of the cases and this was associated with a higher global survival (p = 0.007), a higher recurrence-free survival (p < 0.0001), an absence of associated lesions (p = 0.001), lymph node metastasis (p = 0.0053), and HPV infection (p = 0.034). Furthermore, c-KIT mRNA quantitation revealed higher levels of transcripts in normal samples compared to tumor samples (p = 0,0009). Conclusions Our findings indicate that those vulvar tumors staining positively for c-KIT present better prognosis. Thus, positivity of c-KIT as evaluated by IHC may be a good predictor for use of more conservative surgery techniques and lymph node dissection in vulvar cancer. So part of the essence of our study is to see the possibility of translating our current results from the bench to the bedside. This will help provide patients a more appropriate, less mutilating treatment, in order to keep the maximum physical and psychic quality as possible to these women. PMID:22839358
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Mavroeidis, Leonidas; Metaxa-Mariatou, Vassiliki; Papoudou-Bai, Alexandra; Lampraki, Angeliki Maria; Kostadima, Lida; Tsinokou, Ilias; Zarkavelis, George; Papadaki, Alexandra; Petrakis, Dimitrios; Gκoura, Stefania; Kampletsas, Eleftherios; Nasioulas, George; Batistatou, Anna; Pentheroudakis, George
2018-01-01
Gastrointestinal stromal tumours (GIST) are mesenchymal neoplasms that usually carry an activating mutation in KIT or platelet-derived growth factor receptor alpha ( PDGFRA ) genes with predictive and prognostic significance. We investigated the extended mutational status of GIST in a patient population of north-western Greece in order to look at geopraphic/genotypic distinctive traits. Clinicopathological and molecular data of 38 patients diagnosed from 1996 to 2016 with GIST in the region of Epirus in Greece were retrospectively assessed. Formalin-fixed paraffin-embedded tumours were successfully analysed for mutations in 54 genes with oncogenic potential. Next generation sequencing was conducted by using the Ion AmpliSeqCancer Hotspot Panel V.2 for DNA analysis (Thermofisher Scientific). Among 38 tumours, 24 (63.16%) and seven (18.42%) of the tumours harboured mutations in the KIT and PDGFRA genes, respectively, while seven (18.42%) tumours were negative for either KIT or PDGFRA mutation. No mutations were detected in five (13.16%) cases. Concomitant mutations of BRAF and fibroblast growth factor receptor 3 ( FGFR3 ) genes were observed in two patients with KIT gene mutation. Two patients with KIT / PDGFRA wild-type GIST had mutations in either KRAS or phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA ) genes. There was no significant survival difference regarding the exonic site of mutation in either KIT or PDGFRA gene. The presence of a mutation in pathway effectors downstream of KIT or PDGFRA , such as BRAF , KRAS or PIK3CA , was associated with poor prognosis. Adverse prognosticators were also high mitotic index and the advanced disease status at diagnosis. We report comparable incidence of KIT and PDGFRA mutation in patients with GIST from north-western Greece as compared with cohorts from other regions. Interestingly, we identified rare mutations on RAS , BRAF and PIK3CA genes in patients with poor prognosis.
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Li, Xiangjie; Liu, Na; Liu, Rui; Dong, Zhengmou; Liu, Luchuan; Deng, Manjing
2012-02-01
To compare the consistency of root canal configuration types of mandibular first premolar by using micro-CT and radio visio graphy (RVG). One hundred extracted mandibular first premolars with complete dental root and apex which received no endodontic treatment were randomly selected. Each tooth was radiographed with RVG through a buccolingual and mesiodistal direction, and then scanned with micro-CT and reconstructed. The classifications of the root canal types according to Vertucci's type with the two methods were compared. The canal patterns were classified as type I (67%), type III (3%), type V (18%), type VII (2%), additional type (10%) with micro-CT and canal patterns as type I (71%), type III (2%), type V (23%), type VII (1%), additional type (3%) with RVG. 63% of teeth showed one canal in both micro-CT and RVG. Only 25% of teeth were diagnosed as complex canal by the same canal type in both micro-CT and RVG. The Kappa value between micro-CT and RVG was 0.541 which suggested that the two kinds of methods had intermediate consistency. 82.8% of the premolars with root groove had two or more than two canals. Although RVG can basically reflect the root canal system type of the mandibular first premolars in vitro, it offers poor accuracy images to complex root canals. Micro-CT three-dimensional images could clearly and precisely display the root canal system morphology of the mandibular first pre-molars in vitro.
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Methods and systems for micro transmissions
DOE Office of Scientific and Technical Information (OSTI.GOV)
Stalford, Harold L
2014-12-23
Methods and systems for micro transmissions for a micro machine may comprise an input shaft assembly coupled to a micro actuator, an output shaft assembly coupled to a micro shaft, and one or more power conversion elements operable to convert a first type of movement from the micro actuator into a second, disparate type of movement for the micro shaft.
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Liu, Chao; Ouyang, Wei; Xia, Jingyan; Sun, Xiaoru; Zhao, Liying; Xu, Feng
2018-06-05
Mast cells (MCs) play a key role in immune process response to invading pathogens. This study assessed the involvement of MCs in controlling Staphylococcus aureus infection in a cutaneous infection model of MC-deficient (KitW-sh/W-sh) mice. KitW-sh/W-sh mice developed significantly larger skin lesions after the cutaneous S. aureus challenge, when compared to wild-type (WT) mice, while MC dysfunction reduced the inflammation response to S. aureus. The levels of tumor necrosis factor (TNF)-α in skin tissues were significantly decreased in KitW-sh/W-sh mice upon infection. Moreover, the exogenous administration of MCs or recombinant TNF-α effectively restored the immune response against S. aureus in KitW-sh/W-sh mice via the recruitment of neutrophils to the infected site. These results indicate that the effects of MC deficiency are largely attributed to the decrease in production of TNF-α in cutaneous S. aureus infection. In addition, S. aureus-induced MC activation was dependent on the c-kit receptor-activated phosphoinositide 3-kinase (PI3K)/AKT/P65-nuclear factor (NF-κB) pathway, which was confirmed by treatment with Masitinib (a c-kit receptor inhibitor), Wortmannin (a PI3K inhibitor), and pyrrolidine dithiocarbamate (a NF-κB inhibitor), respectively. The present study identifies the critical role of MCs in the host defense against S. aureus infection.
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Code of Federal Regulations, 2010 CFR
2010-10-01
... 46 Shipping 6 2010-10-01 2010-10-01 false Type. 160.058-2 Section 160.058-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Desalter Kits, Sea Water, for Merchant Vessels § 160.058-2 Type. (a) Desalter...
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Dysregulation of Aldosterone Secretion in Mast Cell-Deficient Mice.
Boyer, Hadrien-Gaël; Wils, Julien; Renouf, Sylvie; Arabo, Arnaud; Duparc, Céline; Boutelet, Isabelle; Lefebvre, Hervé; Louiset, Estelle
2017-12-01
Resident adrenal mast cells have been shown to activate aldosterone secretion in rat and man. Especially, mast cell proliferation has been observed in adrenal tissues from patients with aldosterone-producing adrenocortical adenoma. In the present study, we show that the activity of adrenal mast cells is stimulated by low-sodium diet and correlates with aldosterone synthesis in C57BL/6 and BALB/c mice. We have also investigated the regulation of aldosterone secretion in mast cell-deficient C57BL/6 Kit W-sh/W-sh mice in comparison with wild-type C57BL/6 mice. Kit W-sh/W-sh mice submitted to normal sodium diet had basal plasma aldosterone levels similar to those observed in wild-type animals. Conversely, low-sodium diet unexpectedly induced an exaggerated aldosterone response, which seemed to result from an increase in adrenal renin and angiotensin type 1 receptor expression. Severe hyperaldosteronism was associated with an increase in systolic blood pressure and marked hypokalemia, which favored polyuria. Adrenal renin and angiotensin type 1 receptor overexpression may represent a compensatory mechanism aimed at activating aldosterone production in the absence of mast cells. Finally, C57BL/6 Kit W-sh/W-sh mice represent an unexpected animal model of primary aldosteronism, which has the particularity to be triggered by sodium restriction. © 2017 American Heart Association, Inc.
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Yao, Yining; Yang, Qinrui; Shao, Chengchen; Liu, Baonian; Zhou, Yuxiang; Xu, Hongmei; Zhou, Yueqin; Tang, Qiqun; Xie, Jianhui
2018-01-01
Rare variants are widely observed in human genome and sequence variations at primer binding sites might impair the process of PCR amplification resulting in dropouts of alleles, named as null alleles. In this study, 5 cases from routine paternity testing using PowerPlex ® 21 System for STR genotyping were considered to harbor null alleles at TH01, FGA, D5S818, D8S1179, and D16S539, respectively. The dropout of alleles was confirmed by using alternative commercial kits AGCU Expressmarker 22 PCR amplification kit and AmpFℓSTR ® . Identifiler ® Plus Kit, and sequencing results revealed a single base variation at the primer binding site of each STR locus. Results from the collection of previous reports show that null alleles at D5S818 were frequently observed in population detected by two PowerPlex ® typing systems and null alleles at D19S433 were mostly observed in Japanese population detected by two AmpFℓSTR™ typing systems. Furthermore, the most popular mutation type appeared the transition from C to T with G to A, which might have a potential relationship with DNA methylation. Altogether, these results can provide helpful information in forensic practice to the elimination of genotyping discrepancy and the development of primer sets. Copyright © 2017 Elsevier B.V. All rights reserved.
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Farag, S; van der Kolk, L E; van Boven, H H; van Akkooi, A C J; Beets, G L; Wilmink, J W; Steeghs, N
2018-04-01
Gastrointestinal stromal tumors (GISTs) occur mostly sporadically. GISTs associated with a familial syndrome are very rare and are mostly wild type for KIT and platelet-derived growth factor alpha (PDGFRA). To date 35 kindreds and 8 individuals have been described with GISTs associated with germline KIT mutations. This is the third family described with a germline p.Trp557Arg mutation in exon 11 of the KIT gene. The effect of imatinib in patients harboring a germline KIT mutation has been rarely described. Moreover, in some studies imatinib treatment was withheld considering the lack of evidence for efficacy of this treatment in GIST patients harboring a germline KIT mutation. This paper describes a 52-year old patient with a de novo germline p.Trp557Arg mutation with multiple GISTs throughout the gastrointestinal tract and cutaneous hyperpigmentation. Imatinib treatment showed long-term regression of the GISTs and evident pathological response was seen after resection. Remarkably, the hyperpigmentation of the skin also diminished during imatinib treatment. Genetic screening of the family revealed the same mutation in two daughters, both with similar cutaneous hyperpigmentation. One daughter, aged 23, was diagnosed with multiple small intestine GISTs, which were resected. She was treated with adjuvant imatinib which prompted rapid regression of the cutaneous hyperpigmentation. Imatinib treatment in GIST patients harboring a germline KIT mutation shows favorable and long-term responses in both the tumor and the phenotypical hyperpigmentation.
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Radiosensitization Effect of STI-571 on Pancreatic Cancer Cells In Vitro
DOE Office of Scientific and Technical Information (OSTI.GOV)
Chung, Hye Won; Wen, Jing; Lim, Jong-Baeck
2009-11-01
Purpose: To examine STI-571-induced radiosensitivity in human pancreatic cancer cells in vitro. Methods and Materials: Three human pancreatic cancer cell lines (Bxpc-3, Capan-1, and MiaPaCa-2) exhibiting different expression levels of c-Kit and platelet-derived growth factor receptor beta (PDGFRbeta) and showing different K-ras mutation types were used. For evaluation of the antitumor activity of STI-571 in combination with radiation, clonogenic survival assays, Western blot analysis, and the annexin V/propidium iodide assay with microscopic evaluation by 4',6-diamidino-2-phenylindole were conducted. Results: Dramatic phosphorylated (p)-c-Kit and p-PDGFRbeta attenuation, a modest dose- and time-dependent growth inhibition, and significant radiosensitization were observed after STI-571 treatment inmore » view of apoptosis, although the levels of growth inhibition and increased radiosensitization were different according to cell lines. The grades of radiosensitivity corresponded to the attenuation levels of p-c-Kit and p-PDGFRbeta by STI-571, particularly to those of p-c-Kit, and the radiosensitivity was partially affected by K-ras mutation in pancreatic cancer cells. Among downstream pathways associated with c-Kit or PDGFRbeta, p-PLCgamma was more closely related to radiosensitivity compared with p-Akt1 or p-extracellular signal-regulated kinase 1. Conclusion: STI-571 enhances radiation response in pancreatic cancer cells. This effect is affected by the attenuation levels of p-c-Kit or p-PDGFRbeta, and K-ras mutation status. Among them, p-c-Kit plays more important roles in the radiosensitivity in pancreatic cancer compared with p-PDGFRbeta or K-ras mutation status.« less
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Apollonio, Luigino G; Whittall, Ian R; Pianca, Dennis J; Kyd, Jennelle M; Maher, William A
2007-05-01
The aim of this study was to evaluate the Bio-Quant Direct ELISA assays for amphetamine and methamphetamine in the routine presumptive screening of biological fluids. Standard concentration curves of the target analytes were assayed to assess sensitivity, and known concentrations of common amphetamine-type substances (ephedrine, pseudoephedrine, phentermine), designer analogues (MDA, MDMA, MDEA, MBDB, PMA, 4-MTA, 2CB), and putrefactive amines (phenylethylamine, putrescine, tryptamine, tyramine) were analyzed to determine cross-reactivity. Results of the standard curve studies show the capacity of both Direct ELISA kits to confidently detect down to 3 ng/mL interday (PBS matrix; CVs 6.3-15.5%). Cross-reactivity relative to that of 50 ng/mL preparations of the target compounds demonstrated that the Direct ELISA kit for amphetamine also detected MDA (282%), PMA (265%), 4-MTA (280%), and phentermine (61%), and the Direct ELISA for methamphetamine also assayed positive for MDMA (73%), MDEA (18%), pseudoephedrine (19%), MBDB (8%), and ephedrine (9%). Matrix studies demonstrated that both ELISA kits could be applied to screening of blood, urine, and saliva to a concentration of 6 ng/mL or lower. In conclusion, the Bio-Quant Direct ELISA kits for amphetamine and methamphetamine are fast and accurate and have demonstrated themselves to be useful tools in routine toxicological testing.
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Inokuchi, Shota; Yamashita, Yasuhiro; Nishimura, Kazuma; Nakanishi, Hiroaki; Saito, Kazuyuki
2017-11-01
Phenomena known as null alleles and peak imbalance can occur because of mutations in the primer binding sites used for DNA typing. In these cases, an accurate statistical evaluation of DNA typing is difficult. The estimated likelihood ratio is incorrectly calculated because of the null allele and allele dropout caused by mutation-induced peak imbalance. Although a number of studies have attempted to uncover examples of these phenomena, few reports are available on the human identification kit manufactured by Qiagen. In this study, 196 Japanese individuals who were heterozygous at D2S1360 were genotyped using an Investigator HDplex Kit with optimal amounts of DNA. A peak imbalance was frequently observed at the D2S1360 locus. We performed a sequencing analysis of the area surrounding the D2S1360 repeat motif to identify the cause for peak imbalance. A point mutation (G>A transition) 136 nucleotides upstream from the D2S1360 repeat motif was discovered in a number of samples. The allele frequency of the mutation was 0.0566 in the Japanese population. Therefore, human identification or kinship testing using the Investigator HDplex Kit requires caution because of the higher frequency of single nucleotide polymorphisms at the primer binding site of D2S1360 locus in the Japanese population.
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Mechanical Complication with Broviac Repair Kit in a 4-Year-Old Boy with MEN 2a.
Sesia, Sergio B; Haecker, Frank-Martin; Mayr, Johannes
2009-01-01
Background. Mechanical complications in the use of indwelling central venous catheters (CVCs) such as the Broviac catheter (BC) include kinking, occlusion, dislocation or leaking. We report on a mechanical complication after using a repair kit for the BC. Method. A 4-year old boy, suffering from multiple endocrine neoplasia type 2a (MEN 2a), intestinal aganglionosis (Hirschsprung's disease), and short bowel syndrome, required a BC for home parenteral nutrition. Result. Due to recurrent leakage of the BC, 5 subsequent repairs were necessary within seven months. During one repair a metallic tube belonging to the repair kit was found to have migrated proximally to the skin entrance level within the BC and requiring surgical removal. Conclusion. To our knowledge, this is the first report focusing on such a serious complication using a BC and its repair kit. The proximal migration of this metallic tube constitutes a distinct theoretical risk of endothoracic foreign body embolization.
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STR-typing of ancient skeletal remains: which multiplex-PCR kit is the best?
Harder, Melanie; Renneberg, Rebecca; Meyer, Patrick; Krause-Kyora, Ben; von Wurmb-Schwark, Nicole
2012-01-01
Aim To comparatively test nine commercially available short tandem repeat (STR)-multiplex kits (PowerPlex 16, 16HS, ES, ESI17, ESX17, S5 [all Promega]; AmpFiSTR Identifiler, NGM and SEfiler [all Applied Biosystems]) for their efficiency and applicability to analyze ancient and thus highly degraded DNA samples. Methods Fifteen human skeletal remains from the late medieval age were obtained and analyzed using the nine polymerase chain reaction assays with slightly modified protocols. Data were systematically compared to find the most meaningful and sensitive assay. Results The ESI, ESX, and NGM kits showed the best overall results regarding amplification success, detection rate, identification of heterozygous alleles, sex determination, and reproducibility of the obtained data. Conclusion Since application of these three kits enables the employment of different primer sequences for all the investigated amplicons, a combined application is recommended for best possible and – most importantly – reliable genetic analysis of ancient skeletal material or otherwise highly degraded samples, eg, from forensic cases. PMID:23100203
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Shuttle orbiter TPS flight repair kit development
NASA Technical Reports Server (NTRS)
1979-01-01
The design and application of a TPS repair kit is presented. The repair kit is designed for on orbit use by a crew member working in the manned maneuvering unit (MMU). The kit includes the necessary equipment and materials to accomplish the repair tasks which include the following: HRSI emittance coating repair, damaged tile repair, missing tile repair, and multiple tile repair. Two types of repair materials required to do the small area repair and the large area repair are described. The materials area cure in place, silicone base ablator for small damaged areas and precured ablator tile for repair of larger damaged areas is examined. The cure in place ablator is also used as an adhesive to bond the precured tiles in place. An applicator for the cure in place ablator, designed to contain a two-part silicon compound, mix the two components at correct ratio, and dispense the materials at rates compatible with mission timelines established for the EVA is described.
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A Y-chromosome STR marker should be added to commercial multiplex STR kits.
Oz, Carla; Zaken, Neomi; Amiel, Merav; Zamir, Ashira
2008-07-01
Autosomal short tandem repeat (STR) analysis has become highly relevant in the identification of victims from mass disasters and terrorist attacks. In such events, gender misidentification can be of grave consequences, yet the list reporting amelogenin amplification failure using STR multiplex kits continues to grow. Presented here are three such examples. In the first case, we present two male suspects who demonstrated amelogenin Y-deficient results using two commercial kit procedures. The presence of their Y chromosomes was proven by obtaining a Y-haplotype. The second case demonstrated a profile from a third male suspect where only the Y homolog of the XY pair was amplified. In events such as mass disasters or terrorist attacks, timely and reliable high throughput DNA typing results are essential. As the number of reported cases of amplification failure at the amelogenin gene continues to grow, we suggest that the incorporation of a better gender identification tool in commercial kits is crucial.
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Home bowel cancer tests and informed choice--is current information sufficient?
Howard, K; Salkeld, G
2003-10-01
To evaluate the type of information that is available to purchasers of home-based bowel cancer test kits. Manufacturers, pharmacies and independent testing programs were contacted to obtain faecal occult blood test (FOBT) kits. State cancer organisations were contacted for information on bowel cancer screening. Information on bowel cancer, the FOBT kit, the testing process and potential benefits and harms of the screening process were assessed using guidelines provided by the UK General Medical Council (GMC). FOBT kits and cancer organisation information provided adequate information on the purpose of screening, the screening process itself and potential benefits, but provided no information concerning uncertainties of screening or potential harms. On the basis of both the UK GMC criteria and patient desires for information, the information available at present falls short of being considered adequate for an informed decision to purchase a home-based FOBT. We must ensure adequate and balanced information is available to redress the present information asymmetry to facilitate informed participation in a potentially valuable public health initiative.
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Meex, Cécile; Neuville, Florence; Descy, Julie; Huynen, Pascale; Hayette, Marie-Pierre; De Mol, Patrick; Melin, Pierrette
2012-11-01
In cases of bacteraemia, a rapid species identification of the causal agent directly from positive blood culture broths could assist clinicians in the timely targeting of empirical antimicrobial therapy. For this purpose, we evaluated the direct identification of micro-organisms from BacT/ALERT (bioMérieux) anaerobic positive blood cultures without charcoal using the Microflex matrix-assisted laser desorption/ionization (MALDI) time of flight MS (Bruker), after bacterial extraction by using two different methods: the MALDI Sepsityper kit (Bruker) and an in-house saponin lysis method. Bruker's recommended criteria for identification were expanded in this study, with acceptance of the species identification when the first three results with the best matches with the MALDI Biotyper database were identical, whatever the scores were. In total, 107 monobacterial cultures and six polymicrobial cultures from 77 different patients were included in this study. Among monomicrobial cultures, we identified up to the species level 67 and 66 % of bacteria with the MALDI Sepsityper kit and the saponin method, respectively. There was no significant difference between the two extraction methods. The direct species identification was particularly inconclusive for Gram-positive bacteria, as only 58 and 52 % of them were identified to the species level with the MALDI Sepsityper kit and the saponin method, respectively. Results for Gram-negative bacilli were better, with 82.5 and 90 % of correct identification to the species level with the MALDI Sepsityper kit and the saponin method, respectively. No misidentifications were given by the direct procedures when compared with identifications provided by the conventional method. Concerning the six polymicrobial blood cultures, whatever the extraction method used, a correct direct identification was only provided for one of the isolated bacteria on solid medium in all cases. The analysis of the time-to-result demonstrated a reduction in the turnaround time for identification ranging from 1 h 06 min to 24 h 44 min, when performing the blood culture direct identification in comparison with the conventional method, whatever the extraction method.
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The American Revolution; A Bibliography of Multimedia
ERIC Educational Resources Information Center
Fultz, Norma J.
1975-01-01
This bibliography lists several types of multimedia instructional materials useful in teaching the American Revolution for elementary and secondary students. The following types of media are included: audiotapes, films, filmstrips, kits, phonodiscs, pictures, realia, simulations, slides, and transparencies. (JR)
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Bloß, P., E-mail: bloss@kuz-leipzig.de, E-mail: juettner@kuz-leipzig.de, E-mail: jacob@kuz-leipzig.de, E-mail: loeser@kuz-leipzig.de, E-mail: michaelis@kuz-leipzig.de, E-mail: krajewsky@kuz-leipzig.de; Jüttner, G., E-mail: bloss@kuz-leipzig.de, E-mail: juettner@kuz-leipzig.de, E-mail: jacob@kuz-leipzig.de, E-mail: loeser@kuz-leipzig.de, E-mail: michaelis@kuz-leipzig.de, E-mail: krajewsky@kuz-leipzig.de; Jacob, S., E-mail: bloss@kuz-leipzig.de, E-mail: juettner@kuz-leipzig.de, E-mail: jacob@kuz-leipzig.de, E-mail: loeser@kuz-leipzig.de, E-mail: michaelis@kuz-leipzig.de, E-mail: krajewsky@kuz-leipzig.de
2014-05-15
Micro plastic parts open new fields for application, e. g., to electronics, sensor technologies, optics, and medical engineering. Before micro parts can go to mass production, there is a strong need of having the possibility for testing different designs and materials including material combinations. Hence, flexible individual technical and technological solutions for processing are necessary. To manufacture high quality micro parts, a micro injection moulding machine named formicaPlast based on a two-step plunger injection technology was developed. Resulting from its design, the residence time and the accuracy problems for managing small shot volumes with reproducible high accuracy are uncompromisingly solved.more » Due to their simple geometry possessing smooth transitions and non adherent inner surfaces, the plunger units allow to process 'all' thermoplastics from polyolefines to high performance polymers, optical clear polymers, thermally sensitive bioresorbables, highly filled systems (the so-called powder injection molding PIM), and liquid silicon rubber (LSR, here with a special kit). The applied platform strategy in the 1K and 2K version allows integrating automation for assembling, handling and packaging. A perpendicular arrangement allows encapsulation of inserts, also partially, and integration of this machine into process chains. Considering a wide variety of different parts consisting of different materials, the high potential of the technology is demonstrated. Based on challenging industrial parts from electronic applications (2K micro MID and bump mat, where both are highly structured parts), the technological solutions are presented in more detail.« less
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Genotoxicity of mercury used in chromosome aberration tests.
Akiyama, M; Oshima, H; Nakamura, M
2001-01-01
The purpose of this study was to investigate the genotoxic effects of Hg released from dental amalgams. The chromosome aberration test was conducted using original extracts and their diluted solutions of conventional type amalgam and high copper amalgam. The concentrations of Hg, Cu and Ag in the original extract of high copper amalgam were 17.64, 7.97 and 43.90 microM, respectively. Those in the original extract of conventional type amalgam were 20.63, 7.87 and 14.79 microM, respectively. 10 and 30 microM Hg(2+) were also used for comparison. The frequency of chromosome aberrations was below 5% with 0 microM Hg(2+) and with a triple dilution of high copper amalgam extract, containing 5.88 microM Hg, 14.63 microM Cu and 2.65 microM Ag. However, 9.5% of the cells showed chromosome aberrations with a quadruple dilution of conventional type amalgam, containing 5.15 microM Hg, 3.69 microM Cu and 1.96 microM Ag. The amount of Hg in the quadruple dilution of conventional type amalgam was less than that in the triple dilution of high copper amalgam extract and 10 microM Hg(2+). A concentration of 30 microM Hg(2+) caused 34.5% of the cells to show chromosome aberrations while with a two-thirds dilution of high copper amalgam extract, containing 11.76 microM Hg, 29.26 microM Cu and 5.31 microM Ag, 58.5% of the cells showed chromosome aberrations. A two-thirds dilution of high copper amalgam extract induced more chromosome aberrations than 30 microM Hg(2+), although the amount of Hg was less than 30 microM Hg(2+). A triple dilution of conventional type amalgam extract, original extracts of conventional type amalgam and high copper amalgam and 100 microM Hg(2+) were induced few metaphases. It was revealed that the conventional type amalgam induced chromosome aberrations with quadruple dilution where cell viability was about 80% and that the high copper amalgam induced a high level of chromosome aberrations with the two-thirds dilution. The effects of low level Hg on humans are not clear.
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Chiu, Yvonne E; Dugan, Stefanie; Basel, Donald; Siegel, Dawn H
2013-01-01
Piebaldism is a rare genodermatosis caused by KIT mutations. We report the case of a 5-year-old boy who had the white forelock and leukoderma of piebaldism, but the presence of many café-au-lait macules and axillary and inguinal freckling complicated the diagnosis. Patients with similar cutaneous findings have been previously reported, and their disorder has been attributed to an overlap of piebaldism and neurofibromatosis type 1. Legius syndrome is a recently described syndrome caused by Sprouty-related, Ena/vasodilator-stimulated phosphoprotein homology-1 domain containing protein 1 (SPRED1) mutations that also has multiple café-au-lait macules and intertriginous freckling. Based on our current understanding of KIT and SPRED1 protein interactions, we propose that café-au-lait macules and freckling may be seen in some patients with piebaldism and does not necessarily represent coexistence of neurofibromatosis type 1. © 2012 Wiley Periodicals, Inc.
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Murata, Takashi; Tsuzaki, Kokoro; Nirengi, Shinsuke; Watanabe, Tomokazu; Mizutani, Yukako; Okada, Hayami; Tsukamoto, Masami; Odori, Shinji; Nakagawachi, Reiko; Kawaguchi, Yaeko; Yoshioka, Fumi; Yamada, Kazunori; Shimatsu, Akira; Kotani, Kazuhiko; Satoh-Asahara, Noriko; Sakane, Naoki
2017-07-01
The distributer of the anti-glutamic acid decarboxylase antibody assay kit using radioimmunoassay (RIA) recently announced its discontinuation, and proposed an alternative kit using enzyme-linked immunosorbent assay (ELISA). The aim of the present study was to investigate the diagnostic values of the anti-glutamic acid decarboxylase antibody by RIA and ELISA among type 1 diabetes mellitus patients and control participants. A total of 79 type 1 diabetes mellitus patients and 79 age-matched controls were enrolled and assessed using RIA and ELISA. Sensitivity, specificity, positive predictive values and negative predictive values were calculated for cut-off values (RIA = 1.5 U/mL and ELISA = 5.0 U/mL, respectively). Kappa coefficients were used to test for agreements between the RIA and ELISA methods regarding the diagnosis of type 1 diabetes mellitus. The sensitivity, specificity, positive predictive values, and negative predictive values for diagnosing type 1 diabetes mellitus were 57.0, 97.5, 95.7, and 69.4% by RIA, and 60.8, 100.0, 100.0 and 71.8% by ELISA, respectively. The diagnosis of type 1 diabetes mellitus using the RIA and ELISA methods showed substantial agreement with the kappa values of 0.74 for all participants, and of 0.64 for the acute type; however, there was moderate agreement with the kappa value of 0.56 for the slowly progressive type. The present study suggests that both anti-glutamic acid decarboxylase antibody by RIA and ELISA was useful for diagnosing type 1 diabetes mellitus. However, in the slowly progressive type, the degree of agreement of these two kits was poorer compared with those in all participants or in the acute type. © 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.
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Pracht, M; Mogha, A; Lespagnol, A; Fautrel, A; Mouchet, N; Le Gall, F; Paumier, V; Lefeuvre-Plesse, C; Rioux-Leclerc, N; Mosser, J; Oger, E; Adamski, H; Galibert, M-D; Lesimple, T
2015-08-01
Mutations of BRAF, NRAS and c-KIT oncogenes are preferentially described in certain histological subtypes of melanoma and linked to specific histopathological features. BRAF-, MEK- and KIT-inhibitors led to improvement in overall survival of patients harbouring mutated metastatic melanoma. To assess the prevalence and types of BRAF, NRAS, c-KIT and MITF mutations in cutaneous and mucous melanoma and to correlate mutation status with clinicopathological features and outcome. Clinicopathological features and mutation status of 108 samples and of 98 consecutive patients were, respectively, assessed in one retrospective and one prospective study. Clinicopathological features were correlated with mutation status and the predictive value of these mutations was studied. This work identified significant correlations between BRAF mutations and melanoma occurring on non-chronic sun-damaged skin and superficial spreading melanoma (P < 0.05) on one hand, and between NRAS mutations and nodular melanoma (P < 0.05) on the other hand. Younger age (P < 0.05), microscopic (P < 0.05) and macroscopic (P < 0.05) lymphatic involvement at diagnosis of primary melanoma were significantly linked to BRAF mutations. A mutated status was a positive predictive factor of a response to BRAF inhibitors (OR = 3.44). Mutated melanoma showed a significantly (P = 0.038) higher objective response rate to cytotoxic chemotherapy (26.3%) than wild-type tumours (6.7%). Clinical and pathological characteristics of the primary melanoma differed between wild-type and BRAF- or NRAS-mutated tumours. Patients with BRAF-mutated tumours were younger at diagnosis of primary melanoma. Patients carrying mutations showed better responses better to specific kinase inhibitors and interestingly also to systemic cytotoxic chemotherapy. © 2015 European Academy of Dermatology and Venereology.
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DATATOC: a novel conjugate for kit-type 68Ga labelling of TOC at ambient temperature.
Seemann, Johanna; Waldron, Bradley; Parker, David; Roesch, Frank
2017-01-01
The widespread acceptance and application of 68 Ga-PET depends on our ability to develop radiopharmaceuticals that can be prepared in a convenient and suitable manner. A kit-type labelling protocol provides such characteristics and requires chelators that can be radiolabelled under exceptionally mild conditions. Recently the DATA chelators have been introduced that fulfil these requirements. In continuing their development, the synthesis and radiolabelling of the first DATA bifunctional chelator (BFC) and peptide conjugate are described. A BFC derived from the DATA ligand (2,2'-(6-((carboxymethyl)amino)-1,4-diazepane-1,4-diyl)diacetic acid) has been synthesised in five steps from simple building blocks, with an overall yield of 8 %. DATA M5 -3 t Bu (5-[1,4-Bis-tert-butoxycarbonylmethyl-6-(tert-butoxycarbonylmethyl-methyl-amino)-[1, 4]diazepan-6-yl]-pentanoic acid) has been coupled to [DPhe 1 ][Tyr 3 ]-octreotide (TOC) and the resulting peptide conjugate (DATATOC) radiolabelled with purified 68 Ga derived via four different 68 Ge/ 68 Ga generator post-processing (PP) methods. The stability and lipophilicity of the radiotracer have been assessed and a kit-type formulation for radiolabelling evaluated. 68 Ga-DATATOC has been prepared with a > 95 % radiochemical yield (RCY) within 1 (fractionated and acetone-PP) and 10 min (ethanol- and NaCl-PP) at 23 °C (pH 4.2-4.9, 13 nmol). The radiolabelled peptide is stable in the presence of human serum. Lipophilicity of 68 Ga-DATATOC was calculated as logP = -3.2 ± 0.3, with a HPLC retention time ( t R = 10.4 min) similar to 68 Ga-DOTATOC (logP = -2.9 ± 0.4, t R = 10.3 min). Kit-type labelling from a lyophilised solid using acetone-PP based labelling achieves > 95 % RCY in 10 min at 23 °C. The favourable labelling properties of the DATA chelators have been retained for DATATOC. High radiochemical purity can be achieved at 23 °C in less than 1 min and from a kit formulation. The speed, reliability, ease, flexibility and simplicity with which 68 Ga-DATATOC can be prepared makes it a very attractive alternative to current standards.
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Guo, Fei; Yu, Jiao; Zhang, Lu; Li, Jun
2017-11-01
The ForenSeq™ DNA Signature Prep Kit (ForenSeq Kit) is designed to detect more than 200 forensically relevant markers in a single reaction on the MiSeq FGx™ Forensic Genomics System (MiSeq FGx System), including Amelogenin, 27 autosomal short tandem repeats (A-STRs), 7 X chromosomal STRs (X-STRs), 24 Y chromosomal STRs (Y-STRs) and 94 identity-informative single nucleotide polymorphisms (iSNPs) with the option to contain 22 phenotypic-informative SNPs (pSNPs) and 56 ancestry-informative SNPs (aSNPs). In this study, we evaluated the MiSeq FGx System on three major parts: methodological optimization (DNA extraction, sample quantification, library normalization, diluted libraries concentration, and sample-to-cell arrangement), massively parallel sequencing (MPS) performance (depth of coverage, sequence coverage ratio, and allele coverage ratio), and ForenSeq Kit characteristics (repeatability and concordance, sensitivity, mixture, stability and case-type samples). Results showed that quantitative polymerase chain reaction (qPCR)-based sample quantification and library normalization and the appropriate number of pooled libraries and concentration of diluted libraries provided a greater level of MPS performance and repeatability. Repeatable and concordant genotypes were obtained by the ForenSeq Kit. Full profiles were obtained from ≥100pg input DNA for STRs and ≥200pg for SNPs. A sample with ≥5% minor contributors was considered as a mixture by imbalanced allele coverage ratio distribution, and full profiles from minor contributors were easily detected between 9:1 and 1:9 mixtures with known reference profiles. The ForenSeq Kit tolerated considerable concentrations of inhibitors like ≤200μM hematin and ≤50μg/ml humic acid, and >56% STR profiles and >88% SNP profiles were obtained from ≥200-bp degraded samples. Also, it was adapted to case-type samples. As a whole, the ForenSeq Kit is a well-performed, robust, reliable, reproducible and highly informative assay, and it can fully meet requirements for human identification. Further, sensitive QC indicator and automated sample comparison function in the ForenSeq™ Universal Analysis Software are quite helpful, so that we can concentrate on questionable genotypes and avoid tedious and time-consuming labor to maximum the time spent in data analysis. Copyright © 2017 Elsevier B.V. All rights reserved.
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Hua, Li; Zhu, Guirong; Wei, Jianguo
2018-05-30
Non-small cell lung cancer (NSCLC) is a major type of lung cancer. Drug resistance is a enormous obstacle for cancer treatment. Copious microRNAs (miRNAs) have been demonstrated to be implicated in drug resistance in NSCLC. In the present study, RT-qPCR assay revealed that microRNA-1 (miR-1) expression was downregulated in DDP resistant NSCLC tissues and cells. Western blot assay presented a remarkable increase of LC3B-II/LC3B-I ratio and a notable decline of p62 level in DDP resistant NSCLC cells, while these effects were weakened by miR-1. GFP-LC3 puncta experiment showed that ectopic expression of miR-1 induced a noticeable downregulation of GFP-LC3 positive cell percentage in DDP resistant NSCLC cells. Bioinformatical analysis and luciferase assay revealed that autophagy related 3 (ATG3) was a target of miR-1. Also, western blot and RT-qPCR assays manifested that ATG3 was highly expressed in DDP resistant NSCLC tissues and cells. Additionally, miR-1 inhibited ATG3 expression and ATG3 upregulation abolished miR-1-meidated autophagy inhibition in DDP resistant NSCLC cells. Cell Counting Kit-8 (CCK-8) assay showed that the half maximal inhibitory concentration (IC 50 ) of cisplatin (DDP) was reduced in miR-1-enforced DDP resistant NSCLC cells, but was restored following the overexpression of ATG3. Flow cytometry experiments further showed that miR-1 overexpression induced a significant upregulation of apoptotic rate and ATG3 restoration weakened miR-1-induced apoptosis in DDP resistant NSCLC cells. Collectively, our study validated that miR-1 overexpression improved DDP sensitivity of NSCLC cells by inhibiting ATG3-mediated autophagy, providing a potential therapeutic target for easing chemoresistance of anti-tumor drugs. This article is protected by copyright. All rights reserved.
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Plasma pharmacokinetics and CYP3A12-dependent metabolism of c-kit inhibitor imatinib in dogs.
Ishizuka, M; Nagai, S; Sakamoto, K Q; Fujita, S
2007-05-01
Imatinib is a highly selective tyrosine kinase inhibitor, and is used for the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumours (GISTs) in humans. The aim of this study is to determine the in vitro and in vivo pharmacokinetics of imatinib in dogs and which cytochrome P450 (CYPs) contribute to its metabolism. Imatinib was administered orally or intravenously to dogs and the time of the peak concentration (T(max)) of imatinib was 4-9 h. The mean half-life was 622 +/- 368 min, and the AUC was 1256 +/- 809 microM * min after oral administration. The range of C0 of intravenously injected dogs was 12-24 microM. The half-life and AUC after intravenous injection were 206 +/- 112 min and 1026 +/- 371 microM * min, respectively. Recombinant system of dog CYP3A12 and CYP2C21 showed that CYP3A12 contributed to the metabolism of imatinib. The inhibition of CYP3A-dependent activity using a rat anti-CYP3A antibody or ketoconazole revealed that CYP3A12 plays a major role in the metabolism of imatinib in dog liver microsomes.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Acciarri, R.; Adams, C.; An, R.
The development and operation of Liquid-Argon Time-Projection Chambers for neutrino physics has created a need for new approaches to pattern recognition in order to fully exploit the imaging capabilities offered by this technology. Whereas the human brain can excel at identifying features in the recorded events, it is a significant challenge to develop an automated, algorithmic solution. The Pandora Software Development Kit provides functionality to aid the design and implementation of pattern-recognition algorithms. It promotes the use of a multi-algorithm approach to pattern recognition, in which individual algorithms each address a specific task in a particular topology. Many tens ofmore » algorithms then carefully build up a picture of the event and, together, provide a robust automated pattern-recognition solution. This paper describes details of the chain of over one hundred Pandora algorithms and tools used to reconstruct cosmic-ray muon and neutrino events in the MicroBooNE detector. Metrics that assess the current pattern-recognition performance are presented for simulated MicroBooNE events, using a selection of final-state event topologies.« less
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Acciarri, R.; Adams, C.; An, R.; ...
2018-01-29
The development and operation of Liquid-Argon Time-Projection Chambers for neutrino physics has created a need for new approaches to pattern recognition in order to fully exploit the imaging capabilities offered by this technology. Whereas the human brain can excel at identifying features in the recorded events, it is a significant challenge to develop an automated, algorithmic solution. The Pandora Software Development Kit provides functionality to aid the design and implementation of pattern-recognition algorithms. It promotes the use of a multi-algorithm approach to pattern recognition, in which individual algorithms each address a specific task in a particular topology. Many tens ofmore » algorithms then carefully build up a picture of the event and, together, provide a robust automated pattern-recognition solution. This paper describes details of the chain of over one hundred Pandora algorithms and tools used to reconstruct cosmic-ray muon and neutrino events in the MicroBooNE detector. Metrics that assess the current pattern-recognition performance are presented for simulated MicroBooNE events, using a selection of final-state event topologies.« less
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Kreuz, Peter Cornelius; Krüger, Jan Philipp; Metzlaff, Sebastian; Freymann, Undine; Endres, Michaela; Pruss, Axel; Petersen, Wolf; Kaps, Christian
2015-10-01
To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P < .0001; and PRP-C, P < .0001) stimulated migration of MPCs. All platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP preparations did induce such differentiation. These findings suggest differing outcomes with PRP treatment in stem cell-based cartilage repair. Our findings may help to explain the variability of results in studies examining the use of PRP clinically. Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.
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Comparative evaluation of different extraction and quantification methods for forensic RNA analysis.
Grabmüller, Melanie; Madea, Burkhard; Courts, Cornelius
2015-05-01
Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material. In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol® Reagent, Invitrogen; NucleoSpin® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling. We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of 'RNA integrity number' (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates. By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin® miRNA Kit excelling at DNA co-extraction and RNA results, respectively. Overall, there was no 'best' method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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Agarwal, Shruti; Kazi, Julhash U.; Rönnstrand, Lars
2013-01-01
The receptor tyrosine kinase c-Kit, also known as the stem cell factor receptor, plays a key role in several developmental processes. Activating mutations in c-Kit lead to alteration of these cellular processes and have been implicated in many human cancers such as gastrointestinal stromal tumors, acute myeloid leukemia, testicular seminomas and mastocytosis. Regulation of the catalytic activity of several kinases is known to be governed by phosphorylation of tyrosine residues in the activation loop of the kinase domain. However, in the case of c-Kit phosphorylation of Tyr-823 has been demonstrated to be a late event that is not required for kinase activation. However, because phosphorylation of Tyr-823 is a ligand-activated event, we sought to investigate the functional consequences of Tyr-823 phosphorylation. By using a tyrosine-to-phenylalanine mutant of tyrosine 823, we investigated the impact of Tyr-823 on c-Kit signaling. We demonstrate here that Tyr-823 is crucial for cell survival and proliferation and that mutation of Tyr-823 to phenylalanine leads to decreased sustained phosphorylation and ubiquitination of c-Kit as compared with the wild-type receptor. Furthermore, the mutated receptor was, upon ligand-stimulation, quickly internalized and degraded. Phosphorylation of the E3 ubiquitin ligase Cbl was transient, followed by a substantial reduction in phosphorylation of downstream signaling molecules such as Akt, Erk, p38, Shc, and Gab2. Thus, we propose that activation loop tyrosine 823 is crucial for activation of both the MAPK and PI3K pathways and that its disruption leads to a destabilization of the c-Kit receptor and decreased survival of cells. PMID:23803604
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Garton, Andrew J; Crew, Andrew P A; Franklin, Maryland; Cooke, Andrew R; Wynne, Graham M; Castaldo, Linda; Kahler, Jennifer; Winski, Shannon L; Franks, April; Brown, Eric N; Bittner, Mark A; Keily, John F; Briner, Paul; Hidden, Chris; Srebernak, Mary C; Pirrit, Carrie; O'Connor, Matthew; Chan, Anna; Vulevic, Bojana; Henninger, Dwight; Hart, Karen; Sennello, Regina; Li, An-Hu; Zhang, Tao; Richardson, Frank; Emerson, David L; Castelhano, Arlindo L; Arnold, Lee D; Gibson, Neil W
2006-01-15
OSI-930 is a novel inhibitor of the receptor tyrosine kinases Kit and kinase insert domain receptor (KDR), which is currently being evaluated in clinical studies. OSI-930 selectively inhibits Kit and KDR with similar potency in intact cells and also inhibits these targets in vivo following oral dosing. We have investigated the relationships between the potency observed in cell-based assays in vitro, the plasma exposure levels achieved following oral dosing, the time course of target inhibition in vivo, and antitumor activity of OSI-930 in tumor xenograft models. In the mutant Kit-expressing HMC-1 xenograft model, prolonged inhibition of Kit was achieved at oral doses between 10 and 50 mg/kg and this dose range was associated with antitumor activity. Similarly, prolonged inhibition of wild-type Kit in the NCI-H526 xenograft model was observed at oral doses of 100 to 200 mg/kg, which was the dose level associated with significant antitumor activity in this model as well as in the majority of other xenograft models tested. The data suggest that antitumor activity of OSI-930 in mouse xenograft models is observed at dose levels that maintain a significant level of inhibition of the molecular targets of OSI-930 for a prolonged period. Furthermore, pharmacokinetic evaluation of the plasma exposure levels of OSI-930 at these effective dose levels provides an estimate of the target plasma concentrations that may be required to achieve prolonged inhibition of Kit and KDR in humans and which would therefore be expected to yield a therapeutic benefit in future clinical evaluations of OSI-930.
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Alvarez-Twose, Iván; Matito, Almudena; Morgado, José Mário; Sánchez-Muñoz, Laura; Jara-Acevedo, María; García-Montero, Andrés; Mayado, Andrea; Caldas, Carolina; Teodósio, Cristina; Muñoz-González, Javier Ignacio; Mollejo, Manuela; Escribano, Luis; Orfao, Alberto
2017-01-01
Resistance to imatinib has been recurrently reported in systemic mastocytosis (SM) carrying exon 17 KIT mutations. We evaluated the efficacy and safety of imatinib therapy in 10 adult SM patients lacking exon 17 KIT mutations, 9 of which fulfilled criteria for well-differentiated SM (WDSM). The World Health Organization 2008 disease categories among WDSM patients were mast cell (MC) leukemia (n = 3), indolent SM (n = 3) and cutaneous mastocytosis (n = 3); the remainder case had SM associated with a clonal haematological non-MC disease. Patients were given imatinib for 12 months −400 or 300 mg daily depending on the presence vs. absence of > 30% bone marrow (BM) MCs and/or signs of advanced disease–. Absence of exon 17 KIT mutations was confirmed in highly-purified BM MCs by peptide nucleic acid-mediated PCR, while mutations involving other exons were investigated by direct sequencing of purified BM MC DNA. Complete response (CR) was defined as resolution of BM MC infiltration, skin lesions, organomegalies and MC-mediator release-associated symptoms, plus normalization of serum tryptase. Criteria for partial response (PR) included ≥ 50% reduction in BM MC infiltration and improvement of skin lesions and/or organomegalies. Treatment was well-tolerated with an overall response rate of 50%, including early and sustained CR in four patients, three of whom had extracellular mutations of KIT, and PR in one case. This later patient and all non-responders (n = 5) showed wild-type KIT. These results together with previous data from the literature support the relevance of the KIT mutational status in selecting SM patients who are candidates for imatinib therapy. PMID:28978170
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Alvarez-Twose, Iván; Matito, Almudena; Morgado, José Mário; Sánchez-Muñoz, Laura; Jara-Acevedo, María; García-Montero, Andrés; Mayado, Andrea; Caldas, Carolina; Teodósio, Cristina; Muñoz-González, Javier Ignacio; Mollejo, Manuela; Escribano, Luis; Orfao, Alberto
2017-09-15
Resistance to imatinib has been recurrently reported in systemic mastocytosis (SM) carrying exon 17 KIT mutations. We evaluated the efficacy and safety of imatinib therapy in 10 adult SM patients lacking exon 17 KIT mutations, 9 of which fulfilled criteria for well-differentiated SM (WDSM). The World Health Organization 2008 disease categories among WDSM patients were mast cell (MC) leukemia ( n = 3), indolent SM ( n = 3) and cutaneous mastocytosis ( n = 3); the remainder case had SM associated with a clonal haematological non-MC disease. Patients were given imatinib for 12 months -400 or 300 mg daily depending on the presence vs. absence of > 30% bone marrow (BM) MCs and/or signs of advanced disease-. Absence of exon 17 KIT mutations was confirmed in highly-purified BM MCs by peptide nucleic acid-mediated PCR, while mutations involving other exons were investigated by direct sequencing of purified BM MC DNA. Complete response (CR) was defined as resolution of BM MC infiltration, skin lesions, organomegalies and MC-mediator release-associated symptoms, plus normalization of serum tryptase. Criteria for partial response (PR) included ≥ 50% reduction in BM MC infiltration and improvement of skin lesions and/or organomegalies. Treatment was well-tolerated with an overall response rate of 50%, including early and sustained CR in four patients, three of whom had extracellular mutations of KIT , and PR in one case. This later patient and all non-responders ( n = 5) showed wild-type KIT . These results together with previous data from the literature support the relevance of the KIT mutational status in selecting SM patients who are candidates for imatinib therapy.
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Langer, Christian; Radmacher, Michael D.; Ruppert, Amy S.; Whitman, Susan P.; Paschka, Peter; Mrózek, Krzysztof; Baldus, Claudia D.; Vukosavljevic, Tamara; Liu, Chang-Gong; Ross, Mary E.; Powell, Bayard L.; de la Chapelle, Albert; Kolitz, Jonathan E.; Larson, Richard A.; Marcucci, Guido
2008-01-01
BAALC expression is considered an independent prognostic factor in cytogenetically normal acute myeloid leukemia (CN-AML), but has yet to be investigated together with multiple other established prognostic molecular markers in CN-AML. We analyzed BAALC expression in 172 primary CN-AML patients younger than 60 years of age, treated similarly on CALGB protocols. High BAALC expression was associated with FLT3-ITD (P = .04), wild-type NPM1 (P < .001), mutated CEBPA (P = .003), MLL-PTD (P = .009), absent FLT3-TKD (P = .005), and high ERG expression (P = .05). In multivariable analysis, high BAALC expression independently predicted lower complete remission rates (P = .04) when adjusting for ERG expression and age, and shorter survival (P = .04) when adjusting for FLT3-ITD, NPM1, CEBPA, and white blood cell count. A gene-expression signature of 312 probe sets differentiating high from low BAALC expressers was identified. High BAALC expression was associated with overexpression of genes involved in drug resistance (MDR1) and stem cell markers (CD133, CD34, KIT). Global microRNA-expression analysis did not reveal significant differences between BAALC expression groups. However, an analysis of microRNAs that putatively target BAALC revealed a potentially interesting inverse association between expression of miR-148a and BAALC. We conclude that high BAALC expression is an independent adverse prognostic factor and is associated with a specific gene-expression profile. PMID:18378853
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ABO/Rh Blood-Typing Model: A Problem-Solving Activity
ERIC Educational Resources Information Center
Wake, Carol
2005-01-01
An ARO/Rh Blood-Typing kit useful for students to visualize blood-typing activities and practice problem-solving skills with transfusion reactions is presented. The model also enables students to identify relationships between A, B, and Rh antigens and antibodies in blood and to understand molecular mechanisms involved in transfusion agglutination…
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De Vita, Serena; Schneider, Rebekka K.; Garcia, Michael; Wood, Jenna; Gavillet, Mathilde; Ebert, Benjamin L.; Gerbaulet, Alexander; Roers, Axel; Levine, Ross L.; Mullally, Ann; Williams, David A.
2014-01-01
Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM. PMID:24788138
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Eriksson, Per; Mourkas, Evangelos; González-Acuna, Daniel; Olsen, Björn; Ellström, Patrik
2017-01-01
ABSTRACT Introduction: Advances in the development of nucleic acid-based methods have dramatically facilitated studies of host–microbial interactions. Fecal DNA analysis can provide information about the host’s microbiota and gastrointestinal pathogen burden. Numerous studies have been conducted in mammals, yet birds are less well studied. Avian fecal DNA extraction has proved challenging, partly due to the mixture of fecal and urinary excretions and the deficiency of optimized protocols. This study presents an evaluation of the performance in avian fecal DNA extraction of six commercial kits from different bird species, focusing on penguins. Material and methods: Six DNA extraction kits were first tested according to the manufacturers’ instructions using mallard feces. The kit giving the highest DNA yield was selected for further optimization and evaluation using Antarctic bird feces. Results: Penguin feces constitute a challenging sample type: most of the DNA extraction kits failed to yield acceptable amounts of DNA. The QIAamp cador Pathogen kit (Qiagen) performed the best in the initial investigation. Further optimization of the protocol resulted in good yields of high-quality DNA from seven bird species of different avian orders. Conclusion: This study presents an optimized approach to DNA extraction from challenging avian fecal samples. PMID:29152162
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Fabrication of PDMS architecture
NASA Astrophysics Data System (ADS)
Adam, Tijjani; Hashim, U.
2017-03-01
The study report novel, yet simple and flexible fabrication method for micro channel patterning PDMS thin mold on glass surfaces, the method allows microstructures with critical dimensions to be formed using PDMS. Micro channel production is a two-step process. First, soft photolithography methods are implemented to fabricate a reusable mold. The mold is then used to create the micro channel, which consists of SU8, PDMS and glass. The micro channel design was performed using AutoCAD and the fabrication begins by creating a replicable mold. The mold is created on a glass slide. by spin-coating speed between 500 to 1250rpm with an acceleration of 100 rpm/s for 100 and 15 second ramp up and down speed respectively. Channel flow rate based on concentration were measured by analyzing the recorded flow profiles which was collected from the high powered microscope at. 80µ, 70µm, 50µm for inlet channel 1, 2, 3 respectively the channel flow were compared for flow efficiency at different concentrations and Re. Thus, the simplicity of device structure and fabrication makes it feasible to miniaturize it for the development of point-of-care kits, facilitating its use in both clinical and non-clinical environments. With its simple geometric structure and potential for mass commercial fabrication, the device can be developed to become a portable photo detection sensor that can be use for both environmental and diagnostic application.
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Hersmus, Remko; Stoop, Hans; van de Geijn, Gert Jan; Eini, Ronak; Biermann, Katharina; Oosterhuis, J. Wolter; DHooge, Catharina; Schneider, Dominik T.; Meijssen, Isabelle C.; Dinjens, Winand N. M.; Dubbink, Hendrikus Jan; Drop, Stenvert L. S.; Looijenga, Leendert H. J.
2012-01-01
Activating c-KIT mutations (exons 11 and 17) are found in 10–40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent ∼3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY. PMID:22937135
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Kelly, Lorna; Bryan, Kenneth; Kim, Su Young; Janeway, Katherine A.; Killian, J. Keith; Schildhaus, Hans-Ulrich; Miettinen, Markku; Helman, Lee; Meltzer, Paul S.; van de Rijn, Matt; Debiec-Rychter, Maria; O’Sullivan, Maureen
2013-01-01
In contrast to adult mutant gastrointestinal stromal tumors [GISTs], pediatric/wild-type GISTs remain poorly understood overall, given their lack of oncogenic activating tyrosine kinase mutations. These GISTs, with a predilection for gastric origin in female patients, show limited response to therapy with tyrosine kinase inhibitors and generally pursue a more indolent course, but still may prove fatal. Defective cellular respiration appears to underpin tumor development in these wild-type cases, which as a group lack expression of succinate dehydrogenase [SDH] B, a surrogate marker for respiratory chain metabolism. Yet, only a small subset of the wild-type tumors show mutations in the genes coding for the SDH subunits [SDHx]. To explore additional pathogenetic mechanisms in these wild-type GISTs, we elected to investigate post-transcriptional regulation of these tumors by conducting microRNA (miRNA) profiling of a mixed cohort of 73 cases including 18 gastric pediatric wild-type, 25 (20 gastric, 4 small bowel and 1 retroperitoneal) adult wild-type GISTs and 30 gastric adult mutant GISTs. By this approach we have identified distinct signatures for GIST subtypes which correlate tightly with clinico-pathological parameters. A cluster of miRNAs on 14q32 show strikingly different expression patterns amongst GISTs, a finding which appears to be explained at least in part by differential allelic methylation of this imprinted region. Small bowel and retroperitoneal wild-type GISTs segregate with adult mutant GISTs and express SDHB, while adult wild-type gastric GISTs are dispersed amongst adult mutant and pediatric wild-type cases, clustering in this situation on the basis of SDHB expression. Interestingly, global methylation analysis has recently similarly demonstrated that these wild-type, SDHB-immunonegative tumors show a distinct pattern compared with KIT and PDGFRA mutant tumors, which as a rule do express SDHB. All cases with Carney triad within our cohort cluster together tightly. PMID:23717541
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Kelly, Lorna; Bryan, Kenneth; Kim, Su Young; Janeway, Katherine A; Killian, J Keith; Schildhaus, Hans-Ulrich; Miettinen, Markku; Helman, Lee; Meltzer, Paul S; van de Rijn, Matt; Debiec-Rychter, Maria; O'Sullivan, Maureen
2013-01-01
In contrast to adult mutant gastrointestinal stromal tumors [GISTs], pediatric/wild-type GISTs remain poorly understood overall, given their lack of oncogenic activating tyrosine kinase mutations. These GISTs, with a predilection for gastric origin in female patients, show limited response to therapy with tyrosine kinase inhibitors and generally pursue a more indolent course, but still may prove fatal. Defective cellular respiration appears to underpin tumor development in these wild-type cases, which as a group lack expression of succinate dehydrogenase [SDH] B, a surrogate marker for respiratory chain metabolism. Yet, only a small subset of the wild-type tumors show mutations in the genes coding for the SDH subunits [SDHx]. To explore additional pathogenetic mechanisms in these wild-type GISTs, we elected to investigate post-transcriptional regulation of these tumors by conducting microRNA (miRNA) profiling of a mixed cohort of 73 cases including 18 gastric pediatric wild-type, 25 (20 gastric, 4 small bowel and 1 retroperitoneal) adult wild-type GISTs and 30 gastric adult mutant GISTs. By this approach we have identified distinct signatures for GIST subtypes which correlate tightly with clinico-pathological parameters. A cluster of miRNAs on 14q32 show strikingly different expression patterns amongst GISTs, a finding which appears to be explained at least in part by differential allelic methylation of this imprinted region. Small bowel and retroperitoneal wild-type GISTs segregate with adult mutant GISTs and express SDHB, while adult wild-type gastric GISTs are dispersed amongst adult mutant and pediatric wild-type cases, clustering in this situation on the basis of SDHB expression. Interestingly, global methylation analysis has recently similarly demonstrated that these wild-type, SDHB-immunonegative tumors show a distinct pattern compared with KIT and PDGFRA mutant tumors, which as a rule do express SDHB. All cases with Carney triad within our cohort cluster together tightly.
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NASA Astrophysics Data System (ADS)
Gou, Jun; Niu, Qingchen; Liang, Kai; Wang, Jun; Jiang, Yadong
2018-03-01
Antenna-coupled micro-bridge structure is proven to be a good solution to extend infrared micro-bolometer technology for THz application. Spiral-type antennas are proposed in 25 μm × 25 μm micro-bridge structure with a single separate linear antenna, two separate linear antennas, or two connected linear antennas on the bridge legs, in addition to traditional spiral-type antenna on the support layer. The effects of structural parameters of each antenna on THz absorption of micro-bridge structure are discussed for optimized absorption of 2.52 THz wave radiated by far infrared CO2 lasers. The design of spiral-type antenna with two separate linear antennas for wide absorption peak and spiral-type antenna with two connected linear antennas for relatively stable absorption are good candidates for high absorption at low absorption frequency with a rotation angle of 360* n ( n = 1.6). Spiral-type antenna with extended legs also provides a highly integrated micro-bridge structure with fast response and a highly compatible, process-simplified way to realize the structure. This research demonstrates the design of several spiral-type antenna-coupled micro-bridge structures and provides preferred schemes for potential device applications in room temperature sensing and real-time imaging.
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Gou, Jun; Niu, Qingchen; Liang, Kai; Wang, Jun; Jiang, Yadong
2018-03-05
Antenna-coupled micro-bridge structure is proven to be a good solution to extend infrared micro-bolometer technology for THz application. Spiral-type antennas are proposed in 25 μm × 25 μm micro-bridge structure with a single separate linear antenna, two separate linear antennas, or two connected linear antennas on the bridge legs, in addition to traditional spiral-type antenna on the support layer. The effects of structural parameters of each antenna on THz absorption of micro-bridge structure are discussed for optimized absorption of 2.52 THz wave radiated by far infrared CO 2 lasers. The design of spiral-type antenna with two separate linear antennas for wide absorption peak and spiral-type antenna with two connected linear antennas for relatively stable absorption are good candidates for high absorption at low absorption frequency with a rotation angle of 360*n (n = 1.6). Spiral-type antenna with extended legs also provides a highly integrated micro-bridge structure with fast response and a highly compatible, process-simplified way to realize the structure. This research demonstrates the design of several spiral-type antenna-coupled micro-bridge structures and provides preferred schemes for potential device applications in room temperature sensing and real-time imaging.
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Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning
2014-01-01
The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.
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Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning
2014-01-01
The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection. PMID:24751715
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Tracking calcification in tissue-engineered bone using synchrotron micro-FTIR and SEM.
Deegan, Anthony J; Cinque, Gianfelice; Wehbe, Katia; Konduru, Sandeep; Yang, Ying
2015-02-01
One novel tissue engineering approach to mimic in vivo bone formation is the use of aggregate or micromass cultures. Various qualitative and quantitative techniques, such as histochemical staining, protein assay kits and RT-PCR, have been used previously on cellular aggregate studies to investigate how these intricate arrangements lead to mature bone tissue. However, these techniques struggle to reveal spatial and temporal distribution of proliferation and mineralization simultaneously. Synchrotron-based Fourier transform infrared microspectroscopy (micro-FTIR) offers a unique insight at the molecular scale by coupling high IR sensitivity to organic matter with the high spatial resolution allowed by diffraction limited SR microbeam. This study is set to investigate the effects of culture duration and aggregate size on the dynamics and spatial distribution of calcification in engineered bone aggregates by a combination of micro-FTIR and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX). A murine bone cell line has been used, and small/large bone aggregates have been induced using different chemically treated culture substrates. Our findings suggest that bone cell aggregate culturing can greatly increase levels of mineralization over short culture periods. The size of the aggregates influences mineralisation rates with larger aggregates mineralizing at a faster rate than their smaller counterparts. The micro-FTIR mapping has demonstrated that mineralization in the larger aggregates initiated from the periphery and spread to the centre, whilst the smaller aggregates have more minerals in the centre at the early stage and deposited more in the periphery after further culturing, implying that aggregate size influences calcification distribution and development over time. SEM/EDX data correlates well with the micro-FTIR results for the total mineral content. Thus, synchrotron-based micro-FTIR can accurately track mineralization process/mechanism in the engineered bone.
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Relationship between microRNA-146a expression and plasma renalase levels in hemodialyzed patients
Koch, Wojciech; Kukula-Koch, Wirginia; Gaweł, Kinga; Bednarek-Skublewska, Anna; Małecka-Massalska, Teresa; Milanowski, Janusz; Petkowicz, Beata; Solski, Janusz
2017-01-01
Background microRNA (miRNA) belongs to the non-coding RNAs family responsible for the regulation of gene expression. Renalase is a protein composed of 342 amino acids, secreted by the kidneys and possibly plays an important role in the regulation of sympathetic tone and blood pressure. The aim of the present study was to investigate plasma renalase concentration, and explore the relationship between miRNA-146a-5p expression and plasma renalase levels in hemodialyzed patients. Methods The study population comprised 55 subjects who succumbed to various cardiac events, 27 women and 28 men, aged 65–70 years. The total RNA including miRNA fraction was isolated using QiagenmiRNEasy Serum/Plasma kit according to the manufacturer’s protocol. The isolated miRNAs were analyzed using a quantitative polymerase chain reaction (qRT-PCR) technique. The plasma renalase levels were measured using a commercial ELISA kit. Results In the group of patients with high levels of renalase, higher miRNA-146a expression was found, compared with those with low concentration of renalase. Patients with simultaneous low miRNA-146a expression and high level of renalase were confirmed to deliver a significantly longer survival time compared with other patients. Conclusions miRNA-146a and plasma renalase levels were estimated as independent prognostic factors of hemodialyzed patients’ survival time. Patients with low miRNA-146a expression demonstrated a significantly longer survival time in contrast to the patients with a high expression level of miRNA-146a. Moreover, a significantly longer survival time was found in patients with high renalase activity compared with patients with low activity of the enzyme. PMID:28614373
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Xiao, Jinsong; Liu, Li; Zhong, Zian; Xiao, Cheng; Zhang, Junjian
2015-06-01
Mangiferin, a flavonoid extracted from the leaves of the Anacardiaceae plant, the mango tree, has physiological activity and pharmacological effects in many aspects. The present study aimed to clarify the effect of mangiferin on proliferation and apoptosis of glioma cells and the mechanism of these curative effects of mangiferin. In this experiment, we detected the proliferation using 3-(4,5-dimethylthylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Then, cell apoptosis of U87 glioma cells was measured with the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit, DAPI staining assay and the caspase-3 and caspase-9 activity assay kit. Next, quantitative real-time PCR and gelatin zymography were used to analyze the expression of microRNA-15b (miR-15b) and matrix metalloproteinase-9 (MMP-9), respectively. MMP-9 agonist, miR-15b mimics and anti-miR-15b mimics were added to the U87 glioma cells for elucidating the mechanisms involved in the curative effects of mangiferin. In the present study, mangiferin notably restrained the proliferation and increased the apoptosis of the U87 glioma cells. Meanwhile, mangiferin specifically promoted the expression of miR-15b and suppressed the level of MMP-9 in the U87 glioma cells. miR-15b regulated the expression of MMP-9 in the U87 glioma cells. MMP-9 agonist and anti-miR‑15b reduced the curative effects of mangiferin in the U87 glioma cells. In summary, mangiferin regulates proliferation and apoptosis in glioma cells by induction of miR-15b and inhibition of MMP-9 expression.
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Hellberg, Rosalee S; Martin, Keely G; Keys, Ashley L; Haney, Christopher J; Shen, Yuelian; Smiley, R Derike
2013-12-01
Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Peptide and protein biomarkers for type 1 diabetes mellitus
DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhang, Qibin; Metz, Thomas O.
A method for identifying persons with increased risk of developing type 1 diabetes mellitus, or having type I diabetes mellitus, utilizing selected biomarkers described herein either alone or in combination. The present disclosure allows for broad based, reliable, screening of large population bases. Also provided are arrays and kits that can be used to perform such methods.
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Peptide and protein biomarkers for type 1 diabetes mellitus
Zhang, Qibin; Metz, Thomas O.
2014-06-10
A method for identifying persons with increased risk of developing type 1 diabetes mellitus, or having type I diabetes mellitus, utilizing selected biomarkers described herein either alone or in combination. The present disclosure allows for broad based, reliable, screening of large population bases. Also provided are arrays and kits that can be used to perform such methods.
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A Learning Tool and Program Development for Mechatronics Design Education
NASA Astrophysics Data System (ADS)
Iribe, Masatsugu; Shirahata, Akihiro; Kita, Hiromasa; Sasashige, Yousuke; Dasai, Ryoichi
In this paper we propose a new type educational program for Mechatronics design which contributes to develop the physical sense and problem solving ability of the students who study Mechatronics design. For this program we provide a new handicraft kit of 4-wheeled car which is composed of inexpensive and commonplace parts, and the performance of the assembled 4-wheeled car is sensitive to its assembly arrangement. And then we implemented this program with the handicraft kit to the university freshmen, and verified its effectiveness, and report the results of the program.
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Haplotype data for 23 Y-chromosome markers in four U.S. population groups.
Coble, Michael D; Hill, Carolyn R; Butler, John M
2013-05-01
The PowerPlex Y23 kit contains 23 Y-chromosomal loci including all 17 of the markers in the Yfiler Y-STR kit plus six additional markers: DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643. We have typed 1032 unrelated population samples from four self-declared US groups: African Americans, Asians, Hispanics, and Western European Caucasians. An analysis of the population genetic parameters and the improvement of adding additional Y-STR markers to the dataset are described. Published by Elsevier Ireland Ltd.
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Ebenhan, Thomas; Vorster, Mariza; Marjanovic-Painter, Biljana; Wagener, Judith; Suthiram, Janine; Modiselle, Moshe; Mokaleng, Brenda; Zeevaart, Jan Rijn; Sathekge, Mike
2015-08-14
Prostate-specific membrane antigen (PSMA), a type II glycoprotein, is highly expressed in almost all prostate cancers. By playing such a universal role in the disease, PSMA provides a target for diagnostic imaging of prostate cancer using positron emission tomography/computed tomography (PET/CT). The PSMA-targeting ligand Glu-NH-CO-NH-Lys-(Ahx)-HBED-CC (DKFZ-PSMA-11) has superior imaging properties and allows for highly-specific complexation of the generator-based radioisotope Gallium-68 ((68)Ga). However, only module-based radiolabeling procedures are currently available. This study intended to develop a single vial kit solution to radiolabel buffered DKFZ-PSMA-11 with (68)Ga. A (68)Ge/(68)Ga-generator was utilized to yield (68)GaCl3 and major aspects of the kit development were assessed, such as radiolabeling performance, quality assurance, and stability. The final product was injected into patients with prostate cancer for PET/CT imaging and the kit performance was evaluated on the basis of the expected biodistribution, lesion detection, and dose optimization. Kits containing 5 nmol DKFZ-PSMA-11 showed rapid, quantitative (68)Ga-complexation and all quality measurements met the release criteria for human application. The increased precursor content did not compromise the ability of (68)Ga-DKFZ-PSMA-11 PET/CT to detect primary prostate cancer and its advanced lymphatic- and metastatic lesions. The (68)Ga-DKFZ-PSMA-11 kit is a robust, ready-to-use diagnostic agent in prostate cancer with high diagnostic performance.
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Baumann, U; Chouchakova, N; Gewecke, B; Köhl, J; Carroll, M C; Schmidt, R E; Gessner, J E
2001-07-15
We induced the passive reverse Arthus reaction to IgG immune complexes (IC) at different tissue sites in mice lacking C3 treated or not with a C5aR-specific antagonist, or in mice lacking mast cells (Kit(W)/Kit(W-v) mice), and compared the inflammatory responses with those in the corresponding wild-type mice. We confirmed that IC inflammation of skin can be mediated largely by mast cells expressing C5aR and FcgammaRIII. In addition, we provided evidence for C3-independent C5aR triggering, which may explain why the cutaneous Arthus reaction develops normally in C3(-/-) mice. Furthermore, some, but not all, of the acute changes associated with the Arthus response in the lung were significantly more intense in normal mice than in C3(-/-) or Kit(W)/Kit(W-v) mice, indicating for C3- and mast cell-dependent and -independent components. Finally, we demonstrated that C3 contributed to the elicitation of neutrophils to alveoli, which corresponded to an increased synthesis of TNF-alpha, macrophage-inflammatory protein-2, and cytokine-induced neutrophil chemoattractant. While mast cells similarly influenced alveolar polymorphonuclear leukocyte influx, the levels of these cytokines remained largely unaffected in mast cell deficiency. Together, the phenotypes of C3(-/-) mice and Kit(W)/Kit(W-v) mice suggest that complement and mast cells have distinct tissue site-specific requirements acting by apparently distinct mechanisms in the initiation of IC inflammation.
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Olofsson, Jill Katharina; Mogensen, Helle Smidt; Buchard, Anders; Børsting, Claus; Morling, Niels
2015-05-01
Recently, the Yfiler® Plus PCR Amplification Kit (Yfiler® Plus, Thermo Fisher Scientific, Waltham, MA, USA) was introduced. Yfiler® Plus amplifies 27 Y-chromosomal short tandem repeat loci (Y-STRs) and adds ten new Y-STRs to those analysed with the commonly used AmpFlSTR® Yfiler® PCR Amplification Kit (Yfiler®, Thermo Fisher Scientific, Waltham, MA, USA). Seven of the new Y-STRs are rapidly mutating Y-STRs (RM Y-STRs). In this study, 551 male individuals from Denmark, Greenland and Somalia were typed with Yfiler® Plus. The results were compared to those obtained with Yfiler® in the same individuals. Forensic and population genetic parameters were estimated for Yfiler® Plus. Yfiler® Plus had a higher power of discrimination than Yfiler® in all three populations. Compared to Yfiler®, Yfiler® Plus offers increased power of discrimination, which is obviously an advantage in crime case investigations. However, the inclusion of seven RM Y-STRs in Yfiler® Plus makes it less attractive for relationship testing because of the relatively high combined mutation rate, approximately 15%. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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Lai, Jonathan H; Fleming, Kirsten E; Ly, Thai Yen; Pasternak, Sylvia; Godlewski, Marek; Doucette, Steve; Walsh, Noreen M
2015-09-01
Merkel cell polyomavirus is of oncogenic significance in approximately 80% of Merkel cell carcinomas. Morphological subcategories of the tumor differ in regard to viral status, the rare combined type being uniformly virus negative and the predominant pure type being mainly virus positive. Indications that different biological subsets of the tumor exist led us to explore this diversity. In an Eastern Canadian cohort of cases (75 patients; mean age, 76 years [range, 43-91]; male/female ratio, 43:32; 51 [68%] pure and 24 [34%] combined tumors), we semiquantitatively compared the immunohistochemical expression of 3 cellular proteins (p53, Bcl-2, and c-kit) in pure versus combined groups. Viral status was known in a subset of cases. The significant overexpression of p53 in the combined group (mean [SD], 153.8 [117.8] versus 121.6 [77.9]; P = .01) and the increased epidermal expression of this protein (p53 patches) in the same group lend credence to a primary etiologic role for sun damage in these cases. Expression of Bcl-2 and c-kit did not differ significantly between the 2 morphological groups. A relative increase in c-kit expression was significantly associated with a virus-negative status (median [interquartile range], 100 [60-115] versus 70 [0-100]; P = .03). Emerging data reveal divergent biological pathways in Merkel cell carcinoma, each with a characteristic immunohistochemical profile. Virus-positive tumors (all pure) exhibit high retinoblastoma protein and low p53 expression, whereas virus-negative cases (few pure and all combined) show high p53 and relatively high c-kit expression. The potential biological implications of this dichotomy call for consistent stratification of these tumors in future studies. Copyright © 2015 Elsevier Inc. All rights reserved.
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An evaluation of direct PCR amplification
Hall, Daniel E.; Roy, Reena
2014-01-01
Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kalbasi, Roozbeh Javad, E-mail: rkalbasi@iaush.ac.ir; Mosaddegh, Neda
2011-11-15
Composite poly(N-vinyl-2-pyrrolidone)/KIT-5 (PVP/KIT-5) was prepared by in situ polymerization method and used as a support for palladium nanoparticles obtained through the reduction of Pd(OAc){sub 2} by hydrazine hydrate. The physical and chemical properties of the catalyst were investigated by XRD, FT-IR, UV-vis, TG, BET, SEM, and TEM techniques. The catalytic performance of this novel heterogeneous catalyst was determined for the Suzuki-Miyaura cross-coupling reaction between aryl halides and phenylboronic acid in the presence of water at room temperature. The stability of the nanocomposite catalyst was excellent and could be reused 8 times without much loss of activity in the Suzuki-Miyaura cross-couplingmore » reaction. - Graphical Abstract: Pd-poly(N-vinyl-2-pyrrolidone)/KIT-5 was prepared as an organic-inorganic hybrid catalyst for the Suzuki-Miyaura reaction. The stability of the catalyst was excellent and could be reused 8 times in the Suzuki-Miyaura reaction. Highlights: > Pd-poly(N-vinyl-2-pyrrolidone)/KIT-5 was prepared as a novel nanocomposite. > Nanocomposite was prepared based on a cage-type mesoporous system. > Catalyst showed excellent activity for Suzuki-Miyaura reaction in water. > Stability of the catalyst was excellent and could be reused 8 times.« less
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Training Aids for Online Instruction: An Analysis.
ERIC Educational Resources Information Center
Guy, Robin Frederick
This paper describes a number of different types of training aids currently employed in online training: non-interactive audiovisual presentations; interactive computer-based aids; partially interactive aids based on recorded searches; print-based materials; and kits. The advantages and disadvantages of each type of aid are noted, and a table…
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NASA Astrophysics Data System (ADS)
Tsai, Chien-Chung; Huang, Yi-Chao; Yang, Tsa-Hsien; Chen, Jen-Chieh
2006-01-01
The concentric circles type and saw-tooth type of micro grating device based upon the diffraction theory are proposed in this study. The geometry dimension of micro optical device is 200 × 200 μm2, the interval of grating is 4 μm, and the depth is 0.75 μm. The Micro Array Thermal Actuator, MATA, is applied to drive the micro grating device, and the pre-elevating structure is designed to lift the micro grating device by the residual stress of polysilicon combined with metal. The micro grating device is fabricated by Surface Micromachining for applications and research technology platform, SMart, common process. The incident ray of He-Ne laser focused by a lens which focal length is 250 mm is applied to be the light source for the experiment, and then analyzes the optical information of the outgoing ray. From the experimental results, the basic optical features are examined based upon the concentric circles type and saw-tooth type of micro grating device, respectively. The outgoing ray angle of central spot is 60° in theory. The measurements are 59.475° for the concentric circles type and 59.88° for the saw-tooth type. The outgoing ray angle of the first stripe is 46.9° in theory, and 46.81° for the concentric circles type and 46.67° for the saw-tooth type are measured from the experiment. The variation of outgoing ray angle is smaller than 1% compared the measurement results with theory of diffraction on the central spot and first stripe characteristics. The work successfully demonstrates the micro grating device with highly accurate performance by the verification of optical information. All of the efforts will be contributed to Controlled Blazed Diffraction micro grating device, CBDMG, and that will be the main device of Integrate Opto-Electronics applied on display to develop in the future.
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Szeremeta, Michał; Pepiński, Witold; Niemcunowicz-Janica, Anna; Skawrońska, Małgorzata; Sackiewicz, Adam; Ptaszyńska-Sarosiek, Iwona; Okłota, Magdalena
2010-01-01
The objective of the investigation was evaluation of visualization of human blood and saliva stains with the use of alternative light source for the purpose of genetic identification. Experimental bloodstains on the bright base were the most clearly seen in the natural light and white light, up to blood dilution of 1:600. Complete typeability of AmpFISTR SGM Plus kit profiles was obtained from bloodstains at dilution 1:1500. Partial AmpFISTR SGM Plus kit profiles were typed from bloodstains at dilutions 1:1750 and 1:2000. Experimental saliva stains on the light-colored base were completely invisible in the natural light and white light, while they were visualized at wavelength range 300-415 nm through yellow goggles, and at wavelength range 300-455 nm through orange goggles at saliva dilution 1: 600. Complete typeability of AmpFISTR SGM Plus kit loci was obtained from saliva stains at dilution 1:1750. Partial AmpFISTR SGM Plus kit profiles were typed from saliva stains at dilution 1:2000. The wavelength of 455 nm and orange goggles were the optimal set for visualization of bloodstains on various, noncontrasting materials. Other useful wavelength/combinations of goggles were CSS light/red goggles. In case of saliva, the most useful general condition for visualization of stains on various, non-contrasting materials was with the wavelength set to 300-415 nm, while wearing yellow goggles. Other useful combinations of wavelength/goggles were 300-455 nm/orange or red goggles, and also CSS light/orange or red goggles.
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Corless, Christopher L.; Harrell, Patina; Lacouture, Mario; Bainbridge, Troy; Le, Claudia; Gatter, Ken; White, Clifton; Granter, Scott; Heinrich, Michael C.
2006-01-01
Oncogenic mutations of the receptor tyrosine kinase KIT contribute to the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis (SM), and some cases of acute myelogenous leukemia (AML). The D816V substitution in the activation loop of KIT results in relative resistance to the kinase inhibitor imatinib (Gleevec). Because this mutation occurs in 80 to 95% of adult SM, its detection has diagnostic and predictive significance. Unfortunately, the fraction of mutation-positive cells in clinical SM samples is often below the 20 to 30% threshold needed for detection by direct DNA sequencing. We have developed an allele-specific polymerase chain reaction assay using a mutation-specific primer combined with a wild-type blocking oligonucleotide that amplifies D816V at the level of 1% mutant allele in DNA extracted from formalin-fixed, paraffin-embedded tissue. There were no amplifications among 64 KIT wild-type tumors and cell lines, whereas all D816V-mutant samples (eight AML and 11 mast cell disease) were positive. Other D816 substitutions associated with resistance to imatinib in vitro are rare in SM. Among these D816F was detectable with the assay whereas D816H, D816Y, and D816G did not amplify. Nine biopsies (bone marrow, skin, or colon) with suspected SM were negative by denaturing high performance liquid chromatography and/or DNA sequencing but positive by allele-specific polymerase chain reaction. Thus, the assay may be useful in confirming the diagnosis of SM. PMID:17065430
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Tanno, Toshihiko; Zhang, Peng; Lazarski, Christopher A.; Liu, Yang
2017-01-01
The efficacy of traditional chemotherapy is limited by its toxicity, especially with regard to hematopoiesis. Here we show that miR-26a plays a critical role in protecting mice against chemotherapy-induced myeloid suppression by targeting a proapoptotic protein (Bak1) in hematopoietic stem/progenitor cells (HSPCs). Because c-Kit is expressed at high levels in HSPCs, we designed a microRNA-aptamer chimera that contains miR-26a mimic and c-Kit–targeting aptamer and successfully delivered miR-26a into HSPCs to attenuate toxicity of 5′ fluorouracil (5-FU) and carboplatin. Meanwhile, our in silico analysis revealed widespread and prognosis-associated downregulation of miR-26a in advanced breast cancer and also showed that KIT is overexpressed among basal-like breast cancer cells and that such expression is associated with poor prognosis. Importantly, the miR-26a aptamer effectively repressed tumor growth in vivo and synergized with 5-FU or carboplatin in cancer therapy in the mouse breast cancer models. Thus, targeted delivery of miR-26a suppresses tumor growth while protecting the host against myelosuppression by chemotherapy. PMID:29296753
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49 CFR 173.161 - Chemical kits and first aid kits.
Code of Federal Regulations, 2010 CFR
2010-10-01
... 49 Transportation 2 2010-10-01 2010-10-01 false Chemical kits and first aid kits. 173.161 Section... Class 7 § 173.161 Chemical kits and first aid kits. (a) Chemical kits and First aid kits must conform to... 10 kg. (b) Chemical kits and First aid kits are excepted from the specification packaging...
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Lebelo, Ramokone L; Thys, Sofie; Benoy, Ina; Depuydt, Christophe E; Bogers, John-Paul; Bida, Meshack N; Mphahlele, M Jeffrey
2015-10-01
The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections. © 2015 Wiley Periodicals, Inc.
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Maity, Susmita; Nandi, Srijita; Biswas, Subrata; Sadhukhan, Salil Kumar; Saha, Malay Kumar
2012-11-26
HIV, HBV and HCV pose a major public health problem throughout the world. Detection of infection markers for these agents is a major challenge for testing laboratories in a resource poor setting. As blood transfusion is an important activity saving millions of live every year, it also carries a risk of transfusion transmissible infections caused by these fatal blood borne pathogens if the quality of testing is compromised. Conventional ELISA is regarded as the mostly used screening technique but due to limitations like high cost, unavailability in many blood banks and testing sites, involvement of costly instruments, time taking nature and requirement of highly skilled personnel for interpretation, rapid tests are gaining more importance and warrants comparison of performance. A comparative study between these two techniques has been performed using commercially available diagnostic kits to assess their efficacy for detection of HIV, HBV and HCV infections. Rapid kits were more efficient in specificity with synthetic antigens along with high PPV than ELISA in most cases. Comparison between different ELISA kits revealed that Microlisa HIV and Hepalisa (J. Mitra & Co. Pvt. Ltd.); ERBA LISA HIV1 + 2, ERBA LISA Hepatitis B and ERBA LISA HCV (Transasia Bio-medicals Ltd.) gives uniform result with good performance in terms of sensitivity, specificity, PPV, NPV and efficiency, whereas, Microlisa HCV (J. Mitra & Co. Pvt. Ltd.), Microscreen HBsAg ELISA and INNOVA HCV (Span Diagnostics Ltd.) did not perform well. Rapid kits were also having high degree of sensitivity and specificity (100%) except in HIV Comb and HCV Comb (J. Mitra & Co. Pvt. Ltd.). The kit efficiency didn't vary significantly among different companies and lots in all the cases except for HCV ELISA showing statistically significant variation (p < 0.01) among three kit types. ELISA is a good screening assay for markers of HIV, HBV and HCV infections. Rapid tests are useful for further detection of false positive samples. ELISA seems the appropriate assay in blood bank. For availability of quality commercial diagnostic assays, evaluation of kit may be helpful.
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2012-01-01
Background HIV, HBV and HCV pose a major public health problem throughout the world. Detection of infection markers for these agents is a major challenge for testing laboratories in a resource poor setting. As blood transfusion is an important activity saving millions of live every year, it also carries a risk of transfusion transmissible infections caused by these fatal blood borne pathogens if the quality of testing is compromised. Conventional ELISA is regarded as the mostly used screening technique but due to limitations like high cost, unavailability in many blood banks and testing sites, involvement of costly instruments, time taking nature and requirement of highly skilled personnel for interpretation, rapid tests are gaining more importance and warrants comparison of performance. Results A comparative study between these two techniques has been performed using commercially available diagnostic kits to assess their efficacy for detection of HIV, HBV and HCV infections. Rapid kits were more efficient in specificity with synthetic antigens along with high PPV than ELISA in most cases. Comparison between different ELISA kits revealed that Microlisa HIV and Hepalisa (J. Mitra & Co. Pvt. Ltd.); ERBA LISA HIV1 + 2, ERBA LISA Hepatitis B and ERBA LISA HCV (Transasia Bio-medicals Ltd.) gives uniform result with good performance in terms of sensitivity, specificity, PPV, NPV and efficiency, whereas, Microlisa HCV (J. Mitra & Co. Pvt. Ltd.), Microscreen HBsAg ELISA and INNOVA HCV (Span Diagnostics Ltd.) did not perform well. Rapid kits were also having high degree of sensitivity and specificity (100%) except in HIV Comb and HCV Comb (J. Mitra & Co. Pvt. Ltd.). The kit efficiency didn’t vary significantly among different companies and lots in all the cases except for HCV ELISA showing statistically significant variation (p < 0.01) among three kit types. Conclusions ELISA is a good screening assay for markers of HIV, HBV and HCV infections. Rapid tests are useful for further detection of false positive samples. ELISA seems the appropriate assay in blood bank. For availability of quality commercial diagnostic assays, evaluation of kit may be helpful. PMID:23181517
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Mutant KIT as imatinib-sensitive target in metastatic sinonasal carcinoma.
Dieter, S M; Heining, C; Agaimy, A; Huebschmann, D; Bonekamp, D; Hutter, B; Ehrenberg, K R; Fröhlich, M; Schlesner, M; Scholl, C; Schlemmer, H-P; Wolf, S; Mavratzas, A; Jung, C S; Gröschel, S; von Kalle, C; Eils, R; Brors, B; Penzel, R; Kriegsmann, M; Reuss, D E; Schirmacher, P; Stenzinger, A; Federspil, P A; Weichert, W; Glimm, H; Fröhling, S
2017-01-01
Sinonasal carcinomas (SNCs) comprise various rare tumor types that are characterized by marked histologic diversity and largely unknown molecular profiles, yet share an overall poor prognosis owing to an aggressive clinical course and frequent late-stage diagnosis. The lack of effective systemic therapies for locally advanced or metastatic SNC poses a major challenge to therapeutic decision making for individual patients. We here aimed to identify actionable genetic alterations in a patient with metastatic SNC whose tumor, despite all diagnostic efforts, could not be assigned to any known SNC category and was refractory to multimodal therapy. We used whole-exome and transcriptome sequencing to identify a KIT exon 11 mutation (c.1733_1735del, p.D579del) as potentially druggable target in this patient and carried out cancer hotspot panel sequencing to detect secondary resistance-conferring mutations in KIT. Furthermore, as a step towards clinical exploitation of the recently described signatures of mutational processes in cancer genomes, we established and applied a novel bioinformatics algorithm that enables supervised analysis of the mutational catalogs of individual tumors. Molecularly guided treatment with imatinib in analogy to the management of gastrointestinal stromal tumor (GIST) resulted in a dramatic and durable response with remission of nearly all tumor manifestations, indicating a dominant driver function of mutant KIT in this tumor. KIT dependency was further validated by a secondary KIT exon 17 mutation (c.2459_2462delATTCinsG, p.D820_S821delinsG) that was detected upon tumor progression after 10 months of imatinib treatment and provided a rationale for salvage therapy with regorafenib, which has activity against KIT exon 11/17 mutant GIST. These observations highlight the potential of unbiased genomic profiling for uncovering the vulnerabilities of individual malignancies, particularly in rare and unclassifiable tumors, and underscore that KIT exon 11 mutations represent tractable therapeutic targets across different histologies. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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Ingram, David A.; Yang, Feng-Chun; Travers, Jeffrey B.; Wenning, Mary Jo; Hiatt, Kelly; New, Sheryl; Hood, Antoinette; Shannon, Kevin; Williams, David A.; Clapp, D. Wade
2000-01-01
Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's “two hit” model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1−/− murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W41 mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras–mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W41) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types. PMID:10620616
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Ingram, D A; Yang, F C; Travers, J B; Wenning, M J; Hiatt, K; New, S; Hood, A; Shannon, K; Williams, D A; Clapp, D W
2000-01-03
Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's "two hit" model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1-/- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W(41) mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras-mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W(41)) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.
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49 CFR 173.161 - Chemical kits and first aid kits.
Code of Federal Regulations, 2012 CFR
2012-10-01
... 49 Transportation 2 2012-10-01 2012-10-01 false Chemical kits and first aid kits. 173.161 Section... Class 7 § 173.161 Chemical kits and first aid kits. (a) Applicability. Chemical kits and first aid kits... assigned to the chemical kit and first aid kit as a whole must be the most stringent packing group assigned...
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NASA Astrophysics Data System (ADS)
Zayed, M. A.; El-Rasheedy, El-Gazy A.
2012-03-01
Two simple, sensitive, cheep and reliable spectrophotometric methods are suggested for micro-determination of pseudoephedrine in its pure form and in pharmaceutical preparation (Sinofree Tablets). The first one depends on the drug reaction with inorganic sensitive reagent like molybdate anion in aqueous media via formation of ion-pair mechanism. The second one depends on the drug reaction with π-acceptor reagent like DDQ in non-aqueous media via formation of charge transfer complex. These reactions were studied under various conditions and the optimum parameters were selected. Under proper conditions the suggested procedures were successfully applied for micro-determination of pseudoephedrine in pure and in Sinofree Tablets without interference from excepients. The values of SD, RSD, recovery %, LOD, LOQ and Sandell sensitivity refer to the high accuracy and precession of the applied procedures. The results obtained were compared with the data obtained by an official method, referring to confidence and agreement with DDQ procedure results; but it referred to the more accuracy of the molybdate data. Therefore, the suggested procedures are now successfully being applied in routine analysis of this drug in its pharmaceutical formulation (Sinofree) in Saudi Arabian Pharmaceutical Company (SPIMACO) in Boridah El-Qaseem, Saudi Arabia instead of imported kits had been previously used.
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Iwaniec, Urszula T; Turner, Russell T
2013-03-01
A reciprocal association between bone marrow fat and bone mass has been reported in ovariectomized rodents, suggesting that bone marrow adipogenesis has a negative effect on bone growth and turnover balance. Mice with loss of function mutations in kit receptor (kit(W/W-v)) have no bone marrow adipocytes in tibia or lumbar vertebra. We therefore tested the hypothesis that marrow fat contributes to the development of osteopenia by comparing the skeletal response to ovariectomy (ovx) in growing wild type (WT) and bone marrow adipocyte-deficient kit(W/W-v) mice. Mice were ovx at 4 weeks of age and sacrificed 4 or 10 weeks post-surgery. Body composition was measured at necropsy by dual-energy X-ray absorptiometry. Cortical (tibia) and cancellous (tibia and lumbar vertebra) bone architecture were evaluated by microcomputed tomography. Bone marrow adipocyte size and density, osteoblast- and osteoclast-lined bone perimeters, and bone formation were determined by histomorphometry. Ovx resulted in an increase in total body fat mass at 10 weeks post-ovx in both genotypes, but the response was attenuated in the in kit(W/W-v) mice. Adipocytes were present in bone marrow of tibia and lumbar vertebra in WT mice and bone marrow adiposity increased following ovx. In contrast, marrow adipocytes were not detected in either intact or ovx kit(W/W-v) mice. However, ovx in WT and kit(W/W-v) mice resulted in statistically indistinguishable changes in cortical and cancellous bone mass, cortical and cancellous bone formation rate, and cancellous osteoblast and osteoclast-lined bone perimeters. In conclusion, our findings do not support a causal role for increased bone marrow fat as a mediator of ovx-induced osteopenia in mice. Copyright © 2012 Elsevier Inc. All rights reserved.
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Taguchi, Kazumi; Usawachintachit, Manint; Hamamoto, Shuzo; Unno, Rei; Tzou, David T; Sherer, Benjamin A; Wang, Yongmei; Okada, Atsushi; Stoller, Marshall L; Yasui, Takahiro; Chi, Thomas
2017-09-01
Endoscopic tools have provided versatile examination and treatment for kidney stone procedures. Despite endourologists researching urinary stone disease using endoscopes to collect tissue, this tissue collection method is limited. Endoscopically removed tissues are small in size, restricting the types of genome-based examination possible. We investigated a new method of renal papilla biopsy and RNA extraction to establish a genomic research methodology for kidney stone disease. We conducted a prospective multi-institutional study and collected renal papilla specimens from consecutive percutaneous nephrolithotomy and ureteroscopy (URS) cases performed for removal of upper urinary tract stones. Renal papilla tissue was extracted using ureteroscopic biopsy forceps after stone removal. RNA was extracted using two different extraction kits, and their quantity and quality were examined. Additionally, the impact of biopsy on surgical complications was compared between cases performed with and without biopsy by matched case-control analysis adjusted for age, gender, body mass index, bilaterality, and stone burden. A total of 90 biopsies from 49 patients were performed, and the median duration between specimen collection and RNA extraction was 61 days. Both univariate and multivariate analyses showed BIGopsy ® forceps usage significantly increased the total yield (p = 0.004) and quality (p = 0.001 for A260/280, p = 0.004 for A260/A230) of extracted RNA. Extraction using the RNeasy Micro Kit ® also improved A260/A230, whereas reduced RNA integrity number of extracted RNA by univariate and multivariate analyses (p = 0.002 and p < 0.001, respectively). Moreover, matched case-control study demonstrated that endoscopic renal papilla biopsy caused no significant surgical complications, including bleeding, decreased stone clearance and hematocrit, and renal dysfunction. Biopsies during URS imparted an average of 20 minutes of procedure time over nonbiopsy cases. We demonstrate a safe methodology for optimal RNA extraction of renal papilla tissue. This technique will accelerate advanced genomic studies for kidney stone formers by facilitating larger tissue yields.
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Possible Correlation of Transfusion Transmitted Diseases with Rh type and ABO Blood Group System.
Tyagi, Surabhi; Tyagi, Alok
2013-09-01
Screening of blood is mandatory for transfusion transmitted diseases and is routinely done in the blood banks. As blood is the major source transmission of hepatitis B, hepatitis C, human immunodeficiency virus & many other diseases the hazards can be minimised by effective donor selection and screening. To find out the correlation between the transfusion transmitted diseases and blood groups and the seroprevalence of HIV, HBV, HCV & syphilis among the apparently healthy human blood donors. Study, Setting & Design: This retrospective study was conducted at the blood bank of a tertiary health care teaching centre for a period of four years. All voluntary and replacement donors reporting to the blood bank were screened for HIV-1 & 2, HBsAg, HCV and Syphilis. Anti-HIV -1 & 2, HBsAg & anti - HCV was tested using the appropriate Enzyme-linked immunosorbent assay (ELISA) technique using micro-elisa kit supplied by J.Mitra & Co.Ltd. The seropositive samples were again tested on ELISA kits of RFCL &/or BIORAD for further confirmation & ruling out any false positive or false negative results. The rapid plasma reagain (RPR) test was used for estimation of syphilis infection. The data entry was carried out using Microsoft office excel worksheet and was analysed by percentage and comparison. Total of 6000 donors were screened which included voluntary and replacement donors. Seroprevalence of HIV (0.1833 %), HCV (1.28%), HBsAg (1.5833 %) and syphilis (0.4333 %) was detected. In the study done it was also noted - that the NEGATIVE blood groups were more prone to TTIs. Blood group A negative was more prone to TTIs with HIV, HBsAg and VDRL while blood group B negative was more affected by HCV. Seroprevalence of these infections shows that routine screening is a must for blood and blood product safe transfusion. Do negative blood groups predispose to TTIs? A finding which makes us think….
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Rodil-Garcia, Patricia; Arellanes-Licea, Elvira del Carmen; Montoya-Contreras, Angélica; Salazar-Olivo, Luis A.
2017-01-01
Birth weight is an early predictor for metabolic diseases and microRNAs (miRNAs) are proposed as fetal programming participants. To evaluate the use of dried blood spots (DBS) on newborn screening cards (NSC) as a source of analyzable miRNAs, we optimized a commercial protocol to recover total miRNA from normal birth weight (NBW, n = 17–20), low birth weight (LBW, n = 17–20) and high birth weight (macrosomia, n = 17–20) newborns and analyzed the relative expression of selected miRNAs by stem-loop RT-qPCR. The possible role of miRNAs on the fetal programming of metabolic diseases was explored by bioinformatic tools. The optimized extraction of RNA resulted in a 1.2-fold enrichment of miRNAs respect to the commercial kit. miR-33b and miR-375 were overexpressed in macrosomia 9.8-fold (p < 0.001) and 1.7-fold, (p < 0.05), respectively and miR-454-3p was overexpressed in both LBW and macrosomia (19.7-fold, p < 0.001 and 10.8-fold, p < 0.001, respectively), as compared to NBW. Potential target genes for these miRNAs are associated to cyclic-guanosine monophosphate (cGMP)-dependent protein kinase (PKG), mitogen-activated protein kinase (MAPK), type 2 diabetes, transforming growth factor-β (TGF-β)and Forkhead box O protein (FoxO) pathways. In summary, we improved a protocol for analyzing miRNAs from NSC and provide the first evidence that birth weight modifies the expression of miRNAs associated to adult metabolic dysfunctions. Our work suggests archived NSC are an invaluable resource in the search for fetal programming biomarkers. PMID:29182561
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Effect of micro-oxygenation and wood type on the phenolic composition and color of an aged red wine.
Sánchez-Iglesias, Montserrat; González-Sanjosé, Ma Luisa; Pérez-Magariño, Silvia; Ortega-Heras, Miriam; González-Huerta, Carlos
2009-12-23
Many studies have recently been published focused on the effects of micro-oxygenation on the quality of wines, its application modes, and doses, etc. However, there are still few scientific papers on how previously micro-oxygenated wines perform during storage or barrel aging. This study focused on the evolution of the phenolic composition, especially of anthocyanins, and color, together with astringency and tannins, during micro-oxygenation before barrel aging. In addition, to evaluate whether wine evolution during aging depends on barrel type, wines were aged in four different oak barrel types. Tempranillo wines, some micro-oxygenated before malolactic fermentation and others not, were aged for 12 months in American, French, Central European, and Spanish oak, following wine evolution during that period. The study was carried out for two consecutive vintages. Results showed that all wines evolved similarly; therefore, the micro-oxygenation treatment neither accelerated nor delayed the typical changes of aging. Slightly different evolutions were detected according to the barrel wood type, whether or not the wine was micro-oxygenated. The varied evolutions must therefore be associated with the differences from each oak type (structure, grain and density, composition, etc.).
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Code of Federal Regulations, 2010 CFR
1997-10-01
... 46 Shipping 6 1997-10-01 1997-10-01 false Contents. 160.054-4 Section 160.054-4 EQUIPMENT... Liferafts § 160.054-4 Contents. (a) Individual cartons. Cartons and their contents shall be the type...) Instructions. Instructions for the use of the contents of the first-aid kit shall be printed in legible type...
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Veiga-Lopez, Almudena; Luense, Lacey J; Christenson, Lane K; Padmanabhan, Vasantha
2013-05-01
Bisphenol-A (BPA), a ubiquitous environmental endocrine disrupting chemical, is a component of polycarbonate plastic and epoxy resins. Because of its estrogenic properties, there is increasing concern relative to risks from exposures during critical periods of early organ differentiation. Prenatal BPA treatment in sheep results in low birth weight, hypergonadotropism, and ovarian cycle disruptions. This study tested the hypothesis that gestational exposure to bisphenol A, at an environmentally relevant dose, induces early perturbations in the ovarian transcriptome (mRNA and microRNA). Pregnant Suffolk ewes were treated with bisphenol A (0.5 mg/kg, sc, daily, produced ∼2.6 ng/mL of unconjugated BPA in umbilical arterial samples of BPA treated fetuses approaching median levels of BPA measured in maternal circulation) from days 30 to 90 of gestation. Expression of steroidogenic enzymes, steroid/gonadotropin receptors, key ovarian regulators, and microRNA biogenesis components were measured by RT-PCR using RNA derived from fetal ovaries collected on gestational days 65 and 90. An age-dependent effect was evident in most steroidogenic enzymes, steroid receptors, and key ovarian regulators. Prenatal BPA increased Cyp19 and 5α-reductase expression in day 65, but not day 90, ovaries. Fetal ovarian microRNA expression was altered by prenatal BPA with 45 down-regulated (>1.5-fold) at day 65 and 11 down-regulated at day 90 of gestation. These included microRNAs targeting Sry-related high-mobility-group box (SOX) family genes, kit ligand, and insulin-related genes. The results of this study demonstrate that exposure to BPA at an environmentally relevant dose alters fetal ovarian steroidogenic gene and microRNA expression of relevance to gonadal differentiation, folliculogenesis, and insulin homeostasis.
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The Design of Distributed Micro Grid Energy Storage System
NASA Astrophysics Data System (ADS)
Liang, Ya-feng; Wang, Yan-ping
2018-03-01
Distributed micro-grid runs in island mode, the energy storage system is the core to maintain the micro-grid stable operation. For the problems that it is poor to adjust at work and easy to cause the volatility of micro-grid caused by the existing energy storage structure of fixed connection. In this paper, an array type energy storage structure is proposed, and the array type energy storage system structure and working principle are analyzed. Finally, the array type energy storage structure model is established based on MATLAB, the simulation results show that the array type energy storage system has great flexibility, which can maximize the utilization of energy storage system, guarantee the reliable operation of distributed micro-grid and achieve the function of peak clipping and valley filling.
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Friend or Foe: MicroRNAs in the p53 network.
Luo, Zhenghua; Cui, Ri; Tili, Esmerina; Croce, Carlo
2018-04-10
The critical tumor suppressor gene TP53 is either lost or mutated in more than half of human cancers. As an important transcriptional regulator, p53 modulates the expression of many microRNAs. While wild-type p53 uses microRNAs to suppress cancer development, microRNAs that are activated by gain-of-function mutant p53 confer oncogenic properties. On the other hand, the expression of p53 is tightly controlled by a fine-tune machinery including microRNAs. MicroRNAs can target the TP53 gene directly or other factors in the p53 network so that expression and function of either the wild-type or the mutant forms of p53 is downregulated. Therefore, depending on the wild-type or mutant p53 context, microRNAs contribute substantially to suppress or exacerbate tumor development. Copyright © 2018. Published by Elsevier B.V.
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Iqbal, H. Syed; Solomon, Suniti; Murugavel, K. G.; Solomon, Sunil Suhas; Balakrishnan, P.
2005-01-01
Diagnosis of human immunodeficiency virus (HIV) infection is important for patient management and prevention of new infections. The number of test kits available for the detection of HIV antibodies is unprecedented. In order to identify appropriate test kits, we evaluated a variety of commercial kits manufactured abroad as well as in India. The plasma and serum specimens (n = 264) were collected from individuals attending the Voluntary Counseling and Testing Centre at the YRG Centre for AIDS and Education. The specimens were used to evaluate six commercially available HIV test kits: Enzaids HIV 1+2, HIV-CheX, Murex HIV-1.2.0, Genscreen HIV 1/2 version 2, Vironostika HIV Uni-Form II Ag/Ab, and CombAids RS Advantage. High sensitivities and specificities (≥99%) were observed for the Enzaids, Murex, Vironostika, and CombAids assays. HIV-CheX showed the highest number of false-positive and false-negative results. The Genscreen test also gave many false positives. The study indicated that the Enzaids, Murex, and Vironostika enzyme-linked immunosorbent assay kits and the CombAids RS Advantage rapid assay could be used to achieve acceptable results for the detection of HIV antibodies. A combination of two tests is recommended to optimize the efficiency of HIV antibody testing algorithms, especially when evaluation with an HIV Western blot confirmatory test is not possible. PMID:16339066
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Treatment of mining acidic leachates with indigenous limestone, Zimapan Mexico.
Labastida, I; Armienta, M A; Lara-Castro, R H; Aguayo, A; Cruz, O; Ceniceros, N
2013-11-15
An experimental study to evaluate the potential of using indigenous limestones in a passive system to treat acid mine drainage, at a mining zone of Mexico was carried out. Chemical and mineralogical characteristics of four types of native rocks (KIT1, KIT2, KSS, QZ) showed distinct CaCO3 contents. Synthetic aqueous leachates from an old tailings impoundment had a pH of 2.18, 34 mg/L As, 705 mg/L Fetotal, and 3975 mg/L SO4(2-). To evaluate dissolution behavior of rocks, kinetic batch experiments with an acid Fe-rich solution were performed. Decaying kinetic constants adjusting H(+) concentration to a first order exponential process were: KIT1 (k = 2.89), KIT2 (k = 0.89) and KSS (k = 0.47). Infrared spectrum and XRD of precipitates showed schwertmannite formation. To determine As and heavy metals (Fe, Cd, Zn, Al) removal from the synthetic leachates, batch experiments using KIT1 were developed. Arsenic decreased from 34.00 mg/L to 0.04 mg/L, Fe and Al were totally removed, and concentrations of Zn and Cd decreased 88% and 91% respectively. Analyses by IR and SEM-EDS indicate that co-precipitation with Fe-Hydroxides formed upon leachate interaction with limestone is the main As removal process. Chamosite, identified by XRD may participate in the removal of Al, SiO2 and a fraction of Fe. Copyright © 2012 Elsevier B.V. All rights reserved.
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Kataoka, Yu; Tamaki, Yukimichi; Miyazaki, Takashi
2011-01-01
Wire-type electric discharge machining has been applied to the manufacture of endosseous titanium implants as this computer associated technique allows extremely accurate complex sample shaping with an optimal micro textured surface during the processing. Since the titanium oxide layer is sensitively altered by each processing, the authors hypothesized that this technique also up-regulates biological responses through the synergistic effects of the superficial chemistry and micro topography. To evaluate the respective in vitro cellular responses on the superficial chemistry and micro topography of titanium surface processed by wire-type electric discharge, we used titanium-coated epoxy resin replica of the surface. An oxide layer on the titanium surface processed by wire-type electric discharge activated the initial responses of osteoblastic cells through an integrin-mediated mechanism. Since the mRNA expression of ALP on those replicas was up-regulated compared to smooth titanium samples, the micro topography of a titanium surface processed by wire-type electric discharge promotes the osteogenic potential of cells. The synergistic response of the superficial chemistry and micro topography of titanium processed by wire-type electric discharge was demonstrated in this study.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Tsai, M.; Takeishi, Takashi; Geissler, E.N.
1991-07-15
The authors investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype. Recombinant rat SCF{sup 164} (rrSCF{sup 164}) induced the development of large numbers of dermal mast cells in normal mice in vivo. Many of these mast cells had features of connective tissue-type mast cells (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCF{sup 164} induced the proliferation of cloned interleukin 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mastmore » cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC> BMCMC maintained in rrSCF{sup 164} not only proliferated but also matured. These findings identify SCF as a single cytokine that can induce immature, IL-3-dependent mast cells to mature and to acquire multiple characteristics of CTMC. These findings also directly demonstrate that SCF can regulate the development of a cellular lineage expressing c-kit through effects on both proliferation and maturation.« less
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The genetics of brown coat color and white spotting in domestic yaks (Bos grunniens).
Zhang, M-Q; Xu, X; Luo, S-J
2014-10-01
Domestic yaks (Bos grunniens) exhibit two major coat color variations: a brown vs. wild-type black pigmentation and a white spotting vs. wild-type solid color pattern. The genetic basis for these variations in color and distribution remains largely unknown and may be complicated by a breeding history involving hybridization between yaks and cattle. Here, we investigated 92 domestic yaks from China using a candidate gene approach. Sequence variations in MC1R, PMEL and TYRP1 were surveyed in brown yaks; TYRP1 was unassociated with the coloration and excluded. Recessive mutations from MC1R, or p.Gln34*, p.Met73Leu and possibly p.Arg142Pro, are reported in bovids for the first time and accounted for approximately 40% of the brown yaks in this study. The remaining 60% of brown individuals correlated with a cattle-derived deletion mutation from PMEL (p.Leu18del) in a dominant manner. Degrees of white spotting found in yaks vary from color sidedness and white face, to completely white. After examining the candidate gene KIT, we suggest that color-sided and all-white yaks are caused by the serial translations of KIT (Cs6 or Cs29 ) as reported for cattle. The white-faced phenotype in yaks is associated with the KIT haplotype S(wf) . All KIT mutations underlying the serial phenotypes of white spotting in yaks are identical to those in cattle, indicating that cattle are the likely source of white spotting in yaks. Our results reveal the complex genetic origins of domestic yak coat color as either native in yaks through evolution and domestication or as introduced from cattle through interspecific hybridization. © 2014 Stichting International Foundation for Animal Genetics.
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Stangegaard, Michael; Hjort, Benjamin B; Hansen, Thomas N; Hoflund, Anders; Mogensen, Helle S; Hansen, Anders J; Morling, Niels
2013-05-01
The presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. DNA extraction from fabric for forensic genetic purposes may be challenging due to the occasional presence of PCR inhibitors that may be co-extracted with the DNA. Using 120 forensic trace evidence samples consisting of various types of fabric, we compared three automated DNA extraction methods based on magnetic beads (PrepFiler Express Forensic DNA Extraction Kit on an AutoMate Express, QIAsyphony DNA Investigator kit either with the sample pre-treatment recommended by Qiagen or an in-house optimized sample pre-treatment on a QIAsymphony SP) and one manual method (Chelex) with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable STR-profiles. A total of 480 samples were processed. The highest DNA recovery was obtained with the PrepFiler Express kit on an AutoMate Express while the lowest DNA recovery was obtained using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen. Extraction using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen resulted in the lowest percentage of PCR inhibition (0%) while extraction using manual Chelex resulted in the highest percentage of PCR inhibition (51%). The largest number of reportable STR-profiles was obtained with DNA from samples extracted with the PrepFiler Express kit (75%) while the lowest number was obtained with DNA from samples extracted using a QIAsymphony SP with the sample pre-treatment recommended by Qiagen (41%). Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
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Balbi, Carolina; Piccoli, Martina; Barile, Lucio; Papait, Andrea; Armirotti, Andrea; Principi, Elisa; Reverberi, Daniele; Pascucci, Luisa; Becherini, Pamela; Varesio, Luigi; Mogni, Massimo; Coviello, Domenico; Bandiera, Tiziano; Pozzobon, Michela; Cancedda, Ranieri; Bollini, Sveva
2017-05-01
Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT + hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O 2 versus 1% O 2 preconditioning). The capacity of the c-KIT + hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, Smn F7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT + hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340-1355. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.
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Bernheim, Alain; Toujani, Saloua; Saulnier, Patrick; Robert, Thomas; Casiraghi, Odile; Validire, Pierre; Temam, Stéphane; Menard, Philippe; Dessen, Philippe; Fouret, Pierre
2008-05-01
Adenoid cystic carcinoma (ACC) is a rare but distinctive tumor. Oligonucleotide array comparative genomic hybridization has been applied for cataloging genomic copy number alterations (CNAs) in 17 frozen salivary or bronchial tumors. Only four whole chromosome CNAs were found, and most cases had 2-4 segmental CNAs. No high level amplification was observed. There were recurrent gains at 7p15.2, 17q21-25, and 22q11-13, and recurrent losses at 1p35, 6q22-25, 8q12-13, 9p21, 12q12-13, and 17p11-13. The minimal region of gain at 7p15.2 contained the HOXA cluster. The minimal common regions of deletions contained the CDKN2A/CDKN2B, TP53, and LIMA1 tumor suppressor genes. The recurrent deletion at 8q12.3-13.1 contained no straightforward tumor suppressor gene, but the MIRN124A2 microRNA gene, whose product regulates MMP2 and CDK6. Among unique CNAs, gains harbored CCND1, KIT/PDGFRA/KDR, MDM2, and JAK2. The CNAs involving CCND1, MDM2, KIT, CDKN2A/2B, and TP53 were validated by FISH and/or multiplex ligation-dependent probe amplification. Although most tumors overexpressed cyclin D1 compared with surrounding glands, the only case to overexpress MDM2 had the corresponding CNA. In conclusion, our report suggests that ACC is characterized by a relatively low level of structural complexity. Array CGH and immunohistochemical data implicate MDM2 as the oncogene targeted at 12q15. The gain at 4q12 warrants further exploration as it contains a cluster of receptor kinase genes (KIT/PDGFRA/KDR), whose products can be responsive to specific therapies.
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Kocaagaoglu, H; Aslan, T; Gürbulak, A; Albayrak, H; Taşdemir, Z; Gumus, H
2017-05-01
Different polishing kits may have different effects on the composite resin surfaces. The aim of this study was to evaluate the surface roughness and color stability of four different composites which was applied different polishing technique. Thirty specimens were made for each composite resin group (nanohybrid, GrandioSo-GS; nanohybrid, Clearfil Majesty Esthetic-CME; hybrid, Valux Plus-VP; micro-hybrid, Ruby Comp-RC; [15 mm in diameter and 2 mm height]), with the different monomer composition and particle size from a total of 120 specimens. Each composite group was divided into three subgroups (n = 10). The first subgroup of the each composite subgroups served as control (C) and had no surface treatment. The second subgroup of the each composite resin groups was polished with finishing discs (Bisco Finishing Discs; Bisco Inc., Schaumburg, IL, USA). The third subgroup of the each composite resin was polished with polishing wheel (Enhance and PoGo, Dentsply, Konstanz, Germany). The surface roughness and the color differences measurement of the specimens were made and recorded. The data were compared using Kruskal-Wallis test, and regression analysis was used in order to examine the correlation between surface roughness and color differences of the specimens (α = 0.05). The Kruskal-Wallis test indicated significant difference among the composite resins in terms of ΔE (P < 0.05), and there was no statistically significant difference among composite resins in terms of surface roughness (P > 0.05). Result of the regression analysis indicated statistically significant correlation between Ra and ΔE values (P < 0.05, r2 = 0.74). The findings of the present study have clinical relevance in the choice of polishing kits used.
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Crilly, Richard
2014-01-01
A commercial version of a synthetic single crystal diamond detector (SCDD) in a Schottky diode configuration was recently released as the new type 60019 microDiamond detector (PTW‐Freiburg, Germany). In this study we investigate the dosimetric properties of this detector to independently confirm that findings from the developing group of the SCDDs still hold true for the commercial version of the SCDDs. We further explore if the use of the microDiamond detector can be expanded to high‐energy photon beams of up to 15 MV and to large field measurements. Measurements were performed with an Elekta Synergy linear accelerator delivering 6, 10, and 15 MV X‐rays, as well as 6, 9, 12, 15, and 20 MeV electron beams. The dependence of the microdiamond detector response on absorbed dose after connecting the detector was investigated. Furthermore, the dark current of the diamond detector was observed after irradiation. Results are compared to similar results from measurements with a diamond detector type 60003. Energy dependency was investigated, as well. Photon depth‐dose curves were measured for field sizes 3×3,10×10, and 30×30cm2. PDDs were measured with the Semiflex type 31010 detector, microLion type 31018 detector, P Diode type 60016, SRS Diode type 60018, and the microDiamond type 60019 detector (all PTW‐Freiburg). Photon profiles were measured at a depth of 10 cm. Electron depth‐dose curves normalized to the dose maximum were measured with the 14×14cm2 electron cone. PDDs were measured with a Markus chamber type 23343, an E Diode type 60017 and the microDiamond type 60019 detector (all PTW‐Freiburg). Profiles were measured with the E Diode and microDiamond at half of D90,D90,D70, and D50 depths and for electron cone sizes of 6×6cm2, 14×14cm2, and 20×20cm2. Within a tolerance of 0.5% detector response of the investigated detector was stable without any preirradiation. After preirradition with approximately 250 cGy the detector response was stable within 0.1%. A dark current after irradiation was not observed. The microDiamond detector shows no energy dependence in high energy photon or electron dosimetry. Electron PDD measurements with the E Diode and microDiamond are in good agreement. However, compared to E Diode measurements, dose values in the bremsstrahlungs region are about 0.5% lower when measured with the microDiamond detector. Markus detector measurements agree with E Diode measurements in the bremsstrahlungs region. For depths larger than dmax, depth‐dose curves of photon beams measured with the microDiamond detector are in close agreement to those measured with the microLion detector for small fields and with those measured with a Semiflex 0.125 cc ionization chamber for large fields. Differences are in the range of 0.25% and less. For profile measurements, microDiamond detector measurements agree well with microLion and P Diode measurements in the high‐dose region of the profile and the penumbra region. For areas outside the open field, P Diode measurements are about 0.5%–1.0% higher than microDiamond and microLion measurements. Thus it becomes evident that the investigated diamond detector (type 60019) is suitable for a wide range of applications in high‐energy photon and electron dosimetry and is interesting for relative, as well as absolute, dosimetry. PACS numbers: 00.06, 80.87 PMID:25493512
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Radotinib induces high cytotoxicity in c-KIT positive acute myeloid leukemia cells.
Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk
2017-06-05
Previously, we reported that radotinib, a BCR-ABL1 tyrosine kinase inhibitor, induced cytotoxicity in acute myeloid leukemia (AML) cells. However, the effects of radotinib in the subpopulation of c-KIT-positive AML cells were unclear. We observed that low-concentration radotinib had more potent cytotoxicity in c-KIT-positive cells than c-KIT-negative cells from AML patients. To address this issue, cell lines with high c-KIT expression, HEL92.1.7, and moderate c-KIT expression, H209, were selected. HEL92.1.7 cells were grouped into intermediate and high c-KIT expression populations. The cytotoxicity of radotinib against the HEL92.1.7 cell population with intermediate c-KIT expression was not different from that of the population with high c-KIT expression. When H209 cells were grouped into c-KIT expression-negative and c-KIT expression-positive populations, radotinib induced cytotoxicity in the c-KIT-positive population, but not the c-KIT-negative population. Thus, radotinib induces cytotoxicity in c-KIT-positive cells, regardless of the c-KIT expression intensity. Therefore, radotinib induces significant cytotoxicity in c-KIT-positive AML cells, suggesting that radotinib is a potential target agent for the treatment of c-KIT-positive malignancies including AML. Copyright © 2017 Elsevier B.V. All rights reserved.
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Techni-kits and Techni-kit Building Systems
NASA Technical Reports Server (NTRS)
Callender, E. D.; Hartsough, C.; Morris, R. V.; Yamamoto, Y.
1985-01-01
Techni-kits consists of theories, methods, standards and computer based tools that assist in design of information-intensive systems. Techni-kit "building system" is techni-kit that builds techni-kits.
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Toward the human cellular microRNAome.
McCall, Matthew N; Kim, Min-Sik; Adil, Mohammed; Patil, Arun H; Lu, Yin; Mitchell, Christopher J; Leal-Rojas, Pamela; Xu, Jinchong; Kumar, Manoj; Dawson, Valina L; Dawson, Ted M; Baras, Alexander S; Rosenberg, Avi Z; Arking, Dan E; Burns, Kathleen H; Pandey, Akhilesh; Halushka, Marc K
2017-10-01
MicroRNAs are short RNAs that serve as regulators of gene expression and are essential components of normal development as well as modulators of disease. MicroRNAs generally act cell-autonomously, and thus their localization to specific cell types is needed to guide our understanding of microRNA activity. Current tissue-level data have caused considerable confusion, and comprehensive cell-level data do not yet exist. Here, we establish the landscape of human cell-specific microRNA expression. This project evaluated 8 billion small RNA-seq reads from 46 primary cell types, 42 cancer or immortalized cell lines, and 26 tissues. It identified both specific and ubiquitous patterns of expression that strongly correlate with adjacent superenhancer activity. Analysis of unaligned RNA reads uncovered 207 unknown minor strand (passenger) microRNAs of known microRNA loci and 495 novel putative microRNA loci. Although cancer cell lines generally recapitulated the expression patterns of matched primary cells, their isomiR sequence families exhibited increased disorder, suggesting DROSHA- and DICER1-dependent microRNA processing variability. Cell-specific patterns of microRNA expression were used to de-convolute variable cellular composition of colon and adipose tissue samples, highlighting one use of these cell-specific microRNA expression data. Characterization of cellular microRNA expression across a wide variety of cell types provides a new understanding of this critical regulatory RNA species. © 2017 McCall et al.; Published by Cold Spring Harbor Laboratory Press.
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TCGA's Testicular Germ Cell Tumor Study - TCGA
TCGA network researchers identify molecular characteristics that classify testicular germ cell tumor types, including a separate subset of seminomas defined by KIT mutations. This provides a set of candidate biomarkers for risk stratification and potential therapeutic targeting.
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Implementing inquiry-based kits within a professional development school model
NASA Astrophysics Data System (ADS)
Jones, Mark Thomas
2005-07-01
Implementation of guided inquiry teaching for the first time carries inherent problems for science teachers. Reform efforts on inquiry-based science teaching are often unsustainable and are not sensitive to teachers' needs and abilities as professionals. Professional development schools are meant to provide a research-based partnership between a public school and a university. These collaborations can provide support for the professional development of teachers. This dissertation reports a study focused on the implementation of inquiry-based science kits within the support of one of these collaborations. The researcher describes the difficulties and successful adaptations experienced by science teachers and how a coteaching model provided support. These types of data are needed in order to develop a bottom-up, sustainable process that will allow teachers to implement inquiry-based science. A qualitative methodology with "researcher as participant" was used in this study of two science teachers during 2002--2003. These two teachers were supported by a coteaching model, which included preservice teachers for each teacher as well as a supervising professor. Data were collected from the researcher's direct observations of coteachers' practice. Data were also collected from interviews and reflective pieces from the coteachers. Triangulation of the data on each teacher's case supported the validity of the findings. Case reports were prepared from these data for each classroom teacher. These case reports were used and cross-case analysis was conducted to search for major themes and findings in the study. Major findings described the hurdles teachers encounter, examples of adaptations observed in the teachers' cases and the supportive interactions with their coteachers while implementing the inquiry-based kits. In addition, the data were used to make recommendations for future training and use of the kits and the coteaching model. Results from this study showed that the kit's guided structure of inquiry and the collaboration both affected the inservice teachers in the following ways: The coteaching model supported behavioral and material management issues caused by the implementation of the kits; collaboration with preservice teachers created a "smaller-class-size" effect, which allowed teachers to attend to a smaller number of students for cooperative learning and assessment, and the elementary inservice teachers learned pedagogical strategies and science content from collaborating with secondary preservice teachers in kit use and from the kits' curriculum. Results were used as a self-study for future training and support for implementation of inquiry-based kits.
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Chen, Xi; Chen, Zehua; Li, Zhengya; Zhao, Chen; Zeng, Yuxiao; Zou, Ting; Fu, Caiyun; Liu, Xiaoli; Xu, Haiwei; Yin, Zheng Qin
2016-12-30
Despite diverse pathogenesis, the common pathological change observed in age-related macular degeneration and in most hereditary retinal degeneration (RD) diseases is photoreceptor loss. Photoreceptor replacement by cell transplantation may be a feasible treatment for RD. The major obstacles to clinical translation of stem cell-based cell therapy in RD remain the difficulty of obtaining sufficient quantities of appropriate and safe donor cells and the poor integration of grafted stem cell-derived photoreceptors into the remaining retinal circuitry. Eye-wall c-kit + /stage-specific embryonic antigen 1 (SSEA1) - cells were isolated via fluorescence-activated cell sorting, and their self-renewal and differentiation potential were detected by immunochemistry and flow cytometry in vitro. After labeling with quantum nanocrystal dots and transplantation into the subretinal space of rd1 RD mice, differentiation and synapse formation by daughter cells of the eye-wall c-kit + /SSEA1 - cells were evaluated by immunochemistry and western blotting. Morphological changes of the inner retina of rd1 mice after cell transplantation were demonstrated by immunochemistry. Retinal function of rd1 mice that received cell grafts was tested via flash electroretinograms and the light/dark transition test. Eye-wall c-kit + /SSEA1 - cells were self-renewing and clonogenic, and they retained their proliferative potential through more than 20 passages. Additionally, eye-wall c-kit + /SSEA1 - cells were capable of differentiating into multiple retinal cell types including photoreceptors, bipolar cells, horizontal cells, amacrine cells, Müller cells, and retinal pigment epithelium cells and of transdifferentiating into smooth muscle cells and endothelial cells in vitro. The levels of synaptophysin and postsynaptic density-95 in the retinas of eye-wall c-kit + /SSEA1 - cell-transplanted rd1 mice were significantly increased at 4 weeks post transplantation. The c-kit + /SSEA1 - cells were capable of differentiating into functional photoreceptors that formed new synaptic connections with recipient retinas in rd1 mice. Transplantation also partially corrected the abnormalities of inner retina of rd1 mice. At 4 and 8 weeks post transplantation, the rd1 mice that received c-kit + /SSEA1 - cells showed significant increases in a-wave and b-wave amplitude and the percentage of time spent in the dark area. Grafted c-kit + /SSEA1 - cells restored the retinal function of rd1 mice via regulating neural plasticity and forming new graft-to-host synapses.
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NASA Astrophysics Data System (ADS)
Prukner, Vaclav; Dolezalova, Eva; Simek, Milan
2014-10-01
Highly reactive environment produced by atmospheric-pressure, non-equilibrium plasmas generated by surface dielectric barrier discharges (SDBDs) may be used for inactivation of biologically contaminated surfaces. We investigated decontamination efficiency of reactive environment produced by single/multiple surface streamer micro-discharge driven by amplitude-modulated AC power in coplanar electrode geometry on biologically contaminated surface by Escherichia coli. The discharges were fed by synthetic air with water vapor admixtures at atmospheric pressure, time of treatment was set from 10 second to 10 minutes, diameters of used SDBD electrodes (single and multiple streamer) and homogeneously contaminated disc samples were equal (25 mm), the distance between the electrode and contaminated surface was 2 mm. Both a conventional cultivation and fluorescent method LIVE/DEAD Bacterial Viability kit were applied to estimate counts of bacteria after the plasma treatment. Inactivation was effective and bacteria partly lost ability to grow and became injured and viable/active but non-cultivable (VBNC/ABNC). Work was supported by the MEYS under Project LD13010, VES13 COST CZ (COST Action MP 1101).
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Kobayashi, Masato; Kuroki, Shiori; Kurita, Sena; Miyamoto, Ryo; Tani, Hiroyuki; Tamura, Kyoichi; Bonkobara, Makoto
2017-10-01
Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re‑treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.
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Terada, Tadashi
2013-01-01
Various tumors can arise in the urinary bladder (UB); most common is urothelial carcinoma (UC). UC of the UB have many variants. Other types of carcinomas such as adenocarcinoma (AC) and small cell carcinoma (SmCC) can occur in UB carcinomas. Expression of KIT and PDGFRA has not been reported. A 66-year-old man admitted to our hospital because of hematuria. Cystoscopy revealed papillary invasive tumor and a transurethral bladder tumorectomy (TUR-BT) was performed. The TUR-BT showed UC, AC, SmCC, large cell carcinoma (LCC), and pleomorphic carcinoma (PC). The UC component showed plasmacytoid, spindle, nested, clear cell, acantholytic variants. The AC element showed tubular adenocarcinoma and signet-ring cell carcinoma (Sig). Immunohistochemically, all of these subtypes were positive for cytokeratin (CK) AE1/3, CK CAM5.2, CK34BE12, CK5, CK6, CK7, CK8, CK18, CK19, CK20, EMA, CEA, p63, CA19-9, p53 (positive 45%), MUC1, NSE, NCAM, KIT, PDGFRA, and Ki-67 (87%). They were negative for vimentin, chromogranin, synaptophysin, S100 protein, CD34, CD14, α-smooth muscle actin, CD31, caldesmon, CD138, CD45, κ-chain, λ-chain, MUC2, MUC5AC and MUC6. Mucin histochemistry revealed mucins in AC element including Sig. A molecular genetic analysis using PCR-direct sequencing method identified no mutations of KIT (exons 9, 11, 13, and 17) and PDGFRA (exons 12 and 18) genes. The carcinoma was highly aggressive and invaded into muscular layer. The nuclear grade was very high, and there were numerous lymphovascular permeations were seen. The surface showed carcinoma in situ involving von-Brunn's nests. This case shows that carcinoma of UB can show diverse differentiations into numerous histological types and variants, and can express KIT and PDGFRA. The both genes showed no mutations in the present case.
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Edwards, Lynn; Gibson, Rick; Carson, Shannon Ryan; Sampalli, Tara
2013-01-01
Healthcare is in a constant state of change and evolution driven by a multitude of complex factors and interactions. Consequently, organizations, teams and individuals in healthcare have to habitually realign their working relationships. Furthermore, research has shown that "working together" relationships fail in the absence of a defined framework. In this research, a novel framework and a tool kit for working together have been developed and evaluated. The framework has a formal process to articulate the intended purpose/outcome, clearly align the type of working relationship with the purpose and identify the barriers and facilitators to working relationships in healthcare.
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Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling
Ho, Chia Chi M.; Chhabra, Akanksha; Starkl, Philipp; Schnorr, Peter-John; Wilmes, Stephan; Moraga, Ignacio; Kwon, Hye-Sook; Gaudenzio, Nicolas; Sibilano, Riccardo; Wehrman, Tom S.; Gakovic, Milica; Sockolosky, Jonathan T.; Tiffany, Matthew R.; Ring, Aaron M.; Piehler, Jacob; Weissman, Irving L.; Galli, Stephen J.; Shizuru, Judith A.; Garcia, K. Christopher
2017-01-01
SUMMARY Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion, but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems. PMID:28283060
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Monzavi, Mona; Noumbissi, Sammy; Nowzari, Hessam
2017-04-01
Despite increased popularity of Zirconia dental implants, concerns have been raised regarding low temperature degradation (LTD) and its effect on micro-structural integrity. This study evaluated the effect of LTD on four types of Zirconia dental implants at 0, 30, and 60 years of artificial aging. The impact of aging on t-m transformation and micro crack formation was measured. Accelerated aging at 15 and 30 hours, approximating 30 and 60 years in vivo, aged 36 Zirconia dental implants: Z systems ® (A), Straumann ® (B), Ceraroot ® (C), and Zeramex ® (D). Focused ion beam-scanning electron microscopic analysis determined the micro structural features, phase transformation, and the formation of micro cracks. At 15 hours, type A implant presented with micro cracks and t-m transformation of 0.9 µm and 3.1 µm, respectively. At 30 hours, micro cracks remained shallow (1 µm). At 15 hours, type B implant presented micro cracks (0.7 µm) and grain transformation (1.2 µm). At 30 hours, these features remained superficial at 0.6 and 1.5 µm, respectively. Type C implant presented surface micro cracks of 0.3 µm at 15 hours. The depth of t-m transformation slightly increased to 1.4 µm. At 30 hours, number of micro cracks increased at the surface to an average depth of 1.5 µm. Depth of t-m transformation increased to an average of 2.5 µm. At 15 hours, micro cracks remained superficial (0.8 µm) for type D implant and depth of t-m transformation increased to 2.3 µm. At 30 hours, the depth of micro cracks increased to an average of 1.3 µm followed by increased t-m transformation to a depth of 4.1 µm. Depth of grain transformation remained within 1-4 µm from the surface. The effect of aging was minimal for all Zirconia implants. © 2016 Wiley Periodicals, Inc.
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Obata, Y; Horikawa, K; Takahashi, T; Akieda, Y; Tsujimoto, M; Fletcher, J A; Esumi, H; Nishida, T; Abe, R
2017-01-01
Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase–Akt (PI3K–Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek–Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)’s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs. PMID:28192400
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Peeters, M; Huang, C L; Vonk, L A; Lu, Z F; Bank, R A; Helder, M N; Doulabi, B Zandieh
2016-11-01
Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised.Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560-568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3. © 2016 Peeters et al.
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Peeters, M.; Huang, C. L.; Vonk, L. A.; Lu, Z. F.; Bank, R. A.; Doulabi, B. Zandieh
2016-01-01
Objectives Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Materials and Methods Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. Results No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. Conclusion For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised. Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560–568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3. PMID:27881439
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Mukai, Kaori; Karasuyama, Hajime; Kabashima, Kenji; Kubo, Masato; Galli, Stephen J
2017-05-01
There is evidence that mast cells, basophils, and IgE can contribute to immune responses to parasites; however, the relative levels of importance of these effector elements in parasite immunity are not fully understood. Previous work in Il3 -deficient and c- kit mutant Kit W / W-v mice indicated that interleukin-3 and c-Kit contribute to expulsion of the intestinal nematode Strongyloides venezuelensis during primary infection. Our findings in mast cell-deficient Kit W-sh / W-sh mice and two types of mast cell-deficient mice that have normal c- kit ("Hello Kit ty" and MasTRECK mice) confirmed prior work in Kit W / W-v mice that suggested that mast cells play an important role in S. venezuelensis egg clearance in primary infections. We also assessed a possible contribution of basophils in immune responses to S. venezuelensis By immunohistochemistry, we found that numbers of basophils and mast cells were markedly increased in the jejunal mucosa during primary infections with S. venezuelensis Studies in basophil-deficient Mcpt8 DTR mice revealed a small but significant contribution of basophils to S. venezuelensis egg clearance in primary infections. Studies in mice deficient in various components of immune responses showed that CD4 + T cells and ILC2 cells, IgG, FcRγ, and, to a lesser extent, IgE and FcεRI contribute to effective immunity in primary S. venezuelensis infections. These findings support the conclusion that the hierarchy of importance of immune effector mechanisms in primary S. venezuelensis infection is as follows: CD4 + T cells/ILC2 cells, IgG, and FcRγ>mast cells>IgE and FcεRI>basophils. In contrast, in secondary S. venezuelensis infection, our evidence indicates that the presence of CD4 + T cells is of critical importance but mast cells, antibodies, and basophils have few or no nonredundant roles. Copyright © 2017 American Society for Microbiology.
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Cardiac c-Kit Biology Revealed by Inducible Transgenesis.
Gude, Natalie A; Firouzi, Fareheh; Broughton, Kathleen M; Ilves, Kelli; Nguyen, Kristine P; Payne, Christina R; Sacchi, Veronica; Monsanto, Megan M; Casillas, Alexandria R; Khalafalla, Farid G; Wang, Bingyan J; Ebeid, David E; Alvarez, Roberto; Dembitsky, Walter P; Bailey, Barbara A; van Berlo, Jop; Sussman, Mark A
2018-06-22
Biological significance of c-Kit as a cardiac stem cell marker and role(s) of c-Kit+ cells in myocardial development or response to pathological injury remain unresolved because of varied and discrepant findings. Alternative experimental models are required to contextualize and reconcile discordant published observations of cardiac c-Kit myocardial biology and provide meaningful insights regarding clinical relevance of c-Kit signaling for translational cell therapy. The main objectives of this study are as follows: demonstrating c-Kit myocardial biology through combined studies of both human and murine cardiac cells; advancing understanding of c-Kit myocardial biology through creation and characterization of a novel, inducible transgenic c-Kit reporter mouse model that overcomes limitations inherent to knock-in reporter models; and providing perspective to reconcile disparate viewpoints on c-Kit biology in the myocardium. In vitro studies confirm a critical role for c-Kit signaling in both cardiomyocytes and cardiac stem cells. Activation of c-Kit receptor promotes cell survival and proliferation in stem cells and cardiomyocytes of either human or murine origin. For creation of the mouse model, the cloned mouse c-Kit promoter drives Histone2B-EGFP (enhanced green fluorescent protein; H2BEGFP) expression in a doxycycline-inducible transgenic reporter line. The combination of c-Kit transgenesis coupled to H2BEGFP readout provides sensitive, specific, inducible, and persistent tracking of c-Kit promoter activation. Tagging efficiency for EGFP+/c-Kit+ cells is similar between our transgenic versus a c-Kit knock-in mouse line, but frequency of c-Kit+ cells in cardiac tissue from the knock-in model is 55% lower than that from our transgenic line. The c-Kit transgenic reporter model reveals intimate association of c-Kit expression with adult myocardial biology. Both cardiac stem cells and a subpopulation of cardiomyocytes express c-Kit in uninjured adult heart, upregulating c-Kit expression in response to pathological stress. c-Kit myocardial biology is more complex and varied than previously appreciated or documented, demonstrating validity in multiple points of coexisting yet heretofore seemingly irreconcilable published findings. © 2018 American Heart Association, Inc.
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Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy.
Moen, Aina E F; Tannæs, Tone M; Vatn, Simen; Ricanek, Petr; Vatn, Morten Harald; Jahnsen, Jørgen
2016-06-28
Nucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Various considerations must be taken to lyse the microbial cell wall of each microbe. In the present article, we compare several tissue lysis steps and commercial purification kits, to achieve a joint RNA and DNA purification protocol for the purpose of investigating the microbiota and the microbiota-host interactions in a single colonic mucosal tissue sample. A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA. The protocol also offers an opportunity to investigate other members of a microbiota such as viruses, fungi and micro-eukaryotes, and moreover the possibility to extract data on microbiota and host interactions from one single mucosal biopsy.
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David, Victor A.; Menotti-Raymond, Marilyn; Wallace, Andrea Coots; Roelke, Melody; Kehler, James; Leighty, Robert; Eizirik, Eduardo; Hannah, Steven S.; Nelson, George; Schäffer, Alejandro A.; Connelly, Catherine J.; O’Brien, Stephen J.; Ryugo, David K.
2014-01-01
The Dominant White locus (W) in the domestic cat demonstrates pleiotropic effects exhibiting complete penetrance for absence of coat pigmentation and incomplete penetrance for deafness and iris hypopigmentation. We performed linkage analysis using a pedigree segregating White to identify KIT (Chr. B1) as the feline W locus. Segregation and sequence analysis of the KIT gene in two pedigrees (P1 and P2) revealed the remarkable retrotransposition and evolution of a feline endogenous retrovirus (FERV1) as responsible for two distinct phenotypes of the W locus, Dominant White, and white spotting. A full-length (7125 bp) FERV1 element is associated with white spotting, whereas a FERV1 long terminal repeat (LTR) is associated with all Dominant White individuals. For purposes of statistical analysis, the alternatives of wild-type sequence, FERV1 element, and LTR-only define a triallelic marker. Taking into account pedigree relationships, deafness is genetically linked and associated with this marker; estimated P values for association are in the range of 0.007 to 0.10. The retrotransposition interrupts a DNAase I hypersensitive site in KIT intron 1 that is highly conserved across mammals and was previously demonstrated to regulate temporal and tissue-specific expression of KIT in murine hematopoietic and melanocytic cells. A large-population genetic survey of cats (n = 270), representing 30 cat breeds, supports our findings and demonstrates statistical significance of the FERV1 LTR and full-length element with Dominant White/blue iris (P < 0.0001) and white spotting (P < 0.0001), respectively. PMID:25085922
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David, Victor A; Menotti-Raymond, Marilyn; Wallace, Andrea Coots; Roelke, Melody; Kehler, James; Leighty, Robert; Eizirik, Eduardo; Hannah, Steven S; Nelson, George; Schäffer, Alejandro A; Connelly, Catherine J; O'Brien, Stephen J; Ryugo, David K
2014-08-01
The Dominant White locus (W) in the domestic cat demonstrates pleiotropic effects exhibiting complete penetrance for absence of coat pigmentation and incomplete penetrance for deafness and iris hypopigmentation. We performed linkage analysis using a pedigree segregating White to identify KIT (Chr. B1) as the feline W locus. Segregation and sequence analysis of the KIT gene in two pedigrees (P1 and P2) revealed the remarkable retrotransposition and evolution of a feline endogenous retrovirus (FERV1) as responsible for two distinct phenotypes of the W locus, Dominant White, and white spotting. A full-length (7125 bp) FERV1 element is associated with white spotting, whereas a FERV1 long terminal repeat (LTR) is associated with all Dominant White individuals. For purposes of statistical analysis, the alternatives of wild-type sequence, FERV1 element, and LTR-only define a triallelic marker. Taking into account pedigree relationships, deafness is genetically linked and associated with this marker; estimated P values for association are in the range of 0.007 to 0.10. The retrotransposition interrupts a DNAase I hypersensitive site in KIT intron 1 that is highly conserved across mammals and was previously demonstrated to regulate temporal and tissue-specific expression of KIT in murine hematopoietic and melanocytic cells. A large-population genetic survey of cats (n = 270), representing 30 cat breeds, supports our findings and demonstrates statistical significance of the FERV1 LTR and full-length element with Dominant White/blue iris (P < 0.0001) and white spotting (P < 0.0001), respectively. Copyright © 2014 David et al.
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Federal Register 2010, 2011, 2012, 2013, 2014
2010-12-28
..., CO 80027. mountings, fittings, and other machined metal components for aerospace applications. Foam.... custom packaging kits, gaskets, seals, sheets, blocks, etc., of all types of foam materials. Gulf Fish...
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78 FR 9789 - Airworthiness Directives; Schweizer Aircraft Corporation
Federal Register 2010, 2011, 2012, 2013, 2014
2013-02-12
... Certification Office, Engine & Propeller Directorate, 1600 Stewart Ave., suite 410, Westbury, NY 11590... inspection panel kit and stabilizer mount doublers. The Type Certificate for these helicopters transferred...: Stephen Kowalski, Aviation Safety Engineer, New York Aircraft Certification Office, Engine & Propeller...
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CDC Vital Signs: Making Health Care Safer -- Think Sepsis. Time Matters.
... Press Kit Read the MMWR Science Clips Making Health Care Safer Think sepsis. Time matters. Language: English (US) ... the antibiotic type, dose, and duration are correct. Health care facility CEOs/administrators can Make infection control a ...
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[Trial manufacture of a plunger shield for a disposable plastic syringe].
Murakami, Shigeki; Emoto, Takashi; Mori, Hiroshige; Fujita, Katsuhisa; Kubo, Naoki
2008-08-20
A syringe-type radiopharmaceutical being supplied by a manufacturer has a syringe shield and a plunger shield, whereas an in-hospital labeling radiopharmaceutical is administered by a disposable plastic syringe without the plunger shield. In cooperation with Nihon Medi-Physics Co. Ltd., we have produced a new experimental plunger shield for the disposable plastic syringe. In order to evaluate this shielding effect, we compared the leaked radiation doses of our plunger shield with those of the syringe-type radiopharmaceutical (Medi shield type). Our plunger shield has a lead plate of 21 mm in diameter and 3 mm thick. This shield is equipped with the plunger-end of a disposal plastic syringe. We sealed 99mTc solution into a plastic syringe (Terumo Co.) of 5 ml with our plunger shield and Medi shield type of 2 ml. We measured leaked radiation doses around syringes using fluorescent glass dosimeters (Dose Ace). The number of measure points was 18. The measured doses were converted to 70 microm dose equivalent at 740 MBq of radioactivity. The results of our plunger shield and the Medi shield type were as follows: 4-13 microSv/h and 3-14 microSv/h at shielding areas, 3-545 microSv/h and 6-97 microSv/h at non-shielding areas, 42-116 microSv/h and 88-165 microSv/h in the vicinity of the syringe shield, and 1071 microSv/h and 1243 microSv/h at the front of the needle. For dose rates of shielding areas around the syringe, the shielding effects were approximately the same as those of the Medi shield type. In conclusion, our plunger shield may be useful for reducing finger exposure during the injection of an in-hospital labeled radiopharmaceutical.
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MicroRNAs as Key Effectors in the p53 Network.
Goeman, Frauke; Strano, Sabrina; Blandino, Giovanni
2017-01-01
The guardian of the genome p53 is embedded in a fine-spun network of MicroRNAs. p53 is able to activate or repress directly the transcription of MicroRNAs that are participating in the tumor-suppressive mission of p53. On the other hand, the expression of p53 is under tight control of MicroRNAs that are either targeting directly p53 or factors that are modifying its protein level or activity. Although the most important function of p53 is suggested to be transcriptional regulation, there are several nontranscriptional functions described. One of those regards the modulation of MicroRNA biogenesis. Wild-type p53 is increasing the maturation of selected MicroRNAs from the primary transcript to the precursor MiRNA by interacting with the Microprocessor complex. Furthermore, p53 is modulating the mRNA accessibility for certain MicroRNAs by association with the RISC complex and transcriptional regulation of RNA-binding proteins. In this way p53 is able to remodel the MiRNA-mRNA interaction network. As wild-type p53 is employing MicroRNAs to suppress cancer development, gain-of-function mutant p53 proteins use MicroRNAs to confer oncogenic properties like chemoresistance and the ability to drive metastasis. Like its wild-type counterpart mutant p53 is able to regulate MicroRNAs transcriptionally and posttranscriptionally. Mutant p53 affects the MiRNA processing at two cleavage steps through interfering with the Microprocessor complex and by downregulating Dicer and KSRP, a modulator of MiRNA biogenesis. Thus, MicroRNAs are essential components in the p53 pathway, contributing substantially to combat or enhance tumor development depending on the wild-type or mutant p53 context. © 2017 Elsevier Inc. All rights reserved.
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Zayed, M A; El-Rasheedy, El-Gazy A
2012-03-01
Two simple, sensitive, cheep and reliable spectrophotometric methods are suggested for micro-determination of pseudoephedrine in its pure form and in pharmaceutical preparation (Sinofree Tablets). The first one depends on the drug reaction with inorganic sensitive reagent like molybdate anion in aqueous media via formation of ion-pair mechanism. The second one depends on the drug reaction with π-acceptor reagent like DDQ in non-aqueous media via formation of charge transfer complex. These reactions were studied under various conditions and the optimum parameters were selected. Under proper conditions the suggested procedures were successfully applied for micro-determination of pseudoephedrine in pure and in Sinofree Tablets without interference from excepients. The values of SD, RSD, recovery %, LOD, LOQ and Sandell sensitivity refer to the high accuracy and precession of the applied procedures. The results obtained were compared with the data obtained by an official method, referring to confidence and agreement with DDQ procedure results; but it referred to the more accuracy of the molybdate data. Therefore, the suggested procedures are now successfully being applied in routine analysis of this drug in its pharmaceutical formulation (Sinofree) in Saudi Arabian Pharmaceutical Company (SPIMACO) in Boridah El-Qaseem, Saudi Arabia instead of imported kits had been previously used. Copyright © 2011 Elsevier B.V. All rights reserved.
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Loayza, María Fernanda; Villavicencio, Fernando Xavier; Santander, Stephanie Carolina; Baldeón, Manuel; Ponce, Lourdes Karina; Salvador, Iván; Vivar Díaz, Nicolás
2015-01-01
To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. •Use of at least 25 cut sections of 10-20 μm of diameter and 3 μm thick with more than 10 bacteria in each cut.•Lysis with 30 μL of tissue lysis buffer and 20 μL of proteinase K (PK) with the tube in an upside-down position.•The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.
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Mohammadi, M; Talebkhan, Y; Khalili, G; Mahboudi, F; Massarrat, S; Zamaninia, L; Oghalaei, A
2008-01-01
To evaluate a home-made ELISA kit for detection of Helicobacter pylori (Hp) infection and comparison of its immunologic criteria with those of foreign commercial kits. A home-made IgG ELISA kit was developed using soluble antigenic fractions of Hp proteins. Confirmed sera were tested and serological criteria were evaluated through assessment of 199 serum samples. The accuracy, sensitivity and specificity values of home-made kit were 92, 92 and 90.4%, respectively. These immunologic criteria for Trinity kit were 95.2, 95.2 and 95% in comparison with IBL kit (91.3, 92.2 and 88.5%), BIOHIT kit (72.4, 41.6 and 94.1%) and HelicoBlot2.1 (94.2, 93.4 and 100%). Kappa agreement assessment demonstrated that two of the imported ELISA kits had fair to moderate agreement with the home-made kit while the other one had a poor agreement value. Apart from comparable values between the home-made kit and the most efficient imported kit (Trinity) there was significant cost benefit. Therefore, we recommend the home-made kit as a suitable substitution for detection of Hp infection in the Iranian population.
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Evaluation of the kinase domain of c-KIT in canine cutaneous mast cell tumors
Webster, Joshua D; Kiupel, Matti; Yuzbasiyan-Gurkan, Vilma
2006-01-01
Background Mutations in the c-KIT proto-oncogene have been implicated in the progression of several neoplastic diseases, including gastrointestinal stromal tumors and mastocytosis in humans, and cutaneous mast cell tumors (MCTs) in canines. Mutations in human mastocytosis patients primarily occur in c-KIT exon 17, which encodes a portion of its kinase domain. In contrast, deletions and internal tandem duplication (ITD) mutations are found in the juxtamembrane domain of c-KIT in approximately 15% of canine MCTs. In addition, ITD c-KIT mutations are significantly associated with aberrant KIT protein localization in canine MCTs. However, some canine MCTs have aberrant KIT localization but lack ITD c-KIT mutations, suggesting that other mutations or other factors may be responsible for aberrant KIT localization in these tumors. Methods In order to characterize the prevalence of mutations in the phospho-transferase portion of c-KIT's kinase domain in canine MCTs exons 16–20 of 33 canine MCTs from 33 dogs were amplified and sequenced. Additionally, in order to determine if mutations in c-KIT exon 17 are responsible for aberrant KIT localization in MCTs that lack juxtamembrane domain c-KIT mutations, c-KIT exon 17 was amplified and sequenced from 18 canine MCTs that showed an aberrant KIT localization pattern but did not have ITD c-KIT mutations. Results No mutations or polymorphisms were identified in exons 16–20 of any of the MCTs examined. Conclusion In conclusion, mutations in the phospho-transferase portion of c-KIT's kinase domain do not play an important role in the progression of canine cutaneous MCTs, or in the aberrant localization of KIT in canine MCTs. PMID:16579858
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Vicinanza, Carla; Aquila, Iolanda; Scalise, Mariangela; Cristiano, Francesca; Marino, Fabiola; Cianflone, Eleonora; Mancuso, Teresa; Marotta, Pina; Sacco, Walter; Lewis, Fiona C; Couch, Liam; Shone, Victoria; Gritti, Giulia; Torella, Annalaura; Smith, Andrew J; Terracciano, Cesare Mn; Britti, Domenico; Veltri, Pierangelo; Indolfi, Ciro; Nadal-Ginard, Bernardo; Ellison-Hughes, Georgina M; Torella, Daniele
2017-12-01
Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit pos ) cells. The adult heart indeed contains a heterogeneous mixture of c-kit pos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit pos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit pos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit pos sorting. The blood/endothelial lineage-committed (Lineage pos ) CD45 pos c-kit pos cardiac cells were compared to CD45 neg (Lineage neg /Lin neg ) c-kit pos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit pos cardiac cells are blood/endothelial lineage-committed CD45 pos CD31 pos c-kit pos cells. In contrast, the Lin neg CD45 neg c-kit pos cardiac cell cohort, which represents ⩽10% of the total c-kit pos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit neg and the blood/endothelial lineage-committed c-kit pos cardiac cells. Single Lin neg c-kit pos cell-derived clones, which represent only 1-2% of total c-kit pos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Lin neg c-kit pos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kit pos cardiac cells were injected. Thus, among the cardiac c-kit pos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.
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Micro-Raman spectroscopy for meat type detection
NASA Astrophysics Data System (ADS)
De Biasio, M.; Stampfer, P.; Leitner, R.; Huck, C. W.; Wiedemair, V.; Balthasar, D.
2015-06-01
The recent horse meat scandal in Europe increased the demand for optical sensors that can identify meat type. Micro-Raman spectroscopy is a promising technique for the discrimination of meat types. Here, we present micro-Raman measurements of chicken, pork, turkey, mutton, beef and horse meat test samples. The data was analyzed with different combinations of data normalization and classification approaches. Our results show that Raman spectroscopy can discriminate between different meat types. Red and white meat are easily discriminated, however a sophisticated chemometric model is required to discriminate species within these groups.
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Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.
Chen, Zhongming; Zhu, Wuqiang; Bender, Ingrid; Gong, Wuming; Kwak, Il-Youp; Yellamilli, Amritha; Hodges, Thomas J; Nemoto, Natsumi; Zhang, Jianyi; Garry, Daniel J; van Berlo, Jop H
2017-12-12
Although cardiac c-kit + cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit + cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit + cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit + cells. We used single-cell sequencing and genetic lineage tracing of c-kit + cells to determine whether various pathological stimuli would result in different fates of c-kit + cells. Single-cell sequencing of cardiac CD45 - c-kit + cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit + cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit + cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. These results demonstrate that different pathological stimuli induce different cell fates of c-kit + cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit + cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit + cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit + cells. © 2017 American Heart Association, Inc.
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Hayashi, Yujiro; Asuzu, David T.; Gibbons, Simon J.; Aarsvold, Kirsten H.; Bardsley, Michael R.; Lomberk, Gwen A.; Mathison, Angela J.; Kendrick, Michael L.; Shen, K. Robert; Taguchi, Takahiro; Gupta, Anu; Rubin, Brian P.; Fletcher, Jonathan A.; Farrugia, Gianrico; Urrutia, Raul A.; Ordog, Tamas
2013-01-01
Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes. PMID:24116170
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Yin, Dan-Dan; Luo, Jun-Hui; Zhao, Zhu-Ye; Liao, Ying-Jun; Li, Ying
2018-05-01
Renal interstitial fibrosis is a final pathway that is observed in various types of kidney diseases, including diabetic kidney disease (DKD). The present study investigated the effect of tranilast on renal interstitial fibrosis and the association between its role and mast cell infiltration in a rat model of DKD. A total of 30 healthy 6‑week‑old male Sprague‑Dawley rats were randomly divided into the following four groups: Normal control group; DKD model group; low‑dose tranilast group (200 mg/kg/day); and high‑dose tranilast group (400 mg/kg/day). The morphological alterations of tubulointerstitial fibrosis were evaluated by Masson's trichrome staining, while mast cell infiltration into the renal tubular interstitium was measured by toluidine blue staining and complement C3a receptor 1 (C3aR) immunohistochemical staining (IHC). The expression of fibronectin (FN), collagen I (Col‑I), stem cell factor (SCF) and proto‑oncogene c‑kit (c‑kit) was detected by IHC, western blotting and reverse transcription‑quantitative‑polymerase chain reaction. The results demonstrated that tubulointerstitial fibrosis and mast cell infiltration were observed in DKD model rats, and this was improved dose‑dependently in the tranilast treatment groups. The expression of FN, Col‑I, SCF and c‑kit mRNA and protein was upregulated in the tubulointerstitium of DKD model rats compared with the normal control rats, and tranilast inhibited the upregulated expression of these markers. Furthermore, the degree of SCF and c‑kit expression demonstrated a significant positive correlation with C3aR‑positive mast cells and the markers of renal interstitial fibrosis. The results of the present study indicate that mast cell infiltration may promote renal interstitial fibrosis via the SCF/c‑kit signaling pathway. Tranilast may prevent renal interstitial fibrosis through inhibition of mast cell infiltration mediated through the SCF/c-kit signaling pathway.
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Da Silva Figueiredo Celestino Gomes, Priscila; Panel, Nicolas; Laine, Elodie; Pascutti, Pedro Geraldo; Solary, Eric; Tchertanov, Luba
2014-01-01
The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind. PMID:24828813
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Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells
Abbaspour Babaei, Maryam; Kamalidehghan, Behnam; Saleem, Mohammad; Huri, Hasniza Zaman; Ahmadipour, Fatemeh
2016-01-01
c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c-Kit for future cancer therapy. In addition, it has c-Kit inhibitor drug properties and their functions have been listed in tables and demonstrated in schematic pictures. This review also has collected previous studies that targeted c-Kit as a novel strategy for cancer therapy. This paper further emphasizes the advantages of this approach, as well as the limitations that must be addressed in the future. Finally, although c-Kit is an attractive target for cancer therapy, based on the outcomes of treatment of patients with c-Kit inhibitors, it is unlikely that Kit inhibitors alone can lead to cure. It seems that c-Kit mutations alone are not sufficient for tumorogenesis, but do play a crucial role in cancer occurrence. PMID:27536065
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Cantilever-type Thermal Microactuators Fabricated by SOI-MUMPs with U-type and I-type Configurations
NASA Astrophysics Data System (ADS)
Osada, Takahiro; Ochiai, Kuniyuki; Osada, Kazuki; Muro, Hideo
Recently, the micro fluid systems have been extensively studied, where microactuators such as micro valves fabricated by MEMS technology are essential for realizing these systems. In this paper thermal microactuators with U-type and I-type shapes fabricated by SOI-MUMPs technology have been investigated for optimizing their configurations.
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Vivas, J.; Sáa, A. I.; Tinajas, A.; Barbeyto, L.; Rodríguez, L. A.
2000-01-01
This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Negative type 1S panels with conventional biochemical methods for identifying 85 environmental, clinical, and reference strains of eight Aeromonas species. PMID:10742279
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Kozlowski, Maya; Larose, Louise; Lee, Fai; Le, Duc Mingh; Rottapel, Robert; Siminovitch, Katherine A.
1998-01-01
The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other than SHP-1 may also negatively regulate the coupling of c-Kit engagement to proliferation. PMID:9528781
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Baker, Erin J.; Kellogg, Christina A.
2014-01-01
Coral microbiology is an expanding field, yet there is no standard DNA extraction protocol. Although many researchers depend on commercial extraction kits, no specific kit has been optimized for use with coral samples. Both soil and plant DNA extraction kits from MO BIO Laboratories, Inc., have been used by many research groups for this purpose. MO BIO recently replaced their PowerPlant® kit with an improved PowerPlantPro kit, but it was unclear how these changes would affect the kit’s use with coral samples. In order to determine which kit produced the best results, we conducted a comparison between the original PowerPlant kit, the new PowerPlantPro kit, and an alternative kit, PowerSoil, using samples from several different coral genera. The PowerPlantPro kit had the highest DNA yields, but the lack of 16S rRNA gene amplification in many samples suggests that much of the yield may be coral DNA rather than microbial DNA. The most consistent positive amplifications came from the PowerSoil kit.
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Electronic system for the complex measurement of a Wilberforce pendulum
NASA Astrophysics Data System (ADS)
Kos, B.; Grodzicki, M.; Wasielewski, R.
2018-05-01
The authors present a novel application of a micro-electro-mechanical measurement system to the description of basic physical phenomena in a model Wilberforce pendulum. The composition of the kit includes a tripod with a mounted spring with freely hanging bob, a module GY-521 on the MPU 6050 coupled with an Arduino Uno, which in conjunction with a PC acts as measuring set. The system allows one to observe the swing of the pendulum in real time. Obtained data stays in good agreement with both theoretical predictions and previous works. The aim of this article is to introduce the study of a Wilberforce pendulum to the canon of physical laboratory exercises due to its interesting properties and multifaceted method of measurement.
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de Beer, Jessica L.; Kremer, Kristin; Ködmön, Csaba; Supply, Philip
2012-01-01
Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future. PMID:22170917
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Shi, Xiarong; Sousa, Leiliane P.; Mandel-Bausch, Elizabeth M.; Tome, Francisco; Reshetnyak, Andrey V.; Hadari, Yaron; Schlessinger, Joseph; Lax, Irit
2016-01-01
Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants. PMID:27482095
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Megakaryocytes compensate for Kit insufficiency in murine arthritis.
Cunin, Pierre; Penke, Loka R; Thon, Jonathan N; Monach, Paul A; Jones, Tatiana; Chang, Margaret H; Chen, Mary M; Melki, Imene; Lacroix, Steve; Iwakura, Yoichiro; Ware, Jerry; Gurish, Michael F; Italiano, Joseph E; Boilard, Eric; Nigrovic, Peter A
2017-05-01
The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell-deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1-dependent manner. Transfer of WT but not IL-1-deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1-driven systemic inflammatory disease.
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Ytterbium-doped fibre laser Q-switched by a cantilever-type micro-mirror.
Fabert, Marc; Desfarges-Berthelemot, Agnès; Kermène, Vincent; Crunteanu, Aurelian; Bouyge, David; Blondy, Pierre
2008-12-22
We present an Ytterbium fibre laser operating in the Q-switch regime by using a Micro- Opto- Electro- Mechanical System (MOEMS) of novel design. The cantilever-type micro-mirror is designed to generate short laser pulses with duration between 20 ns and 100 ns at repetition rates ranging from a few kilohertz up to 800 kHz. The bent profile of this new type of MOEMS ensures a high modulation rate of the laser cavity losses while keeping a high actuating frequency.
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Fabrication of piezoelectric ceramic micro-actuator and its reliability for hard disk drives.
Jing, Yang; Luo, Jianbin; Yang, Wenyan; Ju, Guoxian
2004-11-01
A new U-type micro-actuator for precisely positioning a magnetic head in high-density hard disk drives was proposed and developed. The micro-actuator is composed of a U-type stainless steel substrate and two piezoelectric ceramic elements. Using a high-d31 piezoelectric coefficient PMN-PZT ceramic plate and adopting reactive ion etching process fabricate the piezoelectric elements. Reliability against temperature was investigated to ensure the practical application to the drive products. The U-type substrate attached to each side via piezoelectric elements also was simulated by the finite-element method and practically measured by a laser Doppler vibrometer in order to testify the driving mechanics of it. The micro-actuator coupled with two piezoelectric elements featured large displacement of 0.875 microm and high-resonance frequency over 22 kHz. The novel piezoelectric micro-actuators then possess a useful compromise performance to displacement, resonance frequency, and generative force. The results reveal that the new design concept provides a valuable alternative for multilayer piezoelectric micro-actuators.
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Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.
Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T
1991-09-01
Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.
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Forensic genetic SNP typing of low-template DNA and highly degraded DNA from crime case samples.
Børsting, Claus; Mogensen, Helle Smidt; Morling, Niels
2013-05-01
Heterozygote imbalances leading to allele drop-outs and disproportionally large stutters leading to allele drop-ins are known stochastic phenomena related to STR typing of low-template DNA (LtDNA). The large stutters and the many drop-ins in typical STR stutter positions are artifacts from the PCR amplification of tandem repeats. These artifacts may be avoided by typing bi-allelic markers instead of STRs. In this work, the SNPforID multiplex assay was used to type LtDNA. A sensitized SNP typing protocol was introduced, that increased signal strengths without increasing noise and without affecting the heterozygote balance. Allele drop-ins were only observed in experiments with 25 pg of DNA and not in experiments with 50 and 100 pg of DNA. The allele drop-in rate in the 25 pg experiments was 0.06% or 100 times lower than what was previously reported for STR typing of LtDNA. A composite model and two different consensus models were used to interpret the SNP data. Correct profiles with 42-49 SNPs were generated from the 50 and 100 pg experiments, whereas a few incorrect genotypes were included in the generated profiles from the 25 pg experiments. With the strict consensus model, between 35 and 48 SNPs were correctly typed in the 25 pg experiments and only one allele drop-out (error rate: 0.07%) was observed in the consensus profiles. A total of 28 crime case samples were selected for typing with the sensitized SNPforID protocol. The samples were previously typed with old STR kits during the crime case investigation and only partial profiles (0-6 STRs) were obtained. Eleven of the samples could not be quantified with the Quantifiler™ Human DNA Quantification kit because of partial or complete inhibition of the PCR. For eight of these samples, SNP typing was only possible when the buffer and DNA polymerase used in the original protocol was replaced with the AmpFℓSTR(®) SEfiler Plus™ Master Mix, which was developed specifically for challenging forensic samples. All the crime case samples were successfully typed with the SNPforID multiplex assay and the match probabilities ranged from 1.1×10(-15) to 7.9×10(-23). In comparison, four of the samples could not be typed with the AmpFℓSTR(®) SEfiler Plus™ kit and the match probabilities were higher than 10(-7) for another six samples. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
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Gonadoblastoma: evidence for a stepwise progression to dysgerminoma in a dysgenetic ovary.
Pauls, Katharina; Franke, Folker E; Büttner, Reinhard; Zhou, Hui
2005-09-01
Gonadoblastomas are neoplasms of dysgenetic gonads which may undergo regression or become overgrown by malignant germ cell tumors (mGCTs). Since little is known about their relationship to normal gonadal development and mGCTs, we studied the phenotype and antigenic profile of gonadoblastomas in comparison with adjacent dysgerminomas and fetal gonads. Three cases of gonadoblastomas and fetal gonads of both sexes were analyzed using oncofetal markers to M2A-antigen (M2A), germ cell alkaline phosphatase (PLAP/GCAP), receptor tyrosine kinase c-kit (c-kit), and somatic angiotensin converting enzyme (sACE) as well as the proliferation marker MIB-1. Morphologically, microfollicular pattern of gonadoblastomas showed a fetal germ cell organization reminiscent of oocytic clusters of fetal ovaries. They contained both cell types, similar to oocytes (M2A-, GCAP-, c-kit+/-, sACE-) and oogonia (M2A+, GCAP+, c-kit+, sACE+). The percentage of germ cells immunoreactive for oncofetal markers and the proliferation index increased from microfollicular over coronary patterns to adjacent dysgerminomas. Supportive cells of gonadoblastomas showed a uniform phenotype (CK18+, vimentin+, sACE+, alpha-inhibin+, M2A-) but in contrast to fetal germ cells lacked a clear equivalence to fetal tissues. Our results show that gonadoblastomas mimic female fetal ovary and exhibit a stepwise progression from follicular pattern to coronary pattern and finally to dysgerminomas.
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Pediatric emergencies on a US-based commercial airline.
Moore, Brian R; Ping, Jennifer M; Claypool, David W
2005-11-01
The purpose of this investigation was to determine the incidence and character of pediatric emergencies on a US-based commercial airline and to evaluate current in-flight medical kits. In-flight consultations to a major US airline by a member of our staff are recorded in an institutional database. In this observational retrospective review, the database was queried for consultations for all passengers up to 18 years old between January 1, 1995, and December 31, 2002. Consultations were reviewed for type of emergency, use of the medical kit, and unscheduled landings. Two hundred twenty-two pediatric consultations were identified, representing 1 pediatric call per 20,775 flights. The mean age of patients was 6.8 years. Fifty-three emergencies were preflight calls, and 169 were in-flight pediatric consultations. The most common in-flight consultations concerned infectious disease (45 calls, 27%), neurological (25 calls, 15%), and respiratory tract (22 calls, 13%) emergencies. The emergency medical kit was used for 60 emergencies. Nineteen consultations (11%) resulted in flight diversions (1/240,000 flights), most commonly because of in-flight neurological (9) and respiratory tract (5) emergencies. International flights had a higher incidence than domestic flights of consultations and diversions for pediatric emergencies. The most common in-flight pediatric emergencies involved infectious diseases and neurological and respiratory tract problems. Emergency medical kits should be expanded to include pediatric medications.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Atchudan, R.; Department of Chemistry, CEG Campus, Anna University, Chennai 600025; Joo, Jin., E-mail: joojin@knu.ac.kr
2013-06-01
Highlights: ► Tailored 3D cubic Ni/KIT-6 with large pores was synthesized successfully. ► The new hybrid g-CNTs in large scale were synthesized using Ni/KIT-6 by CVD method. ► The use of mesoporous material by CVD method would be an ideal choice to prepare g-CNTs at reasonable cost. ► This type of g-CNTs might be a new avenue for nano-electronic applications. - Abstract: The new hybrid of graphenated carbon nanotubes (g-CNTs) was superior to either CNTs or graphene. Mesoporous 3D cubic Ni/KIT-6 were synthesized hydrothermally through organic template route and then were used as catalytic template for the production of g-CNTsmore » using acetylene as a carbon precursor by chemical vapor deposition (CVD) method. The deposited new hybrid carbon materials were purified and analyzed by various physico-chemical techniques such as XRD, TGA, SEM, TEM and Raman spectroscopy techniques. The graphitization of CNTs was confirmed by TGA and HRTEM studies. Thermal stability, surface morphology, and structural morphology of these materials were revealed by TGA, SEM and TEM analysis, respectively. Moreover, the tailored mesoporous Ni/KIT-6 molecular sieves were found to possess better quality and massive quantity of g-CNTs produced compared to other catalytic template route.« less
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Development of an add-on kit for scanning confocal microscopy (Conference Presentation)
NASA Astrophysics Data System (ADS)
Guo, Kaikai; Zheng, Guoan
2017-03-01
Scanning confocal microscopy is a standard choice for many fluorescence imaging applications in basic biomedical research. It is able to produce optically sectioned images and provide acquisition versatility to address many samples and application demands. However, scanning a focused point across the specimen limits the speed of image acquisition. As a result, scanning confocal microscope only works well with stationary samples. Researchers have performed parallel confocal scanning using digital-micromirror-device (DMD), which was used to project a scanning multi-point pattern across the sample. The DMD based parallel confocal systems increase the imaging speed while maintaining the optical sectioning ability. In this paper, we report the development of an add-on kit for high-speed and low-cost confocal microscopy. By adapting this add-on kit to an existing regular microscope, one can convert it into a confocal microscope without significant hardware modifications. Compared with current DMD-based implementations, the reported approach is able to recover multiple layers along the z axis simultaneously. It may find applications in wafer inspection and 3D metrology of semiconductor circuit. The dissemination of the proposed add-on kit under $1000 budget could also lead to new types of experimental designs for biological research labs, e.g., cytology analysis in cell culture experiments, genetic studies on multicellular organisms, pharmaceutical drug profiling, RNA interference studies, investigation of microbial communities in environmental systems, and etc.
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Deng, Feng; Zhang, Lei; Zhang, Yi; Song, Jin-lin; Fan, Yuboa
2007-07-01
To compare and analyze the stress distribution at the micro-implant-bone interface based on the different micro-implant-bone conditioned under orthodontic load, and to optimize the design of micro implant's neck. An adult skull with all tooth was scanned by spiral CT, and the data were imported into computer for three-dimensional reconstruction with software Mimics 9.0. The three dimensional finite element models of three micro-implant-bone interfaces(initial stability, full osseointegration and fibrous integration) were analyzed by finite element analysis software ABAQUS6.5. The primary stress distributions of different micro-implant-bone conditions were evaluated when 2N force was loaded. Then the diameter less than 1.5 mm of the micro implant's neck was added with 0.2 mm, to compare the stress distribution of the modified micro-implant-bone interface with traditional type. The stress mostly concentrated on the neck of micro implant and the full osseointegration interface in all models showed the lowest strain level. Compared with the traditional type, the increasing diameter neck of the micro implant obviously decreased the stress level in all the three conditions. The micro-implant-bone interface and the diameter of micro implant's neck both are the important influence factors to the stress distribution of micro implant.
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Khuda, Sefat E; Sharma, Girdhari M; Gaines, Dennis; Do, Andrew B; Pereira, Marion; Chang, Michael; Ferguson, Martine; Williams, Kristina M
2016-08-01
Since the number of recalls involving undeclared allergens is commonly associated with bakery and snack foods, we aimed to determine the frequency of egg allergens in a large number of these products using two commercial enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no egg identified on the product label or which had an egg precautionary statement. Among all samples, egg protein was detected in 5% of products using a Morinaga (MO) kit and 1% of products using a R-Biopharm (RB) kit. For bakery samples, egg protein was detected in 6% of 363 samples with no precautionary labelling (6% by MO and 1% by RB kit) and 12% of 80 samples which had precautionary labelling. For snack samples, egg protein was detected in 2% of 371 samples with no precautionary labelling (2% by MO and < 1% by RB kit) and 5% of 21 samples which had precautionary labelling. The disagreement rates between two methods were 5.2% for bakery products and 2.6% for snack products. The sample repeatability was at an acceptable level for bakery (< 12.5%) and snack foods (< 7.5%) for each method. The relative standard deviation between test kits was high (103.1%) for bakery foods. Four bakery products without precautionary labelling had a higher level of egg protein per serving compared with the eliciting dose (ED10 of 3.7 mg protein) for egg allergic patients. These results highlight the fact that detection methodology plays a vital role for accurate labelling control and mitigation of risk for egg allergic consumers.
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Göde, Sercan; Turhal, Göksel; Tarhan, Ceyda; Yaman, Banu; Kandiloğlu, Gülşen; Öztürk, Kerem; Kaya, İsa; Midilli, Raşit; Karcı, Bülent
2017-05-05
Mucosal melanoma is a rare malignancy arising from melanocytes of the mucosal surfaces. The pattern and frequency of oncogenic mutations and histopathological biomarkers have a role on distinct tumour behaviour and survival. To assess the rate of C-KIT positivity and its effect on survival of surgically treated sinonasal malignant melanoma patients with other histopathological biomarkers and clinical features. Retrospective cross-sectional study. Seventeen sinonasal malignant melanoma patients with a mean age of 65.41 (39-86) years were included. Overall survival and disease-specific survival rates were calculated. The impact of age, gender, stage and extent of the disease, type of surgery, and adjuvant therapies were also taken into consideration. The effect of mitotic index, pigmentation, S100, HMB-45, Melan-A and C-KIT on survival were evaluated. Median tumour size was 20 mm (interquartile range=27.5 mm). Pigmentation was present in 7 (41.2%) cases. Median number of mitoses per millimetre squared was 11 (interquartile range=13). Melan A was positive in 7 (41.2%) patients, ulceration was present in 6 cases (35.3%), and necrosis was present in (47.1%) 8 cases. Six patients (35.3%) were positive for S100, 14 (82.4%) specimens stained positive for HMB-45 and C-KIT (CD117) was positive in 9 cases (52.9%). Three patients (16.7%) developed distant metastasis. Five year overall and disease free survival rates were 61.4% and 43.8%, respectively. Although C-KIT positive sinonasal malignant melanoma patients (52.9%) can be candidates for targeted tumour therapies, the studied clinical or histopathological features along with C-KIT seem to have no significant effect on survival in a small group of patients with sinonasal malignant melanoma.
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Germline mutations of KIT in gastrointestinal stromal tumor (GIST) and mastocytosis.
Ke, Hengning; Kazi, Julhash U; Zhao, Hui; Sun, Jianmin
2016-01-01
Somatic mutations of KIT are frequently found in mastocytosis and gastrointestinal stromal tumor (GIST), while germline mutations of KIT are rare, and only found in few cases of familial GIST and mastocytosis. Although ligand-independent activation is the common feature of KIT mutations, the phenotypes mediated by various germline KIT mutations are different. Germline KIT mutations affect different tissues such as interstitial cells of Cajal (ICC), mast cells or melanocytes, and thereby lead to GIST, mastocytosis, or abnormal pigmentation. In this review, we summarize germline KIT mutations in familial mastocytosis and GIST and discuss the possible cellular context dependent transforming activity of KIT mutations.
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The challenges of lean manufacturing implementation in kitting assembly
NASA Astrophysics Data System (ADS)
Fansuri, A. F. H.; Rose, A. N. M.; Nik Mohamed, N. M. Z.; Ahmad, H.
2017-10-01
Literature studies shows that lean manufacturing goes way back with the original founder Eli Whitney in year 1799. The main purpose of lean manufacturing is to identify and eliminate waste in production. The application of lean manufacturing can be carried out in any industrial processes with regards to the understanding of lean principles, theories and practices. Kitting is one of the important aspects in a successful production. The continuous supply of materials from store to production has to be systematic and able to achieve lean standard for it to be successful. The objective of this paper is to review the implementation of lean manufacturing in kitting assembly. Previous papers show that, the implementation of lean manufacturing in kitting assembly may be beneficial to the organization such as reduce in space occupancy, part shortages, lead time and manpower. Based on previous research, some industries may tend to change between kitting and line stocking which are due to lack of understanding when implementing kitting and causes longer lead time and materials overflow in store. With a proper understanding on what to kit, where to kit, how to kit, why to kit and who kits the material with a standardised process flow may ensure the success of kitting.
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Pim1 kinase regulates c-Kit gene translation.
An, Ningfei; Cen, Bo; Cai, Houjian; Song, Jin H; Kraft, Andrew; Kang, Yubin
2016-01-01
Receptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown. We demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1 -/- mice and Pim1 -/- 2 -/- 3 -/- triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1 -/- and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit 35 S methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level. Our study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.
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NASA Astrophysics Data System (ADS)
Robati, Masoud
This Doctorate program focuses on the evaluation and improving the rutting resistance of micro-surfacing mixtures. There are many research problems related to the rutting resistance of micro-surfacing mixtures that still require further research to be solved. The main objective of this Ph.D. program is to experimentally and analytically study and improve rutting resistance of micro-surfacing mixtures. During this Ph.D. program major aspects related to the rutting resistance of micro-surfacing mixtures are investigated and presented as follow: 1) evaluation of a modification of current micro-surfacing mix design procedures: On the basis of this effort, a new mix design procedure is proposed for type III micro-surfacing mixtures as rut-fill materials on the road surface. Unlike the current mix design guidelines and specification, the new mix design is capable of selecting the optimum mix proportions for micro-surfacing mixtures; 2) evaluation of test methods and selection of aggregate grading for type III application of micro-surfacing: Within the term of this study, a new specification for selection of aggregate grading for type III application of micro-surfacing is proposed; 3) evaluation of repeatability and reproducibility of micro-surfacing mixture design tests: In this study, limits for repeatability and reproducibility of micro-surfacing mix design tests are presented; 4) a new conceptual model for filler stiffening effect on asphalt mastic of micro-surfacing: A new model is proposed, which is able to establish limits for minimum and maximum filler concentrations in the micro-surfacing mixture base on only the filler important physical and chemical properties; 5) incorporation of reclaimed asphalt pavement and post-fabrication asphalt shingles in micro-surfacing mixture: The effectiveness of newly developed mix design procedure for micro-surfacing mixtures is further validated using recycled materials. The results present the limits for the use of RAP and RAS amount in micro-surfacing mixtures; 6) new colored micro-surfacing formulations with improved durability and performance: The significant improvement of around 45% in rutting resistance of colored and conventional micro-surfacing mixtures is achieved through employing low penetration grade bitumen polymer modified asphalt emulsion stabilized using nanoparticles.
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Quantum cascade light emitting diodes based on type-2 quantum wells
NASA Technical Reports Server (NTRS)
Lin, C. H.; Yang, R. Q.; Zhang, D.; Murry, S. J.; Pei, S. S.; Allerman, A. A.; Kurtz, S. R.
1997-01-01
The authors have demonstrated room-temperature CW operation of type-2 quantum cascade (QC) light emitting diodes at 4.2 (micro)m using InAs/InGaSb/InAlSb type-2 quantum wells. The type-2 QC configuration utilizes sequential multiple photon emissions in a staircase of coupled type-2 quantum wells. The device was grown by molecular beam epitaxy on a p-type GaSb substrate and was compared of 20 periods of active regions separated by digitally graded quantum well injection regions. The maximum average output power is about 250 (micro)W at 80 K, and 140 (micro)W at 300 K at a repetition rate of 1 kHz with a duty cycle of 50%.
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Yamasaki, Yasushi; Uedo, Noriya; Kanzaki, Hiromitsu; Kato, Minoru; Hamada, Kenta; Aoi, Kenji; Tonai, Yusuke; Matsuura, Noriko; Kanesaka, Takashi; Yamashina, Takeshi; Akasaka, Tomofumi; Hanaoka, Noboru; Takeuchi, Yoji; Higashino, Koji; Ishihara, Ryu; Tomita, Yasuhiko; Iishi, Hiroyasu
2017-01-01
Magnifying narrow-band imaging (M-NBI) can reportedly help predict the presence and distribution of atrophy and intestinal metaplasia in the gastric corpus. However, the micro-mucosal pattern of the antrum shown by M-NBI differs from that of the corpus. We studied the distribution and histology of the micro-mucosal pattern in the antrum based on magnifying endoscopy. Endoscopic images of the greater curvature of the antrum were evaluated in 50 patients with chronic atrophic fundic gastritis (CAFG). The extent of CAFG was evaluated by autofluorescence imaging. The micro-mucosal pattern was evaluated by M-NBI and classified into groove and white villiform types. The localization of white villiform type mucosa was classified into three types in relation to the areae gastricae : null, central, and segmental types. Biopsies were taken from regions showing different micro-mucosal patterns. Associations among the extent of CAFG, micro-mucosal pattern, and histology were examined. As the extent of CAFG increased, the proportion of white villiform type mucosa increased, whereas that of groove type mucosa decreased (P=0.022). In patients with extensive CAFG, most of the areae gastricae was composed of the segmental or central type of white villiform type mucosa (P=0.044). The white villiform type mucosa had significantly higher grades of atrophy (P=0.002) and intestinal metaplasia (P<0.001) than did the groove type mucosa. White villiform type mucosa is indicative of atrophy and intestinal metaplasia in the gastric antrum. It extends to the whole or central part of the areae gastricae as CAFG becomes more extensive.
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Early kit mortality and growth in farmed mink are affected by litter size rather than nest climate.
Schou, T M; Malmkvist, J
2017-09-01
We investigated the effects of nest box climate on early mink kit mortality and growth. We hypothesised that litters in warm nest boxes experience less hypothermia-induced mortality and higher growth rates during the 1st week of life. This study included data from 749, 1-year-old breeding dams with access to nesting materials. Kits were weighed on days 1 and 7, dead kits were collected daily from birth until day 7 after birth, and nest climate was measured continuously from days 1 to 6. We tested the influences of the following daily temperature (T) and humidity (H) parameters on the number of live-born kit deaths and kit growth: T mean, T min, T max, T var (fluctuation) and H mean. The nest microclimate experienced by the kits was buffered against the ambient climate, with higher temperatures and reduced climate fluctuation. Most (77.0%) live-born kit deaths in the 1st week occurred on days 0 and 1. Seven of 15 climate parameters on days 1 to 3 had significant effects on live-born kit mortality. However, conflicting effects among days, marginal effects and late effects indicated that climate was not the primary cause of kit mortality. Five of 30 climate parameters had significant effects on kit growth. Few and conflicting effects indicated that the climate effect on growth was negligible. One exception was that large nest temperature fluctuations on day 1 were associated with reduced deaths of live-born kit (P<0.001) and increased kit growth (P=0.003). Litter size affected kit vitality; larger total litter size at birth was associated with greater risks of kit death (P<0.001) and reduced growth (P<0.001). The number of living kits in litters had the opposite effect, as kits in large liveborn litters had a reduced risk of death (P<0.001) and those with large mean litter size on days 1 to 7 had increased growth (P=0.026). Nest box temperature had little effect on early kit survival and growth, which could be due to dams' additional maternal behaviour. Therefore, we cannot confirm that temperature is the primary reason for kit mortality, under the conditions of plenty straw access for maternal nest building. Instead, prenatal and/or parturient litter size is the primary factor influencing early kit vitality. The results indicate that the focus should be on litter size and dam welfare around the times of gestation and birth to increase early kit survival in farmed mink.
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Carvalho, Maria Isabel; Pires, Isabel; Dias, Marlene; Prada, Justina; Gregório, Hugo; Lobo, Luis; Queiroga, Felisbina
2015-01-01
In this study 80 malignant CMT were submitted to immunohistochemical detection of CD3, c-kit, VEGF, and CD31, together with clinicopathological parameters of tumor aggressiveness. CD3+ T-cells and c-kit overexpression revealed a positive correlation with VEGF (r = 0.503, P < 0.0001; r = 0.284, P = 0.023 for CD3 and c-kit, resp.) and CD31 (r = 0.654, P < 0.0001; r = 0.365, P = 0.003 for CD3 and c-kit, resp.). A significant association (P = 0.039) and a positive correlation (r = 0.263, P = 0.039) between CD3 and c-kit were also observed. High CD3/VEGF, c-kit/VEGF, and CD3/c-kit tumors were associated with elevated grade of malignancy (P < 0.0001 for all groups), presence of intravascular emboli (P < 0.0001 for CD3/VEGF and CD3/c-kit; P = 0.002 for c-kit/VEGF), and presence of lymph node metastasis (P < 0.0001 for all groups). Tumors with high CD3/VEGF (P = 0.006), c-kit/VEGF (P < 0.0001), and CD3/c-kit (P = 0.002) were associated with poor prognosis. Interestingly high c-kit/VEGF tumors retained their significance by multivariate analysis arising as independent prognostic factor. PMID:26346272
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Sensitive and reliable detection of Kit point mutation Asp 816 to Val in pathological material
Kähler, Christian; Didlaukat, Sabine; Feller, Alfred C; Merz, Hartmut
2007-01-01
Background Human mastocytosis is a heterogenous disorder which is linked to a gain-of-function mutation in the kinase domain of the receptor tyrosine kinase Kit. This D816V mutation leads to constitutive activation and phosphorylation of Kit with proliferative disorders of mast cells in the peripheral blood, skin, and spleen. Most PCR applications used so far are labour-intensive and are not adopted to daily routine in pathological laboratories. The method has to be robust and working on such different materials like archival formalin-fixed, paraffin-embedded tissue (FFPE) and blood samples. Such a method is introduced in this publication. Methods The Kit point mutation Asp 816 to Val is heterozygous which means a problem in detection by PCR because the wild-type allele is also amplified and the number of cells which bear the point mutation is in most of the cases low. Most PCR protocols use probes to block the wild-type allele during amplification with more or less satisfying result. This is why point-mutated forward primers were designed and tested for efficiency in amplification of the mutated allele. Results One primer combination (A) fits the most for the introduced PCR assay. It was able just to amplify the mutated allele with high specificity from different patient's materials (FFPE or blood) of varying quality and quantity. Moreover, the sensitivity for this assay was convincing because 10 ng of DNA which bears the point mutation could be detected in a total volume of 200 ng of DNA. Conclusion The PCR assay is able to deal with different materials (blood and FFPE) this means quality and quantity of DNA and can be used for high-througput screening because of its robustness. Moreover, the method is easy-to-use, not labour-intensive, and easy to realise in a standard laboratory. PMID:17900365
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Cuomo, Raphael E; Miner, Angela; Mackey, Tim K
2015-10-23
Previous studies have examined marketing characteristics of e-cigarettes sold online and others have examined e-cigarettes pricing in retail (non-Internet) settings. This study expands on these findings by examining pricing and marketing characteristics of interest among e-cigarette online vendors. Structured web searches were conducted from August-September 2014 to identify popular e-cigarette Internet vendors. We then collected pricing data (e-cigarette starter kits and disposables), sales tax collection policies and other vendor marketing characteristics. Average price for each product category was then compared with marketing characteristics using linear regression for continuous variables and independent t-tests for binary variables. Our searches yielded 44 e-cigarette Internet vendors of which 77% (n = 34) sold a total of 238 starter kit offerings (Mprice = $55.89). Half (n = 22) sold disposable types of e-cigarettes (Mprice = $7.17 p/e-cigarette) at a price lower than reported elsewhere in retail settings. Average disposable e-cigarette prices were also significantly higher for vendors displaying more health warning notices (P = 0.001). Only 46% disclosed sales tax collection policies and only 39% collected sales tax in their state of business. This study expands on current understanding of e-cigarette pricing and availability online and finds variation in e-cigarette pricing may be influenced by type of product, use of online health warnings and vendor sales tax collection policies. It also finds that e-cigarette online access and availability may be impacted by a combination of pricing and marketing strategies uniquely different from e-cigarette retail settings that requires further study and targeted policy-making. [Cuomo RE, Miner A, Mackey TK. Pricing and sales tax collection policies for e-cigarette starter kits and disposable products sold online. Drug Alcohol Rev 2015]. © 2015 Australasian Professional Society on Alcohol and other Drugs.
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Maruyama, Haruhiko; Kaneko, Michiko; Otake, Taiga; Kano, Rui; Yamaya, Yoshiki; Watari, Toshihiro; Hasegawa, Atsuhiko; Kamata, Hiroshi
2014-03-01
A disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) is a von Willebrand factor (vWF)-cleaving protease. Deficiencies in ADAMTS13 activity are known to cause thrombotic diseases in human beings. The present study evaluated whether the human ADAMTS13 activity enzyme-linked immunosorbent assay (ELISA) kit containing human vWF73 (a minimal substrate) and anti-N10 antibody (which specifically recognizes the decapeptide of the C-terminal edge of cleaved vWF by human ADAMTS13) is applicable to the measurement of canine plasma ADAMTS13 activity. Human vWF73 fused with a GST-tag and a His-tag (GST-hvWF73-His) was reacted with recombinant canine (rc)ADAMTS13, canine plasma, and human plasma, and then used in Western blotting using anti-N10 antibody. Linearity and intra- and interassay reproducibility of the human ADAMTS13 activity ELISA kit in canine plasma were further evaluated. Finally, plasma ADAMTS13 activity was measured in 13 healthy dogs and 6 dogs with bacteremia using the human ADAMTS13 activity ELISA kit. Cleaved products with a 28-kDa GST-hvWF73-His were detected specifically in rcADAMTS13 as well as in human ADAMTS13, and also in canine plasma by anti-N10 antibody, showing excellent linearity. Intra-assay coefficient of variation (CV) was 3.0-12.4%, and interassay CV was 11.5-12.5%. The ADAMTS13 activity was significantly lower in dogs with bacteremia than in healthy dogs (P = 0.0025). The current study revealed that the human ADAMTS13 activity ELISA kit is applicable for measurement of canine plasma ADAMTS13 activity to elucidate the pathology of thrombotic diseases in dogs.
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Prakash, Sonam; Sarran, Lisa; Socci, Nicholas; DeMatteo, Ronald P; Eisenstat, Jonathan; Greco, Alba M; Maki, Robert G; Wexler, Leonard H; LaQuaglia, Michael P; Besmer, Peter; Antonescu, Cristina R
2005-04-01
Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the intestinal tract that typically occur in adults over the age of 40 years. GISTs in younger patients are rare and not well characterized. The objective was to define the characteristics of GISTs in children and young adults (<30 years old). Clinicopathologic and molecular features, including KIT/PDGFRA genotype, in GISTs from 5 children and 10 young adults were analyzed. Gene expression analysis was performed on 5 gastric tumor samples from 2 children, 2 gastric tumors from young adults, and 10 gastric GISTs from older adults using an U133A Affymetrix platform (22,000 genes). All five pediatric GISTs occurred in girls, involved the stomach as multiple nodules, showed predominantly an epithelioid morphology, often involved lymph nodes, and lacked KIT or PDGFRA mutations. Although all five patients developed recurrence (four in the liver, three in the peritoneum, and two in both sites), four are still alive with disease. Of the 10 GISTs in young adults, half occurred in the small bowel and had spindle cell morphology, and one case had lymph node metastasis. KIT mutations were identified in seven cases, four in exon 11 and three in exon 9. Seven patients developed recurrence, and at last follow-up two patients had died of disease. Gene expression analysis showed high expression of PHKA1, FZD2, NLGN4, IGF1R, and ANK3 in the pediatric and young adult versus older adult cases. GISTs that occur in children are a separate clinicopathologic and molecular subset with predilection for girls, multifocal gastric tumors, and wild-type KIT/PDGFRA genotype. In contrast, GISTs in young adults are a more heterogeneous group, including cases that resemble either the pediatric or the older adult-type tumors. The distinct gene expression profile suggests avenues for investigation of pathogenesis and potential therapeutic strategies.
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Armas, Federica; Camperio, Cristina; Coltella, Luana; Selvaggini, Serena; Boniotti, Maria Beatrice; Pacciarini, Maria Lodovica; Di Marco Lo Presti, Vincenzo; Marianelli, Cinzia
2017-08-04
Highly discriminatory genotyping strategies are essential in molecular epidemiological studies of tuberculosis. In this study we evaluated, for the first time, the efficacy of the repetitive sequence-based PCR (rep-PCR) DiversiLab Mycobacterium typing kit over spoligotyping, 12-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and embB single nucleotide polymorphism (SNP) analysis for Mycobacterium bovis typing. A total of 49 M. bovis animal isolates were used. DNA was extracted and genomic DNA was amplified using the DiversiLab Mycobacterium typing kit. The amplified fragments were separated and detected using a microfluidics chip with Agilent 2100. The resulting rep-PCR-based DNA fingerprints were uploaded to and analysed using web-based DiversiLab software through Pearson's correlation coefficient. Rep-PCR DiversiLab grouped M. bovis isolates into ten different clusters. Most isolates sharing identical spoligotype, MIRU-VNTR profile or embB gene polymorphism were grouped into different rep-PCR clusters. Rep-PCR DiversiLab displayed greater discriminatory power than spoligotyping and embB SNP analysis but a lower resolution power than the 12-locus MIRU-VNTR analysis. MIRU-VNTR confirmed that it is superior to the other PCR-based methods tested here. In combination with spoligotyping and 12-locus MIRU-VNTR analysis, rep-PCR improved the discriminatory power for M. bovis typing.
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Dolan, Paul; Rudisill, Caroline
2014-01-01
Financial incentives have been used in a variety of settings to motivate behaviors that might not otherwise be undertaken. They have been highlighted as particularly useful in settings that require a single behavior, such as appointment attendance or vaccination. They also have differential effects based on socioeconomic status in some applications (e.g. smoking). To further investigate these claims, we tested the effect of providing different types of non-cash financial incentives on the return rates of chlamydia specimen samples amongst 16–24 year-olds in England. In 2011 and 2012, we ran a two-stage randomized experiment involving 2988 young people (1489 in Round 1 and 1499 in Round 2) who requested a chlamydia screening kit from Freetest.me, an online and text screening service run by Preventx Limited. Participants were randomized to control, or one of five types of financial incentives in Round 1 or one of four financial incentives in Round 2. We tested the effect of five types of incentives on specimen sample return; reward vouchers of differing values, charity donation, participation in a lottery, choices between a lottery and a voucher and including vouchers of differing values in the test kit prior to specimen return. Financial incentives of any type, did not make a significant difference in the likelihood of specimen return. The more deprived individuals were, as calculated using Index of Multiple Deprivation (IMD), the less likely they were to return a sample. The extent to which incentive structures influenced sample return was not moderated by IMD score. Non-cash financial incentives for chlamydia testing do not seem to affect the specimen return rate in a chlamydia screening program where test kits are requested online, mailed to requestors and returned by mail. They also do not appear more or less effective in influencing test return depending on deprivation level. PMID:24373390
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Dolan, Paul; Rudisill, Caroline
2014-03-01
Financial incentives have been used in a variety of settings to motivate behaviors that might not otherwise be undertaken. They have been highlighted as particularly useful in settings that require a single behavior, such as appointment attendance or vaccination. They also have differential effects based on socioeconomic status in some applications (e.g. smoking). To further investigate these claims, we tested the effect of providing different types of non-cash financial incentives on the return rates of chlamydia specimen samples amongst 16-24 year-olds in England. In 2011 and 2012, we ran a two-stage randomized experiment involving 2988 young people (1489 in Round 1 and 1499 in Round 2) who requested a chlamydia screening kit from Freetest.me, an online and text screening service run by Preventx Limited. Participants were randomized to control, or one of five types of financial incentives in Round 1 or one of four financial incentives in Round 2. We tested the effect of five types of incentives on specimen sample return; reward vouchers of differing values, charity donation, participation in a lottery, choices between a lottery and a voucher and including vouchers of differing values in the test kit prior to specimen return. Financial incentives of any type, did not make a significant difference in the likelihood of specimen return. The more deprived individuals were, as calculated using Index of Multiple Deprivation (IMD), the less likely they were to return a sample. The extent to which incentive structures influenced sample return was not moderated by IMD score. Non-cash financial incentives for chlamydia testing do not seem to affect the specimen return rate in a chlamydia screening program where test kits are requested online, mailed to requestors and returned by mail. They also do not appear more or less effective in influencing test return depending on deprivation level. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.
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A Simple and Efficient Method of Extracting DNA from Aged Bones and Teeth.
Liu, Qiqi; Liu, Liyan; Zhang, Minli; Zhang, Qingzhen; Wang, Qiong; Ding, Xiaoran; Shao, Liting; Zhou, Zhe; Wang, Shengqi
2018-05-01
DNA is often difficult to extract from old bones and teeth due to low levels of DNA and high levels of degradation. This study established a simple yet efficient method for extracting DNA from 20 aged bones and teeth (approximately 60 years old). Based on the concentration and STR typing results, the new method of DNA extraction (OM) developed in this study was compared with the PrepFiler™ BTA Forensic DNA Extraction Kit (BM). The total amount of DNA extracted using the OM method was not significantly different from that extracted using the commercial kit (p > 0.05). However, the number of STR loci detected was significantly higher in the samples processed using the OM method than using the BM method (p < 0.05). This study aimed to establish a DNA extraction method for aged bones and teeth to improve the detection rate of STR typing and reduce costs compared to the BM technique. © 2017 American Academy of Forensic Sciences.
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Antibody targeting KIT as pretransplantation conditioning in immunocompetent mice.
Xue, Xingkui; Pech, Nancy K; Shelley, W Christopher; Srour, Edward F; Yoder, Mervin C; Dinauer, Mary C
2010-12-09
Inherited hematologic defects that lack an in vivo selective advantage following gene correction may benefit from effective yet minimally toxic cytoreduction of endogenous hematopoietic stem cells (HSCs) prior to transplantation of gene-modified HSCs. We studied the efficacy of administering a novel sequential treatment of parenteral ACK2, an antibody that blocks KIT, followed by low-dose irradiation (LD-IR) for conditioning of wild-type and X-linked chronic granulomatous disease (X-CGD) mice. In wild-type mice, combining ACK2 and LD-IR profoundly decreased endogenous competitive long-term HSC repopulating activity, and permitted efficient and durable donor-derived HSC engraftment after congenic transplantation. ACK2 alone was ineffective. The combination of ACK2 and LD-IR was also effective conditioning in X-CGD mice for engraftment of X-CGD donor HSCs transduced ex vivo with a lentiviral vector. We conclude that combining ACK2 with LD-IR is a promising approach to effectively deplete endogenous HSCs and facilitate engraftment of transplanted donor HSCs.
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Development and Validation of a Kit to Measure Drink Antioxidant Capacity Using a Novel Colorimeter.
Priftis, Alexandros; Stagos, Dimitrios; Tzioumakis, Nikolaos; Konstantinopoulos, Konstantinos; Patouna, Anastasia; Papadopoulos, Georgios E; Tsatsakis, Aristides; Kouretas, Dimitrios
2016-08-30
Measuring the antioxidant capacity of foods is essential, as a means of quality control to ensure that the final product reaching the consumer will be of high standards. Despite the already existing assays with which the antioxidant activity is estimated, new, faster and low cost methods are always sought. Therefore, we have developed a novel colorimeter and combined it with a slightly modified DPPH assay, thus creating a kit that can assess the antioxidant capacity of liquids (e.g., different types of coffee, beer, wine, juices) in a quite fast and low cost manner. The accuracy of the colorimeter was ensured by comparing it to a fully validated Hitachi U-1900 spectrophotometer, and a coefficient was calculated to eliminate the observed differences. In addition, a new, user friendly software was developed, in order to render the procedure as easy as possible, while allowing a central monitoring of the obtained results. Overall, a novel kit was developed, with which the antioxidant activity of liquids can be measured, firstly to ensure their quality and secondly to assess the amount of antioxidants consumed with the respective food.
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Feyerabend, Thorsten B; Weiser, Anne; Tietz, Annette; Stassen, Michael; Harris, Nicola; Kopf, Manfred; Radermacher, Peter; Möller, Peter; Benoist, Christophe; Mathis, Diane; Fehling, Hans Jörg; Rodewald, Hans-Reimer
2011-11-23
Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy. Copyright © 2011 Elsevier Inc. All rights reserved.
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Bienek, Diane R; Charlton, David G
2012-05-01
Being able to test for the presence of blood pathogens at forward locations could reduce morbidity and mortality in the field. Rapid, user-friendly blood typing kits for detecting Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), and Hepatitis B Virus (HBV) were evaluated to determine their accuracy after storage at various temperatures/humidities. Rates of positive tests of control groups, experimental groups, and industry standards were compared (Fisher's exact chi2, p < or = 0.05). Compared to the control group, 2 of 10 HIV detection devices were adversely affected by exposure to high temperature/high humidity or high temperature/low humidity. With one exception, none of the environmentally exposed HCV or HBV detection devices exhibited significant differences compared to those stored under control conditions. For HIV, HCV, and HBV devices, there were differences compared to the industry standard. Collectively, this evaluation of pathogen detection kits revealed that diagnostic performance varies among products and storage conditions, and that the tested products cannot be considered to be approved for use to screen blood, plasma, cell, or tissue donors.
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Costa Casagrande, T A; de Oliveira Barros, L M; Fukumasu, H; Cogliati, B; Chaible, L M; Dagli, M L Z; Matera, J M
2015-03-01
This study investigated the correlation between KIT gene expression determined by immunohistochemistry and real-time polymerase chain reaction (RT-PCR) and the rate of tumour recurrence and tumour-related deaths in dogs affected with mast cell tumour (MCT). Kaplan-Meier curves were constructed to compare tumour recurrence and tumour-related death between patients. The log-rank test was used to check for significant differences between curves. KIT-I, KIT-II and KIT-III staining patterns were observed in 9 (11.11%), 50 (61.73%) and 22 (27.16%) tumours, respectively. Tumour recurrence rates and tumour-related deaths were not associated with KIT staining patterns (P = 0278, P > 0.05), KIT (P = 0.289, P > 0.05) or KIT ligand (P = 0.106, P > 0.05) gene expression. Despite the lack of association between KIT staining pattern and patient survival time, the results suggest a correlation between aberrant KIT localization and increased proliferative activity of MCTs. RT-PCR seems to be a sensible method for quantitative detection of KIT gene expression in canine MCT, although expressions levels are not correlated with prognosis. © 2013 Blackwell Publishing Ltd.
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Targeting c-KIT (CD117) by dasatinib and radotinib promotes acute myeloid leukemia cell death.
Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jeong, Yoo Kyung; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk
2017-11-10
Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90β (HSP90β) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90β expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90β showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.
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Singh, Satyendra; Klarmann, Kimberly D.; Coppola, Vincenzo; Keller, Jonathan R.; Tessarollo, Lino
2016-01-01
c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies. PMID:27835883
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Puverel, Sandrine; Kiris, Erkan; Singh, Satyendra; Klarmann, Kimberly D; Coppola, Vincenzo; Keller, Jonathan R; Tessarollo, Lino
2016-12-20
c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.
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Jiang, Feng; Hu, Wei; Zhang, Bicheng; Xu, Jing; Shui, Yongjie; Zhou, Xiaofeng; Ren, Xiaoqiu; Chen, Xiaozhong; Shen, Li; Wei, Qichun
2016-10-01
In the era of intensity-modulated radiotherapy, distant metastasis is currently the main cause of treatment failure for nasopharyngeal carcinoma (NPC). Additional therapeutic strategies are required to control the metastasis and improve survival. One strategy is targeted therapy, for example against c-Kit. In the current study, the frequency of c-Kit expression was determined immunohistochemically in 106 NPC patients. c-Kit expression changes during the course of radiation therapy were detected in 41 cases via weekly biopsy. Twelve cases (11.3%) had c-Kit expression scores of 3 + and 16 (15.1%) had scores of 2 + . Thus, c-Kit overexpression (2 + or 3 + ) was observed in 28 (26.4%) patients. There were 35 (33.0%) and 43 (40.6%) patients with c-Kit expression scores of 1 + and 0, respectively. Furthermore, a trend of decreased c-Kit expression was observed after commencing radiotherapy according to the 41 NPC patients who were biopsied weekly. Therefore, c-Kit overexpression was identified to be common in NPC, and evaluating c-Kit as a therapeutic target for metastatic NPC via c-Kit overexpression subsequent to first line treatment may be of interest. To the best of our knowledge, the present study is the first to demonstrate a trend of decreased c-Kit expression during the course of radiotherapy.
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NASA Astrophysics Data System (ADS)
Astutik, J.
2017-02-01
Food additives are materials that can not be separated from the lives of students and the community. Based on the preliminary questionnaire, it indicates the lack of kit supporting material additives in some schools and communities. The research objectives of this development are (1) to develop Kit experiment (SAYOFU KIT) and supplementary books to improve student learning outcomes in the classroom and public awareness on food additives (2) to describe the feasibility and potential effectiveness of SAYOFU KIT developed (3) to analyze the practice of SAYOFU KIT and benefits for students and the community. This development study uses 4-D models Thiagarajan, et al (1974). Through some stages, they are: defining, designing, developing and disseminating which involes the students and community. The developed SAYOFU KIT includes additives sample kit, borax test kit, curcumin test kit, formaldehyde test kit, modification heater to the identification of dyes and dye test paper. The study is conducted at SMP Plus Hidayatul Mubtadiin, and TKIT Al Uswah. The products are validated by experts and education practitioners. Qualitative data processing uses descriptive method, whereas quantitative data by using the N-gain. The average yield of expert validation of SAYOFU KIT with supplementary books 76.50% teacher’s book and 76.30% student’s book are eligible. The average yield of 96.81% validation of educational practitioners criteria, piloting a small group of 83.15%, and 82.89% field trials are very decent. The average yield on the student questionnaire responses SAYOFU kit and supplementary book is 87.6% with the criteria very well worth it. N-Gain 0:56 cognitive achievement with the criteria enough. The results of the public poll showed 95% feel the benefits SAYOFU kits for testing food. Based from description indicates that SAYOFU Kit developed feasible, practical, useful to support inquiry learning and improve student learning outcomes as well as public awareness of food additives.
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[Development of surgical antibioprophylaxis kits: evaluation of the impact on prescribing habits].
Aouizerate, P; Guizard, M
2002-01-01
In our hospital, surgical antibioprophylaxis (ATBP) was too often administered too late, thus raising the infectious risk. Antibiotic stocks of the anaesthesia department were also systematically used, instead of nominal prescriptions of these drugs. The pharmacy could neither charge antibiotics to each surgical department nor quantify and differentiate ATBP from curative antibiotic therapy. The pharmacy and anaesthesia departments therefore set out to standardize surgical ATBP, in order to adapt this treatment to each surgical indication, and particularly in the case of allergy to beta-lactamase antibiotics (second line treatment kits). Consequently, prescription forms were developed and supplied to each surgery department, as well as ATBP kits. The kits were prepared and distributed by the pharmacy, and comprised boxes containing antibiotics in sufficient quantities to respect the protocols approved by the French Society of Anaesthesia and Resuscitation (SFAR). A protocol describing prescriptions, dispensation and administration has been presented to physicians and nurses. Fifteen surgical departments were included in our study and 30 different kits were prepared. From 1998 to 2001, 5586 surgical operations required administration of a kit (second line treatment kits in 5% of cases): 1848 (33%) in visceral surgery; 764 (13.8%) in urology; 802 (14%) in orthopaedics; 13 (0.2%) in vascular and thoracic surgery; 1236 (22%) in ear-nose-throat (ENT), periodontics and ophtalmology, and 923 (17%) in gynaecology and obstetrics. 93% of filled prescriptions forms were spontaneously returned to the pharmacy, the others were obtained during the renewal of kit stocks. The cost (over 4 years) of ATBP was quantified: 157,871 F for the 15 departments included, 26,123 F in visceral surgery, 13,520 F in urology, 73,741 F in orthopaedics, 569 F in vascular surgery, 39,720 F in ENT/ophthalmology/periodontics and 4,198 F in gynaecology and obstetrics. According to the Altemeier classification, 2226 class I, 3151 class II, and 209 class III surgical operations were performed. Since the kits have been brought into use, the committee for the protection against nosocomial infections (CLIN) has observed a reduction in the incidence of post-operative infections, according to the Altemeier classification: from 1.6% to 0.5% in class I, from 6.5% to 4.3% in class II, and from 11% to 8.5% in class III. The difference was statistically significant only for classes I (p < 0.01) and II (p < 0.001), and unchanged for class III (p = 0.3). No analysis was carried out for class IV (curative treatments). Both nurses and physicians have greatly appreciated the implementation of this organization. The advantage in terms of post-operative infections, administration exhaustiveness and stock management is obvious. The prescribed kits were systematically appropriate for the surgical interventions. In orthopaedics, cefamandole was used over 24 h (188 kits) in ligament plasty and osteotomy, or for 48 h (499 kits) in prosthetic surgery; 24 amoxicillin/clavulanic acid (first line) and 9 clindamycin/gentamicin (second line) single dose kits have been prescribed in traumatic indications. In ophthalmology, kits were only prescribed in endophtalmitis (24 ofloxacin/fosfomycin single amount kits), implant replacement or cornea graft (1076 ofloxacin 24 h kits) and cataract surgery in diabetic patients (12 ofloxacin single amount kits). In ENT and periodontics, 124 surgical operations required cefazolin single dose kits. In vascular surgery, 5 pefloxacin/gentamicin 48 h kits and 1 amoxicillin/clavulanic acid 48 h kit were used in contaminated limb amputation, 1 cefamandole 48 h kit in class I surgery and 1 vancomycin 24 h kit (betalactamase antibiotic allergy); in thoracic surgery, 1 cefamandole 24 h kit was used for a thoracic wound. In visceral surgery, 9 different kits have been used, depending on the opening (class II) or not (class I) of the digestive tract. 797 cefazolin (first line) and 68 clindamycin/gentamicin (second line) single dose kits were used in class I surgery, and 689 amoxicillin/clavulanic acid single dose (SD) kits in class II surgery. Specific protocols consisted of 18 ceftriaxone/metronidazole and 48 metronidazole/gentamicin SD kits in oesophagus surgery, 11 ceftriaxone and 17 gentamicin SD kits in biliary endoscopy, 137 metronidazole SD kits in proctology and 34 amoxicillin/gentamicin 6 h kits for prevention of endocarditis. In urology, 133 cefotaxime and 20 pefloxacin/gentamicin SD kits were precribed in renal lithiasis, 102 amoxicillin/clavulanic acid SD kits in cystectomy, 27 amoxicillin/gentamicin 6 h kits in endocarditis prevention and 58 cefamandole SD kits in all other indications. In gynaecology and obstetrics, 534 cefazoline and 19 clindamycin/gentamicin (second line) SD kits were used, and 370 doxycyclin SD kits were prescribed in pregnancy termination. Some departments (orthopaedics and visceral surgery) adapted the protocols to their needs, specifically with regard to treatment duration. However, these situations were quickly corrected. A constant follow-up and update of this system, associated with routine audits, should allow the maintenance and possibly the improvement of these results, hence shortening treatment duration.
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Singh, S V; Singh, A V; Singh, R; Sharma, S; Shukla, N; Misra, S; Singh, P K; Sohal, J S; Kumar, H; Patil, P K; Misra, P; Sandhu, K S
2008-09-01
Present pilot study is the first attempt in the country to estimate sero-prevalence of Bovine Johne's disease (BJD) by screening cattle and buffaloes representing large population belonging to farmer's and farm herds in the home tracts (Uttar Pradesh (UP) and Punjab) of Hariana cattle and Murrah buffaloes in North India. Indigenous and in-house plate ELISA kit (using protoplasmic antigen from native Mycobacterium avium subsp. paratuberculosis 'Bison type' strain of goat origin), originally developed for goats and sheep was standardized in bovines and used for screening. For this study, 33 villages of south and west UP were randomly selected and surveyed from 2001 to 2003. There were 7943 farmer's families having 38,251 livestock, including cattle, buffaloes, goats and sheep (per family 4.8% livestock). Numerically, buffaloes and cattle were 54.7% and 22.1%, respectively. Serum samples were collected from 726 animals (4.2% of 16, 981 livestock with 4375 farmer's families) located in 33 randomly surveyed villages. Serum samples (699), submitted to Epidemiology Department of Veterinary College (Punjab Agricultural University, Ludhiana), in the year 2004 by farmer's and organized farm herds (Buffaloes, 372, Cattle, 327), were screened by this ELISA kit. Soluble protoplasmic antigen was prepared from Map (S 5) 'Bison type' strain isolated from a terminally sick goat with Johne's disease. Of the total 1425 bovine (Buffaloes and cattle) serum samples screened using indigenous ELISA kit, sero-prevalence of Johne's disease was 29.0% (28.6% in buffalo and 29.8% in cattle) in Northern India. State-wise sero-prevalence was 31.9% and 23.3% in UP and Punjab, respectively. In UP, of the 601 randomly sampled buffaloes, sero-prevalence was 40.3% (16.6% in young and 40.9% adults) and 25.5% (10.5% in young and 26.3% adults) in south and west UP, respectively. Of the 125 cattle screened, sero-prevalence was 42.6% (nil in young and 44.4% adults) and 30.0% (nil in young and 30.6% adults) in south and west UP, respectively. Of the 699 serum samples screened from Ludhiana, Punjab, sero-prevalence of BJD was 23.0%. Sero-prevalence was 23.3% (12.1% in young and 24.4% in adults) and 26.9% (27.2% in young and 26.8% in adults) in buffaloes and cattle, respectively. High prevalence of BJD in buffaloes in native tract of Murrah breed, and Hariana breed of cattle correlated with poor per-animal productivity and BJD may be the major cause. Indigenous ELISA kit was rapid, economic and sensitive test for large-scale screening of buffaloes and cattle population against incurable BJD.
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49 CFR 173.165 - Polyester resin kits.
Code of Federal Regulations, 2012 CFR
2012-10-01
... 49 Transportation 2 2012-10-01 2012-10-01 false Polyester resin kits. 173.165 Section 173.165... Polyester resin kits. (a) Except for transportation by aircraft, polyester resin kits consisting of a base... resin kits consisting of a base material component (Class 3, Packing Group II or III) and an activator...
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Islam, Rafiqul; Kar, Sumit; Islam, Clarinda; Farmen, Raymond
2018-06-01
There has been an increased use of commercial kits for biomarker measurement, commensurate with the increased demand for biomarkers in drug development. However, in most cases these kits do not meet the quality attributes for use in regulated environment. The process for adaptation of these kits can be frustrating, time consuming and resource intensive. In addition, a lack of harmonized guidance for the validation of biomarker poses a significant challenge in the adaptation of kits in a regulated environment. The purpose of this perspective is to propose a tiered approach to commercial drug development kits with clearly defined quality attributes and to demonstrate how these kits can be adapted to perform analytical validation in a regulated environment.
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The Use of Kits in the Analysis of Tissue Lipids Requires Validation.
Rider, T; LeBoeuf, R C; Tso, Patrick; Jandacek, R J
2016-04-01
The ready availability and ease of use of kits for the measurement of serum lipids has greatly facilitated these measurements. In many cases it would be convenient to use these kits in the determination of lipid concentrations in tissues. The successful application of serum kits in tissue analysis requires that two important issues be considered. First, the solvent system for the extraction of the lipids and the solvent used for analysis by the kit must be compatible with the reactions in the kit. Second, the concentration range in the analyzed solution must be within the range for which the kit is used. We report here that lipids in liver and adipose tissues may be significantly underestimated by the use of some kits. We recommend that the use of kits for tissue analysis of lipids be validated for the specific analysis.
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Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site.
Herbst, R; Munemitsu, S; Ullrich, A
1995-01-19
The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v-kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C-terminal 50 amino acids by five unrelated residues. Examinations of individual v-kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity.
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Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo
2017-01-01
Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.
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Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo
2017-01-01
Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells. PMID:28403213
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Analysis of Altered Micro RNA Expression Profiles in Focal Cortical Dysplasia IIB.
Li, Lin; Liu, Chang-Qing; Li, Tian-Fu; Guan, Yu-Guang; Zhou, Jian; Qi, Xue-Ling; Yang, Yu-Tao; Deng, Jia-Hui; Xu, Zhi-Qing David; Luan, Guo-Ming
2016-04-01
Focal cortical dysplasia type IIB is a commonly encountered subtype of developmental malformation of the cerebral cortex and is often associated with pharmacoresistant epilepsy. In this study, to investigate the molecular etiology of focal cortical dysplasia type IIB, the authors performed micro ribonucleic acid (RNA) microarray on surgical specimens from 5 children (2 female and 3 male, mean age was 73.4 months, range 50-112 months) diagnosed of focal cortical dysplasia type IIB and matched normal tissue adjacent to the lesion. In all, 24 micro RNAs were differentially expressed in focal cortical dysplasia type IIB, and the microarray results were validated using quantitative real-time polymerase chain reaction (PCR). Then the putative target genes of the differentially expressed micro RNAs were identified by bioinformatics analysis. Moreover, biological significance of the target genes was evaluated by investigating the pathways in which the genes were enriched, and the Hippo signaling pathway was proposed to be highly related with the pathogenesis of focal cortical dysplasia type IIB. © The Author(s) 2015.
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Multiple-frame IR photo-recorder KIT-3M
DOE Office of Scientific and Technical Information (OSTI.GOV)
Roos, E; Wilkins, P; Nebeker, N
2006-05-15
This paper reports the experimental results of a high-speed multi-frame infrared camera which has been developed in Sarov at VNIIEF. Earlier [1] we discussed the possibility of creation of the multi-frame infrared radiation photo-recorder with framing frequency about 1 MHz. The basis of the photo-recorder is a semiconductor ionization camera [2, 3], which converts IR radiation of spectral range 1-10 micrometers into a visible image. Several sequential thermal images are registered by using the IR converter in conjunction with a multi-frame electron-optical camera. In the present report we discuss the performance characteristics of a prototype commercial 9-frame high-speed IR photo-recorder.more » The image converter records infrared images of thermal fields corresponding to temperatures ranging from 300 C to 2000 C with an exposure time of 1-20 {micro}s at a frame frequency up to 500 KHz. The IR-photo-recorder camera is useful for recording the time evolution of thermal fields in fast processes such as gas dynamics, ballistics, pulsed welding, thermal processing, automotive industry, aircraft construction, in pulsed-power electric experiments, and for the measurement of spatial mode characteristics of IR-laser radiation.« less
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Li, Jianjun; Zhang, Yinghui; Wang, Xiuchao; Zhao, Ruibo
2017-01-01
The expression level and roles of microRNA-497 (miR-497) have been frequently reported in previous studies on cancer. However, its expression, function and associated molecular mechanisms in retinoblastoma remain unknown. In the present study, miR-497 expression levels in human retinoblastoma tissues, normal retinal tissues and retinoblastoma cell lines were determined using reverse transcription-quantitative polymerase chain reaction. In addition, a Cell Counting Kit-8 assay, cell migration assay, cell invasion assay, western blot analysis and Dual-Luciferase reporter assay were used to explore the expression, functions and molecular mechanisms of miR-497 in human retinoblastoma. It was demonstrated that miR-497 was significantly downregulated in retinoblastoma tissues and cell lines compared with normal retinal tissues. Ectopic expression of miR-497 decreased the proliferation, migration and invasion of retinoblastoma cells. Furthermore, VEGFA was verified as a potential direct target of miR-497 in vitro. Taken together, the results indicate that miR-497 functions as a tumor suppressor in the carcinogenesis and progression of retinoblastoma via targeting VEGFA. miR-497 should be investigated as a potential therapeutic target for the treatment of retinoblastoma. PMID:28588740
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Alvarez, M Lucrecia
2014-01-01
Different target prediction algorithms have been developed to provide a list of candidate target genes for a given animal microRNAs (miRNAs). However, these computational approaches provide both false-positive and false-negative predictions. Therefore, the target genes of a specific miRNA identified in silico should be experimentally validated. In this chapter, we describe a step-by-step protocol for the experimental validation of a direct miRNA target using a faster Dual Firefly-Renilla Luciferase Reporter Assay. We describe how to construct reporter plasmids using the simple, fast, and highly efficient cold fusion cloning technology, which does not require ligase, phosphatase, or restriction enzymes. In addition, we provide a protocol for co-transfection of reporter plasmids with either miRNA mimics or miRNA inhibitors in human embryonic kidney 293 (HEK293) cells, as well as a description on how to measure Firefly and Renilla luciferase activity using the Dual-Glo Luciferase Assay kit. As an example of the use of this technology, we will validate glucose-6-phosphate dehydrogenase (G6PD) as a direct target of miR-1207-5p.
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Grzyb, Katarzyna; Rychłowski, Michał; Biegniewska, Anna; Skorkowski, Edward F
2003-02-01
Creatine kinase (CK, ATP creatine phosphotransferase, EC 2.7.3.2) is an enzyme participating in ATP regeneration, which is the primary source of energy in living organisms. We demonstrated that CK from herring spermatozoa has high activity ( approximately 452 micromol/min/g of fresh semen) and has a different electrophoretic mobility from isoenzymes present in skeletal muscle. In our study, we investigated toxic effect of tributyltin (TBT) on herring spermatozoa using a specific sperm viability kit to observe live and dead sperm cells with a confocal microscope. Treatment of herring spermatozoa with TBT caused a time-dependent decrease of viability: 35% nonviable cells with 5 microM TBT and more than 90% nonviable cells with 10 microM TBT after 6 h exposure. We also monitored CK release from damaged spermatozoa into surrounding medium containing different concentrations of TBT. The higher concentration of TBT was used the more CK release from spermatozoa was observed. We suggest that CK could be a good biomarker of sperm cell membranes degradation in the case when lactate dehydrogenase release from permeabilized cells is not possible for rapid determination of the effect of TBT.
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MicroRNAs expression profile in solid and unicystic ameloblastomas.
Setién-Olarra, A; Marichalar-Mendia, X; Bediaga, N G; Aguirre-Echebarria, P; Aguirre-Urizar, J M; Mosqueda-Taylor, A
2017-01-01
Odontogenic tumors (OT) represent a specific pathological category that includes some lesions with unpredictable biological behavior. Although most of these lesions are benign, some, such as the ameloblastoma, exhibit local aggressiveness and high recurrence rates. The most common types of ameloblastoma are the solid/multicystic (SA) and the unicystic ameloblastoma (UA); the latter considered a much less aggressive entity as compared to the SA. The microRNA system regulates the expression of many human genes while its deregulation has been associated with neoplastic development. The aim of the current study was to determine the expression profiles of microRNAs present in the two most common types of ameloblastomas. MicroRNA expression profiles were assessed using TaqMan® Low Density Arrays (TLDAs) in 24 samples (8 SA, 8 UA and 8 control samples). The findings were validated using quantitative RTqPCR in an independent cohort of 19 SA, 8 UA and 19 dentigerous cysts as controls. We identified 40 microRNAs differentially regulated in ameloblastomas, which are related to neoplastic development and differentiation, and with the osteogenic process. Further validation of the top ranked microRNAs revealed significant differences in the expression of 6 of them in relation to UA, 7 in relation to SA and 1 (miR-489) that was related to both types. We identified a new microRNA signature for the ameloblastoma and for its main types, which may be useful to better understand the etiopathogenesis of this neoplasm. In addition, we identified a microRNA (miR-489) that is suggestive of differentiating among solid from unicystic ameloblastoma.
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MicroRNAs expression profile in solid and unicystic ameloblastomas
Setién-Olarra, A.; Bediaga, N. G.; Aguirre-Echebarria, P.; Aguirre-Urizar, J. M.; Mosqueda-Taylor, A.
2017-01-01
Objectives Odontogenic tumors (OT) represent a specific pathological category that includes some lesions with unpredictable biological behavior. Although most of these lesions are benign, some, such as the ameloblastoma, exhibit local aggressiveness and high recurrence rates. The most common types of ameloblastoma are the solid/multicystic (SA) and the unicystic ameloblastoma (UA); the latter considered a much less aggressive entity as compared to the SA. The microRNA system regulates the expression of many human genes while its deregulation has been associated with neoplastic development. The aim of the current study was to determine the expression profiles of microRNAs present in the two most common types of ameloblastomas. Material & methods MicroRNA expression profiles were assessed using TaqMan® Low Density Arrays (TLDAs) in 24 samples (8 SA, 8 UA and 8 control samples). The findings were validated using quantitative RTqPCR in an independent cohort of 19 SA, 8 UA and 19 dentigerous cysts as controls. Results We identified 40 microRNAs differentially regulated in ameloblastomas, which are related to neoplastic development and differentiation, and with the osteogenic process. Further validation of the top ranked microRNAs revealed significant differences in the expression of 6 of them in relation to UA, 7 in relation to SA and 1 (miR-489) that was related to both types. Conclusion We identified a new microRNA signature for the ameloblastoma and for its main types, which may be useful to better understand the etiopathogenesis of this neoplasm. In addition, we identified a microRNA (miR-489) that is suggestive of differentiating among solid from unicystic ameloblastoma. PMID:29053755
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Micro Corona Ionizer as an Ozone Source for Bacterial Cell Lysis
NASA Astrophysics Data System (ADS)
Lee, Eun-Hee; Lim, Hyun Jeong; Chua, Beelee; Son, Ahjeong
2015-04-01
DNA extraction is a critical process of DNA assays including polymerase chain reaction (PCR), microarrays, molecular cloning, and DNA hybridization which has been well established and can be implemented by commercial kits. DNA extraction involves cell lysis, precipitation, and purification through the combination of physical and chemical processes. Cell lysis is essential to high DNA recovery yield which can be achieved via a variety of physical, chemical, and enzymatic methods. However, these methods were originally developed for bioassays that were labor intensive, time consuming, and vulnerable to contamination and inhibition. Here, we proposed to employ a micro corona ionizer as an ozone source to lyse bacterial cells. Ozone has been well known and used as a disinfectant which allows cell lysis and DNA extraction. Previously, we have shown that a micro corona ionizer is capable of generating a significant amount of ozone. In this study, we employed the micro corona ionizer for the bacterial cell lysis which consists of a 50 μm diameter cantilever wire as the discharge cathode and a 50 μm thick copper foil as anode. Applied voltages varied from 1900 to 2200 V with corresponding corona currents from 16 to 28 μA. The resultant ozone (concentration > 0.14 ppm) generated from the micro corona ionizer was bubbled into the sample via a miniature pump. We demonstrated the cell lysis of Pseudomonas putida as the target bacterium using the micro corona ionizer. At a flow rate of 38 ml/min and applied corona voltage of 2000 V, 98.5 ± 0.2% lysis (normalized to sonication result) was achieved after 10 min. In comparison, untreated and air-treated samples showed normalized % lysis of 11.9 ± 2.4 and 36.1 ± 1.7%, respectively. We also showed that the cell lysis efficiency could be significantly increased by increasing the flow rate and the applied corona voltage. By comparing the experimental results for continuous and pulsed treatment, we verified that the percentage of lysis is primarily determined by the total ozone treatment time.
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30 CFR 7.403 - Application requirements.
Code of Federal Regulations, 2013 CFR
2013-07-01
... APPROVAL OF MINING PRODUCTS TESTING BY APPLICANT OR THIRD PARTY Electric Cables, Signaling Cables, and Cable Splice Kits § 7.403 Application requirements. (a) Electric cables and signaling cables. A single... product: (1) Product information: (i) Cable type (for example, G or G-GC). (ii) Construction (for example...
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30 CFR 7.403 - Application requirements.
Code of Federal Regulations, 2012 CFR
2012-07-01
... APPROVAL OF MINING PRODUCTS TESTING BY APPLICANT OR THIRD PARTY Electric Cables, Signaling Cables, and Cable Splice Kits § 7.403 Application requirements. (a) Electric cables and signaling cables. A single... product: (1) Product information: (i) Cable type (for example, G or G-GC). (ii) Construction (for example...
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30 CFR 7.403 - Application requirements.
Code of Federal Regulations, 2014 CFR
2014-07-01
... APPROVAL OF MINING PRODUCTS TESTING BY APPLICANT OR THIRD PARTY Electric Cables, Signaling Cables, and Cable Splice Kits § 7.403 Application requirements. (a) Electric cables and signaling cables. A single... product: (1) Product information: (i) Cable type (for example, G or G-GC). (ii) Construction (for example...
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Single-cell analysis of the fate of c-kit-positive bone marrow cells
NASA Astrophysics Data System (ADS)
Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D.; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa
2017-10-01
The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.
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Single-cell analysis of the fate of c-kit-positive bone marrow cells.
Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa
2017-01-01
The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.
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[Isolation and identification methods of enterobacteria group and its technological advancement].
Furuta, Itaru
2007-08-01
In the last half-century, isolation and identification methods of enterobacteria groups have markedly improved by technological advancement. Clinical microbiology tests have changed overtime from tube methods to commercial identification kits and automated identification. Tube methods are the original method for the identification of enterobacteria groups, that is, a basically essential method to recognize bacterial fermentation and biochemical principles. In this paper, traditional tube tests are discussed, such as the utilization of carbohydrates, indole, methyl red, and citrate and urease tests. Commercial identification kits and automated instruments by computer based analysis as current methods are also discussed, and those methods provide rapidity and accuracy. Nonculture techniques of nucleic acid typing methods using PCR analysis, and immunochemical methods using monoclonal antibodies can be further developed.
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Su, Feng; Zhang, Wentian; Liu, Jianfang
2015-01-01
It has been validated that c-kit positive (c-kit+) cells in infarcted myocardium are from bone marrow (BM). Given the recent study that in the heart, estrogen receptor alpha (ERα) is involved in adaptive mechanisms by supporting cardiomyocytes survival via post-infarct cardiac c-kit+ cells, we tested a novel hypothesis that membrane ERα (mERа) supports survival of BM c-kit+ cells and enhance protective paracrine function for cardiac repair. Our data showed that myocardial infarction (MI) leads to an increase in c-kit+ first in bone marrow and then specifically within the infarcted myocardium. Also up-regulated mERа in post-infarct BM c-kit+ cells was found in day 3 post MI. In vitro co-culture system, mERа+ enhances the beneficial effects of BM c-kit+ cells by increasing their viability and reducing apoptosis. Post-infarct c-kit+ mERа+ cells population expresses predominant ERα and holds self-renewal as well as cardiac differentiation potentials after MI. In vivo, BM c-kit+ cells reduced infarct size, fibrosis and improved cardiac function. In conclusion, BM c-kit+ mERа+ exerted significantly cardiac protection after MI. A potential important implication of this study is that the manipulation of BM c-kit+ stem cells with ERа-dependent fashion may be helpful in recovering functional performance after cardiac tissue injury. PMID:26191121
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Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Veach, Darren; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R; DeMatteo, Ronald P; Besmer, Peter
2017-10-03
Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic Kit V558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant Kit V558Δ/+ mice, double-mutant Kit V558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in Kit V558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing Kit V558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant Kit V558Δ;T669I/+ tumors.
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Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q.; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R.; DeMatteo, Ronald P.; Besmer, Peter
2017-01-01
Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors. PMID:28923937
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Tomkinson, A.; Raeburn, D.
1996-01-01
1. We have examined the effects of the isoenzyme-selective phosphodiesterase (PDE) inhibitors, vinpocetine (type 1), siguazodan (type 3), rolipram (type 4) and zaprinast (type 5) and the non-selective PDE inhibitor enprofylline on methacholine (MCh) contractile concentration-response curves on guinea-pig and rat isolated ileum. 2. In guinea-pig ileum, vinpocetine (10-300 microM), zaprinast (1-300 microM) and enprofylline (100-1000 microM) produced a concentration-dependent depression of the maximum response (Emax) to MCh only without effect on the MCh EC50 values (rank order of potency: zaprinast > vinpocetine > enprofylline). In contrast, siguazodan (10-300 microM) and rolipram (10-300 microM) produced a rightward displacement of the MCh concentration-response curve (increase in EC50: rank order; rolipram > siguazodan), with effects on the MCh maximum seen only at higher concentrations. 3. In the rat ileum, vinpocetine (10-300 microM), zaprinast (0.1-300 microM) and enprofylline (100-1000 microM) caused depression of the MCh maximum contraction (rank order: zaprinast > vinpocetine > enprofylline). Low concentrations of rolipram and siguazodan had no significant effect on the MCh maximum. In the presence of higher concentrations (> 100 microM) of rolipram and siguazodan, a maximum response was not achieved at the highest concentration of MCh tested. As in the guinea-pig ileum, only rolipram (10-300 microM) and siguazodan (10-300 microM) produced a significant, concentration-dependent, rightward displacement of the MCh concentration-response curve (increase in EC50: rank order: rolipram > siguazodan). 4. In the guinea-pig ileum, isoprenaline (0.1 microM) produced a rightward displacement (approximately 3 fold) of the MCh concentration-response curve, accompanied by a significant depression of the maximum response. Increasing the isoprenaline concentration (1 microM) had no further effect on either parameter. Sodium nitroprusside (SNP, > or = 10 microM) produced a concentration-dependent depression of the MCh maximum without an effect on the EC50. 5. In the rat ileum, isoprenaline (1 microM) produced a concentration-dependent rightward displacement (approximately 2.8 fold) of the MCh concentration-response curve with depression of the MCh maximum at higher (> or = 100 microM) concentrations. SNP produced depression of the MCh maximum at a concentration of 10 microM and above. Effects on the MCh EC50 were seen only at 100 and 300 microM. 6. In guinea-pig ileum, isoprenaline (0.1 microM) in combination with rolipram (10 microM) further increased the MCh EC50 and reduced the MCh maximum. The combination of SNP (10 microM) with zaprinast (0.1 microM) produced no further significant effect than SNP alone. 7. In rat ileum, isoprenaline (1 microM) in combination with rolipram (10 microM) further increased the EC50 and reduced the maximum. SNP (10 microM) had no significant effect on either the MCh maximum or EC50. A combination with zaprinast (1 microM) had no further effect. 8. In conclusion, all the PDE inhibitors tested produced a concentration-dependent inhibition of the MCh concentration-response curve, indicating a modulator role for the PDE isoenzymes in gastrointestinal smooth muscle contractility. The PDE inhibitors that elevate cyclic GMP produced a depression of the MCh maximum response only, whilst those that elevate cyclic AMP produced a rightward displacement of the MCh concentration-response curve. This was confirmed by the use of isoprenaline and SNP. This difference in the type of inhibition produced by these PDE isoenzyme inhibitors may reflect a different intracellular site/mechanism by which the cyclic AMP- and cyclic GMP-activated kinases act functionally to antagonize the contractile response. PMID:8864552
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21 CFR 868.1100 - Arterial blood sampling kit.
Code of Federal Regulations, 2010 CFR
2010-04-01
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...
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40 CFR 745.88 - Recognized test kits.
Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Recognized test kits. 745.88 Section... Renovation § 745.88 Recognized test kits. (a) Effective June 23, 2008, EPA recognizes the test kits that have... publicizes its recognition of the first test kit that meets both the negative response and positive response...
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21 CFR 868.1100 - Arterial blood sampling kit.
Code of Federal Regulations, 2014 CFR
2014-04-01
... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...
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21 CFR 868.1100 - Arterial blood sampling kit.
Code of Federal Regulations, 2013 CFR
2013-04-01
... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...
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21 CFR 868.1100 - Arterial blood sampling kit.
Code of Federal Regulations, 2011 CFR
2011-04-01
... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...
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21 CFR 868.1100 - Arterial blood sampling kit.
Code of Federal Regulations, 2012 CFR
2012-04-01
... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Arterial blood sampling kit. 868.1100 Section 868...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Diagnostic Devices § 868.1100 Arterial blood sampling kit. (a) Identification. An arterial blood sampling kit is a device, in kit form, used to obtain arterial blood samples...
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46 CFR 121.710 - First-aid kits.
Code of Federal Regulations, 2012 CFR
2012-10-01
... 46 Shipping 4 2012-10-01 2012-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents and instructions. For equivalent...
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19 CFR 122.132 - Sealing of aircraft liquor kits.
Code of Federal Regulations, 2010 CFR
2010-04-01
... 19 Customs Duties 1 2010-04-01 2010-04-01 false Sealing of aircraft liquor kits. 122.132 Section... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.132 Sealing of aircraft liquor kits. (a) Sealing required. Aircraft liquor kits shall be sealed on board the aircraft by crewmembers...
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46 CFR 184.710 - First-aid kits.
Code of Federal Regulations, 2012 CFR
2012-10-01
... 46 Shipping 7 2012-10-01 2012-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents...
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21 CFR 870.1350 - Catheter balloon repair kit.
Code of Federal Regulations, 2012 CFR
2012-04-01
... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Catheter balloon repair kit. 870.1350 Section 870... repair kit. (a) Identification. A catheter balloon repair kit is a device used to repair or replace the balloon of a balloon catheter. The kit contains the materials, such as glue and balloons, necessary to...
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21 CFR 870.1350 - Catheter balloon repair kit.
Code of Federal Regulations, 2013 CFR
2013-04-01
... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Catheter balloon repair kit. 870.1350 Section 870... repair kit. (a) Identification. A catheter balloon repair kit is a device used to repair or replace the balloon of a balloon catheter. The kit contains the materials, such as glue and balloons, necessary to...
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46 CFR 184.710 - First-aid kits.
Code of Federal Regulations, 2013 CFR
2013-10-01
... 46 Shipping 7 2013-10-01 2013-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents...
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46 CFR 121.710 - First-aid kits.
Code of Federal Regulations, 2014 CFR
2014-10-01
... 46 Shipping 4 2014-10-01 2014-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents and instructions. For equivalent...
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19 CFR 122.132 - Sealing of aircraft liquor kits.
Code of Federal Regulations, 2011 CFR
2011-04-01
... 19 Customs Duties 1 2011-04-01 2011-04-01 false Sealing of aircraft liquor kits. 122.132 Section... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.132 Sealing of aircraft liquor kits. (a) Sealing required. Aircraft liquor kits shall be sealed on board the aircraft by crewmembers...
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21 CFR 870.1350 - Catheter balloon repair kit.
Code of Federal Regulations, 2014 CFR
2014-04-01
... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Catheter balloon repair kit. 870.1350 Section 870... repair kit. (a) Identification. A catheter balloon repair kit is a device used to repair or replace the balloon of a balloon catheter. The kit contains the materials, such as glue and balloons, necessary to...
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46 CFR 121.710 - First-aid kits.
Code of Federal Regulations, 2011 CFR
2011-10-01
... 46 Shipping 4 2011-10-01 2011-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents and instructions. For equivalent...
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46 CFR 184.710 - First-aid kits.
Code of Federal Regulations, 2014 CFR
2014-10-01
... 46 Shipping 7 2014-10-01 2014-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents...
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46 CFR 121.710 - First-aid kits.
Code of Federal Regulations, 2013 CFR
2013-10-01
... 46 Shipping 4 2013-10-01 2013-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents and instructions. For equivalent...
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19 CFR 122.132 - Sealing of aircraft liquor kits.
Code of Federal Regulations, 2013 CFR
2013-04-01
... 19 Customs Duties 1 2013-04-01 2013-04-01 false Sealing of aircraft liquor kits. 122.132 Section... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.132 Sealing of aircraft liquor kits. (a) Sealing required. Aircraft liquor kits shall be sealed on board the aircraft by crewmembers...
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19 CFR 122.132 - Sealing of aircraft liquor kits.
Code of Federal Regulations, 2012 CFR
2012-04-01
... 19 Customs Duties 1 2012-04-01 2012-04-01 false Sealing of aircraft liquor kits. 122.132 Section... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.132 Sealing of aircraft liquor kits. (a) Sealing required. Aircraft liquor kits shall be sealed on board the aircraft by crewmembers...
-
46 CFR 184.710 - First-aid kits.
Code of Federal Regulations, 2011 CFR
2011-10-01
... 46 Shipping 7 2011-10-01 2011-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents...
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19 CFR 122.132 - Sealing of aircraft liquor kits.
Code of Federal Regulations, 2014 CFR
2014-04-01
... 19 Customs Duties 1 2014-04-01 2014-04-01 false Sealing of aircraft liquor kits. 122.132 Section... OF THE TREASURY AIR COMMERCE REGULATIONS Aircraft Liquor Kits § 122.132 Sealing of aircraft liquor kits. (a) Sealing required. Aircraft liquor kits shall be sealed on board the aircraft by crewmembers...
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DEMONSTRATION BULLETIN: CLOR-N-SOIL PCB TEST KIT L2000 PCB/CHLORIDE ANALYZER - DEXSIL CORP.
DEXSIL CORP(Environmental Test Kits)The Dexsil Corporation (Dexsil) produces two test kits that detect polychlorinated biphenyls (PCB) in soil: the Dexsil Clor-N-Soil PCB Screening Kit, and the Dexsil L2000 PCB/Chloride Analyzer. The Dexsil Clor-N-Soil PCB Screening Kit extr...
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46 CFR 184.710 - First-aid kits.
Code of Federal Regulations, 2010 CFR
2010-10-01
... 46 Shipping 7 2010-10-01 2010-10-01 false First-aid kits. 184.710 Section 184.710 Shipping COAST... CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT Miscellaneous § 184.710 First-aid kits. A vessel must carry either a first-aid kit approved under approval series 160.041 or a kit with equivalent contents...
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Investigation of c-KIT and Ki67 expression in normal, preneoplastic and neoplastic canine prostate.
Fonseca-Alves, Carlos Eduardo; Kobayashi, Priscilla Emiko; Palmieri, Chiara; Laufer-Amorim, Renée
2017-12-06
c-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines. Eleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive. The present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer.
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Disruption of c-Kit Signaling in Kit(W-sh/W-sh) Growing Mice Increases Bone Turnover.
Lotinun, Sutada; Krishnamra, Nateetip
2016-08-16
c-Kit tyrosine kinase receptor has been identified as a regulator of bone homeostasis. The c-Kit loss-of-function mutations in WBB6F1/J-Kit(W/W-v) mice result in low bone mass. However, these mice are sterile and it is unclear whether the observed skeletal phenotype is secondary to a sex hormone deficiency. In contrast, C57BL/6J-Kit(W-sh)/(W-sh) (W(sh)/W(sh)) mice, which carry an inversion mutation affecting the transcriptional regulatory elements of the c-Kit gene, are fertile. Here, we showed that W(sh)/W(sh) mice exhibited osteopenia with elevated bone resorption and bone formation at 6- and 9-week-old. The c-Kit W(sh) mutation increased osteoclast differentiation, the number of committed osteoprogenitors, alkaline phosphatase activity and mineralization. c-Kit was expressed in both osteoclasts and osteoblasts, and c-Kit expression was decreased in W(sh)/W(sh)osteoclasts, but not osteoblasts, suggesting an indirect effect of c-Kit on bone formation. Furthermore, the osteoclast-derived coupling factor Wnt10b mRNA was increased in W(sh)/W(sh) osteoclasts. Conditioned medium from W(sh)/W(sh) osteoclasts had elevated Wnt10b protein levels and induced increased alkaline phosphatase activity and mineralization in osteoblast cultures. Antagonizing Wnt10b signaling with DKK1 or Wnt10b antibody inhibited these effects. Our data suggest that c-Kit negatively regulates bone turnover, and disrupted c-Kit signaling couples increased bone resorption with bone formation through osteoclast-derived Wnt 10 b.
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Exacerbated experimental autoimmune encephalomyelitis in mast-cell-deficient Kit W-sh/W-sh mice.
Piconese, Silvia; Costanza, Massimo; Musio, Silvia; Tripodo, Claudio; Poliani, Pietro L; Gri, Giorgia; Burocchi, Alessia; Pittoni, Paola; Gorzanelli, Andrea; Colombo, Mario P; Pedotti, Rosetta
2011-04-01
Mast cell (MC)-deficient c-Kit mutant Kit(W/W-v) mice are protected against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, suggesting a detrimental role for MCs in this disease. To further investigate the role of MCs in EAE, we took advantage of a recently characterized model of MC deficiency, Kit(W-sh/W-sh). Surprisingly, we observed that myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced chronic EAE was exacerbated in Kit(W-sh/W-sh) compared with Kit(+/+) mice. Kit(W-sh/W-sh) mice showed more inflammatory foci in the central nervous system (CNS) and increased T-cell response against myelin. To understand whether the discrepant results obtained in Kit(W-sh/W-sh) and in Kit(W/W-v) mice were because of the different immunization protocols, we induced EAE in these two strains with varying doses of MOG(35-55) and adjuvants. Although Kit(W-sh/W-sh) mice exhibited exacerbated EAE under all immunization protocols, Kit(W/W-v) mice were protected from EAE only when immunized with high, but not low, doses of antigen and adjuvants. Kit(W-sh/W-sh) mice reconstituted systemically, but not in the CNS, with bone marrow-derived MCs still developed exacerbated EAE, indicating that protection from disease could be exerted by MCs mainly in the CNS, and/or by other cells possibly dysregulated in Kit(W-sh/W-sh) mice. In summary, these data suggest to reconsider MC contribution to EAE, taking into account the variables of using different experimental models and immunization protocols.
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Business Pattern of Distributed Energy in Electric Power System Reformation
NASA Astrophysics Data System (ADS)
Liang, YUE; Zhuochu, LIU; Jun, LI; Siwei, LI
2017-05-01
Under the trend of the electric power system revolution, the operation mode of micro power grid that including distributed power will be more diversified. User’s demand response and different strategies on electricity all have great influence on the operation of distributed power grid. This paper will not only research sensitive factors of micro power grid operation, but also analyze and calculate the cost and benefit of micro power grid operation upon different types. Then it will build a tech-economic calculation model, which applies to different types of micro power grid under the reformation of electric power system.
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Discovery of amido-benzisoxazoles as potent c-Kit inhibitors
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kunz, Roxanne K.; Rumfelt, Shannon; Chen, Ning
2010-01-12
Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.
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Mazzia, C; Porcher, C; Julé, Y; Christen, M O; Henry, M
2000-05-01
C-kit immunocytochemistry was performed on ultrathin sections of human distal colon. Our attention was focused on relationships between c-kit immunoreactive interstitial cells (c-kit ICs) and muscular cells and nervous elements located in the external muscular layers of the colonic wall. C-kit ICs established membrane apposition with both nerve fibers and smooth muscle cells of, respectively, the longitudinal and circular muscle layers, the myenteric area, and the extremus submucosus plexus. C-kit ICs also surrounded the external submucosus plexus and established membrane appositions with nerve elements located inside the myenteric ganglia. These membrane appositions were observed either at the level of the c-kit IC bodies or at that of their cytoplasmic processes. In some cases, membrane appositions were observed concomitantly between the c-kit ICs, nerve fibers, and smooth muscle cells. In all the regions studied, the c-kit ICs were also found to be located in the close vicinity of blood vessels and to have established close contacts with non-immunoreactive fibroblast-like cells. The results of the present study shed essential light on the relationships of c-kit ICs with the neighboring muscle cells and nerve elements, and confirm that the intercalated c-kit ICs well fit with the so-called "interstitial cells of Cajal".
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Analysis of Platelet-Rich Plasma Extraction
Fitzpatrick, Jane; Bulsara, Max K.; McCrory, Paul Robert; Richardson, Martin D.; Zheng, Ming Hao
2017-01-01
Background: Platelet-rich plasma (PRP) has been extensively used as a treatment in tissue healing in tendinopathy, muscle injury, and osteoarthritis. However, there is variation in methods of extraction, and this produces different types of PRP. Purpose: To determine the composition of PRP obtained from 4 commercial separation kits, which would allow assessment of current classification systems used in cross-study comparisons. Study Design: Controlled laboratory study. Methods: Three normal adults each donated 181 mL of whole blood, some of which served as a control and the remainder of which was processed through 4 PRP separation kits: GPS III (Biomet Biologics), Smart-Prep2 (Harvest Terumo), Magellan (Arteriocyte Medical Systems), and ACP (Device Technologies). The resultant PRP was tested for platelet count, red blood cell count, and white blood cell count, including differential in a commercial pathology laboratory. Glucose and pH measurements were obtained from a blood gas autoanalyzer machine. Results: Three kits taking samples from the “buffy coat layer” were found to have greater concentrations of platelets (3-6 times baseline), while 1 kit taking samples from plasma was found to have platelet concentrations of only 1.5 times baseline. The same 3 kits produced an increased concentration of white blood cells (3-6 times baseline); these consisted of neutrophils, leukocytes, and monocytes. This represents high concentrations of platelets and white blood cells. A small drop in pH was thought to relate to the citrate used in the sample preparation. Interestingly, an unexpected increase in glucose concentrations, with 3 to 6 times greater than baseline levels, was found in all samples. Conclusion: This study reveals the variation of blood components, including platelets, red blood cells, leukocytes, pH, and glucose in PRP extractions. The high concentrations of cells are important, as the white blood cell count in PRP samples has frequently been ignored, being considered insignificant. The lack of standardization of PRP preparation for clinical use has contributed at least in part to the varying clinical efficacy in PRP use. Clinical Relevance: The variation of platelet and other blood component concentrations between commercial PRP kits may affect clinical treatment outcomes. There is a need for standardization of PRP for clinical use. PMID:28210651
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Terada, Tadashi
2014-10-01
The molecular mechanisms of ductal plate (DP) development and differentiation (DD) in human fetal livers (HFLs) are unclear. The author immunohistochemically investigated expressions of NCAM, KIT, KIT, PDGFRA, and neuroendocrine antigens in 32 HFLs. The processes of human intrahepatic bile duct (IBD) DD could be categorized into four stages: DP, remodeling DP, remodeled DP, and mature IBD. NCAM was always expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. The biliary elements were positive for cytokeratin (CK)7, 8, 18, and 19. The hepatoblasts were positive for CK8 and CD18, but negative for CK7 and CK19; however, periportal hepatoblasts showed biliary-type CKs (CK7 and CK19). NCAM was always expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. KIT was occasionally (12/32 cases) expressed in DP and remodeling DP, but not in remodeled DP and mature IBD. NCAM expression was also seen in some hepatoblasts and hematopoietic cells and neurons. KIT was also expressed in some hepatoblasts, hematopoietic cells, and mast cells. MET and PDGFRA were strongly expressed in DP, remodeling DP, remodeled DP, and mature IBD. MET and PDGFRA were also strongly expressed in hepatoblasts and hematopoietic cells. MET and PDGFRA were not expressed in portal mesenchyme, portal veins, sinusoids, and hepatic veins. DP showed immunoreactive chromogranin, synaptophysin, neuron-specific enolase (NSE), and CD56. Expressions of chromogranin and CD56 were infrequently seen in remodeling DP. No expressions of these four neuroendocrine antigens were seen in remodeled DP and mature IBD. The nerve fibers were consistently positive for chromogranin, synaptophysin, NSE, and CD56 in the portal mesenchyme in the stages of remodeling DP, remodeled DP, and mature IBDs. The data suggest that NCAM, KIT/stem cell factor-signaling, NSE, hepatocyte growth factor/MET signaling, PDGFα/PDGFRA signaling, chromogranin, synaptophysin, and CD56 play important roles in DD of biliary cells of HFL. They also suggest that the DP cells having neuroendocrine molecules give rise to hepatic stem/progenitor cells. © 2014 Wiley Periodicals, Inc.
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Fontanesi, Luca; Vargiolu, Manuela; Scotti, Emilio; Latorre, Rocco; Faussone Pellegrini, Maria Simonetta; Mazzoni, Maurizio; Asti, Martina; Chiocchetti, Roberto; Romeo, Giovanni; Clavenzani, Paolo; De Giorgio, Roberto
2014-01-01
The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated ("mega") cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ=0.00 LOD =75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5-10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon.
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Fontanesi, Luca; Vargiolu, Manuela; Scotti, Emilio; Latorre, Rocco; Faussone Pellegrini, Maria Simonetta; Mazzoni, Maurizio; Asti, Martina; Chiocchetti, Roberto; Romeo, Giovanni; Clavenzani, Paolo; De Giorgio, Roberto
2014-01-01
The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated (“mega”) cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ = 0.00 LOD = 75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5–10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon. PMID:24736498
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46 CFR 121.710 - First-aid kits.
Code of Federal Regulations, 2010 CFR
2010-10-01
... 46 Shipping 4 2010-10-01 2010-10-01 false First-aid kits. 121.710 Section 121.710 Shipping COAST... SYSTEMS AND EQUIPMENT Miscellaneous § 121.710 First-aid kits. A vessel must carry either a first-aid kit... kits, the contents must be stowed in a suitable, watertight container that is marked “First-Aid Kit”. A...
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Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes
Liu, Qiaozhen; Yang, Rui; Huang, Xiuzhen; Zhang, Hui; He, Lingjuan; Zhang, Libo; Tian, Xueying; Nie, Yu; Hu, Shengshou; Yan, Yan; Zhang, Li; Qiao, Zengyong; Wang, Qing-Dong; Lui, Kathy O; Zhou, Bin
2016-01-01
Cardiac cells marked by c-Kit or Kit, dubbed cardiac stem cells (CSCs), are in clinical trials to investigate their ability to stimulate cardiac regeneration and repair. These studies were initially motivated by the purported cardiogenic activity of these cells. Recent lineage tracing studies using Kit promoter to drive expression of the inducible Cre recombinase showed that these CSCs had highly limited cardiogenic activity, inadequate to support efficient cardiac repair. Here we reassess the lineage tracing data by investigating the identity of cells immediately after Cre labeling. Our instant lineage tracing approach identifies Kit-expressing cardiomyocytes, which are labeled immediately after tamoxifen induction. In combination with long-term lineage tracing experiments, these data reveal that the large majority of long-term labeled cardiomyocytes are pre-existing Kit-expressing cardiomyocytes rather than cardiomyocytes formed de novo from CSCs. This study presents a new interpretation for the contribution of Kit+ cells to cardiomyocytes and shows that Kit genetic lineage tracing over-estimates the cardiogenic activity of Kit+ CSCs. PMID:26634606
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Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yuzawa,S.; Opatowsky, Y.; Zhang, Z.
2007-01-01
Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4more » interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.« less
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Parichy, D M; Ransom, D G; Paw, B; Zon, L I; Johnson, S L
2000-07-01
Developmental mechanisms underlying traits expressed in larval and adult vertebrates remain largely unknown. Pigment patterns of fishes provide an opportunity to identify genes and cell behaviors required for postembryonic morphogenesis and differentiation. In the zebrafish, Danio rerio, pigment patterns reflect the spatial arrangements of three classes of neural crest-derived pigment cells: black melanocytes, yellow xanthophores and silver iridophores. We show that the D. rerio pigment pattern mutant panther ablates xanthophores in embryos and adults and has defects in the development of the adult pattern of melanocyte stripes. We find that panther corresponds to an orthologue of the c-fms gene, which encodes a type III receptor tyrosine kinase and is the closest known homologue of the previously identified pigment pattern gene, kit. In mouse, fms is essential for the development of macrophage and osteoclast lineages and has not been implicated in neural crest or pigment cell development. In contrast, our analyses demonstrate that fms is expressed and required by D. rerio xanthophore precursors and that fms promotes the normal patterning of melanocyte death and migration during adult stripe formation. Finally, we show that fms is required for the appearance of a late developing, kit-independent subpopulation of adult melanocytes. These findings reveal an unexpected role for fms in pigment pattern development and demonstrate that parallel neural crest-derived pigment cell populations depend on the activities of two essentially paralogous genes, kit and fms.
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WACOP (Westside Area Career/Occupation Project) Media Catalog for 1973, 1974.
ERIC Educational Resources Information Center
Glur, John; Catalano, Ruth
The catalog lists various commercially prepared media materials available on loan from the Westside Area Career Occupation Project (WACOP) Media Center for evaluation prior to school or district purchase. Among the types of media specified are: books, kits, cassettes, films, instructional units, transparencies, posters, pamphlets, and magazines.…
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Immunological Prevention of Spontaneous Mammary Carcinoma in Transgenic Mice
2001-08-01
respectively, and a sera Mouse Typer Iso- TUBO cell inhibition was found in BALB/c mice injected with typing kit (Bio-Rad, Richmond , CA) as previously described...Curr. Opin. Immunol., 6: 41. Lapp6, M. A., and Prehn . R. T. Immunologic surveillance at the macroscopic level: 414-419. 1994. nonselective
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Code of Federal Regulations, 2010 CFR
2012-10-01
... 46 Shipping 6 2012-10-01 2012-10-01 false Contents. 160.054-4 Section 160.054-4 Shipping COAST...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-4 Contents. (a) Individual cartons. Cartons and their contents shall be the type indicated in Specification...
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Code of Federal Regulations, 2010 CFR
2005-10-01
... 46 Shipping 6 2005-10-01 2004-10-01 true Contents. 160.054-4 Section 160.054-4 Shipping Coast...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Kits, First-Aid, for Inflatable Liferafts § 160.054-4 Contents. (a) Individual cartons. Cartons and their contents shall be the type indicated in Specification...
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Pneumatics. Student's Manual [and] Instructor's Guide.
ERIC Educational Resources Information Center
Notgrass, Troy
The student's manual in this two-part instructional kit is designed to help the following types of students learn introductory technical material related to pneumatics: (1) groups of trade and industrial students with a variety of vocational objectives and (2) homogeneous groups in which the learning levels of individual students vary…
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Advisory List of Computer-Related Materials.
ERIC Educational Resources Information Center
North Carolina State Dept. of Public Instruction, Raleigh. Div. of Educational Media.
Materials appropriate for instruction in the use of computers in the intermediate grades, junior high schools, and high schools are listed in this advisory list by type of media: activity cards; books; books (for teacher use); books (supplementary texts); book (workbook); book (workbook) with disks; filmstrips (sound); kit (including disks, guide…
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78 FR 52511 - Procurement List; Additions and Deletions
Federal Register 2010, 2011, 2012, 2013, 2014
2013-08-23
..., W7NX USPFO Activity PA ARNG, Annville, PA. Service Type/Location: Integrated Prime Vendor Supply Chain... of PM FSS, does not have a current contract for the provision of Integrated Prime Vendor Supply Chain... kitting or other supply chain management services required were obtained through the DLA depot system. The...
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Cataloging, Processing, Administering AV Materials. A Model for Wisconsin Schools.
ERIC Educational Resources Information Center
Little, Robert D., Ed.
The objective of this cataloging manual is to recommend specific methods for cataloging audiovisual materials for use in individual school media centers. The following types of audiovisual aids are included: educational games, filmstrips, flat graphics, kits, models, motion pictures, realia, records, slides, sound filmstrips, tapes,…
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Effect of planecta and ROSE™ on the frequency characteristics of blood pressure-transducer kits.
Fujiwara, Shigeki; Kawakubo, Yoshifumi; Mori, Satoshi; Tachihara, Keiichi; Toyoguchi, Izumi; Yokoyama, Takeshi
2015-12-01
Pressure-transducer kits have frequency characteristics such as natural frequency and damping coefficient, which affect the monitoring accuracy. The aim of the present study was to investigate the effect of planecta ports and a damping device (ROSE™, Argon Medical Devices, TX, USA) on the frequency characteristics of pressure-transducer kits. The FloTrac sensor kit (Edwards Lifesciences, CA, USA) and the DTXplus transducer kit (Argon Medical Devices) were prepared with planecta ports, and their frequency characteristics were tested with or without ROSE™. The natural frequency and damping coefficient of each kit were obtained using frequency characteristics analysis software and evaluated by plotting them on the Gardner's chart. By inserting a planecta port, the natural frequency markedly decreased in both the FloTrac sensor kit (from 40 to 22 Hz) and the DTXplus transducer kit (from 35 to 22 Hz). In both kits with one planecta port, the damping coefficient markedly increased by insertion of ROSE™ from 0.2 to 0.5, optimising frequency characteristics. In both kits with two planecta ports, however, the natural frequency decreased from 22 to 12 Hz. The damping coefficient increased from 0.2 to 0.8 by insertion of ROSE™; however, optimisation was not achieved even by ROSE™ insertion. Planecta ports decrease the natural frequency of the kit. ROSE™ is useful to optimise the frequency characteristics in the kits without or with one planecta port. However, optimisation is difficult with two or more planecta ports, even with the ROSE™ device.
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NASA Technical Reports Server (NTRS)
Schneider, Michelle
2003-01-01
This viewgraph representation provides an overview of the Telescience Resource Kit. The Telescience Resource Kit is a pc-based telemetry and command system that will be used by scientists and engineers to monitor and control experiments located on-board the International Space Station (ISS). Topics covered include: ISS Payload Telemetry and Command Flow, kit computer applications, kit telemetry capabilities, command capabilities, and training/testing capabilities.
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Chauvot de Beauchêne, Isaure; Allain, Ariane; Panel, Nicolas; Laine, Elodie; Trouvé, Alain; Dubreuil, Patrice; Tchertanov, Luba
2014-01-01
Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D) localized in crucial regulatory segments, the juxtamembrane region (JMR) and the activation (A-) loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts. PMID:25079768
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NASA Astrophysics Data System (ADS)
Trost, Nico; Jiménez, Javier; Imke, Uwe; Sanchez, Victor
2014-06-01
TWOPORFLOW is a thermo-hydraulic code based on a porous media approach to simulate single- and two-phase flow including boiling. It is under development at the Institute for Neutron Physics and Reactor Technology (INR) at KIT. The code features a 3D transient solution of the mass, momentum and energy conservation equations for two inter-penetrating fluids with a semi-implicit continuous Eulerian type solver. The application domain of TWOPORFLOW includes the flow in standard porous media and in structured porous media such as micro-channels and cores of nuclear power plants. In the latter case, the fluid domain is coupled to a fuel rod model, describing the heat flow inside the solid structure. In this work, detailed profiling tools have been utilized to determine the optimization potential of TWOPORFLOW. As a result, bottle-necks were identified and reduced in the most feasible way, leading for instance to an optimization of the water-steam property computation. Furthermore, an OpenMP implementation addressing the routines in charge of inter-phase momentum-, energy- and mass-coupling delivered good performance together with a high scalability on shared memory architectures. In contrast to that, the approach for distributed memory systems was to solve sub-problems resulting by the decomposition of the initial Cartesian geometry. Thread communication for the sub-problem boundary updates was accomplished by the Message Passing Interface (MPI) standard.
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The Liquid Argon Software Toolkit (LArSoft): Goals, Status and Plan
DOE Office of Scientific and Technical Information (OSTI.GOV)
Pordes, Rush; Snider, Erica
LArSoft is a toolkit that provides a software infrastructure and algorithms for the simulation, reconstruction and analysis of events in Liquid Argon Time Projection Chambers (LArTPCs). It is used by the ArgoNeuT, LArIAT, MicroBooNE, DUNE (including 35ton prototype and ProtoDUNE) and SBND experiments. The LArSoft collaboration provides an environment for the development, use, and sharing of code across experiments. The ultimate goal is to develop fully automatic processes for reconstruction and analysis of LArTPC events. The toolkit is based on the art framework and has a well-defined architecture to interface to other packages, including to GEANT4 and GENIE simulation softwaremore » and the Pandora software development kit for pattern recognition. It is designed to facilitate and support the evolution of algorithms including their transition to new computing platforms. The development of the toolkit is driven by the scientific stakeholders involved. The core infrastructure includes standard definitions of types and constants, means to input experiment geometries as well as meta and event- data in several formats, and relevant general utilities. Examples of algorithms experiments have contributed to date are: photon-propagation; particle identification; hit finding, track finding and fitting; electromagnetic shower identification and reconstruction. We report on the status of the toolkit and plans for future work.« less
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Hodi, F Stephen; Corless, Christopher L; Giobbie-Hurder, Anita; Fletcher, Jonathan A; Zhu, Meijun; Marino-Enriquez, Adrian; Friedlander, Philip; Gonzalez, Rene; Weber, Jeffrey S; Gajewski, Thomas F; O'Day, Steven J; Kim, Kevin B; Lawrence, Donald; Flaherty, Keith T; Luke, Jason J; Collichio, Frances A; Ernstoff, Marc S; Heinrich, Michael C; Beadling, Carol; Zukotynski, Katherine A; Yap, Jeffrey T; Van den Abbeele, Annick D; Demetri, George D; Fisher, David E
2013-09-10
Amplifications and mutations in the KIT proto-oncogene in subsets of melanomas provide therapeutic opportunities. We conducted a multicenter phase II trial of imatinib in metastatic mucosal, acral, or chronically sun-damaged (CSD) melanoma with KIT amplifications and/or mutations. Patients received imatinib 400 mg once per day or 400 mg twice per day if there was no initial response. Dose reductions were permitted for treatment-related toxicities. Additional oncogene mutation screening was performed by mass spectroscopy. Twenty-five patients were enrolled (24 evaluable). Eight patients (33%) had tumors with KIT mutations, 11 (46%) with KIT amplifications, and five (21%) with both. Median follow-up was 10.6 months (range, 3.7 to 27.1 months). Best overall response rate (BORR) was 29% (21% excluding nonconfirmed responses) with a two-stage 95% CI of 13% to 51%. BORR was significantly greater than the hypothesized null of 5% and statistically significantly different by mutation status (7 of 13 or 54% KIT mutated v 0% KIT amplified only). There were no statistical differences in rates of progression or survival by mutation status or by melanoma site. The overall disease control rate was 50% but varied significantly by KIT mutation status (77% mutated v 18% amplified). Four patients harbored pretreatment NRAS mutations, and one patient acquired increased KIT amplification after treatment. Melanomas that arise on mucosal, acral, or CSD skin should be assessed for KIT mutations. Imatinib can be effective when tumors harbor KIT mutations, but not if KIT is amplified only. NRAS mutations and KIT copy number gain may be mechanisms of therapeutic resistance to imatinib.
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Khatiwoda, Prasana; Proeschold-Bell, Rae Jean; Meade, Christina S; Park, Lawrence P; Proescholdbell, Scott
2018-01-01
BACKGROUND Naloxone-an opioid antagonist that reverses the effects of opioids-is increasingly being distributed in non-medical settings. We sought to identify the facilitators of, and barriers to, opioid users using naloxone kits in North Carolina. METHODS In 2015, we administered a 15-item survey to a convenience sample of 100 treatment seekers at 4 methadone/buprenorphine Medication Assisted Therapy (MAT) clinics in North Carolina. RESULTS Seventy-four percent of participants reported having ever gotten a naloxone kit; this percentage was higher for females (81%) than males (63%) ( P = .06). The primary reason given for not having a kit was not knowing where to get one. Only 6% had heard of kits from the media and only 5% received one from a medical provider. Among kit recipients, 56% of both females and males reported mostly or sometimes carrying the kit, with additional participants reporting always. Reasons for not carrying a kit were no longer being around drugs, forgetting it, and the kit being too large. Men discussed the difficulties of carrying the naloxone kits, which are currently too large to fit in a pocket. Ninety-four percent of naloxone users reported intending to call emergency services in case of an overdose emergency. LIMITATIONS Study limitations included a small sample, participants limited to MAT clinics, and a predominantly white sample. CONCLUSIONS MAT treatment seekers reported a willingness to carry and use naloxone kits. Education, outreach, media, and medical providers need to promote naloxone kits. A smaller kit may increase the likelihood of men carrying one. ©2018 by the North Carolina Institute of Medicine and The Duke Endowment. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nobuhisa, Ikuo; Ohtsu, Naoki; Okada, Seiji
The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45{sup low} c-Kit{sup +} cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45{sup low} c-Kit{sup -} cells that showed a granulocyte morphology;more » CD45{sup high} c-Kit{sup low/-} that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45{sup low} c-Kit{sup +} cells from the AGM culture had the abilities to reproduce CD45{sup low} c-Kit{sup +} cells and differentiate into CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} cells, whereas CD45{sup low} c-Kit{sup -} and CD45{sup high} c-Kit{sup low/-} did not produce CD45{sup low} c-Kit{sup +} cells. Furthermore, CD45{sup low} c-Kit{sup +} cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45{sup low} c-Kit{sup +} cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.« less
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Foda, Abd Al-Rahman Mohammad; Mohamed, Mie Ali
2015-10-01
c-KIT and DOG-1 are 2 highly expressed proteins in gastrointestinal stromal tumors. Few studies had investigated c-KIT, but not DOG-1, expression in colorectal carcinoma (CRC). This study aims to investigate expressions of c-KIT and DOG-1 in colorectal mucinous carcinoma and nonmucinous carcinoma using manual tissue microarray technique. In this work, we studied tumor tissue specimens from 150 patients with colorectal mucinous (MA) and nonmucinous adenocarcinoma (NMA). High-density manual tissue microarrays were constructed using modified mechanical pencil tip technique, and immunohistochemistry for c-KIT and DOG-1 was done. We found that aberrant c-KIT expression was detected in 12 cases (8%); 6 cases (4%) showed strong expression. Aberrant DOG-1 expression was detected in 15 cases (10%); among them, only 4 cases (2.7%) showed strong expression. Nonmucinous adenocarcinoma showed a significantly high expression of c-KIT, but not DOG-1, than MA. Aberrant c-KIT and DOG-1 expressions were significantly unrelated but were associated with excessive microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. In conclusion, aberrant c-KIT and DOG-1 expressions in CRC are rare events, either in NMA or MA. Nonmucinous adenocarcinoma showed a significantly higher expression of c-KIT, but not DOG-1, than MA. The expressions of both in CRC are significantly unrelated but are associated with microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. So, c-KIT and DOG-1 immunostaining is not a cost-effective method of identifying patients with CRC who may benefit from treatment with tyrosine kinase inhibitors. Copyright © 2015 Elsevier Inc. All rights reserved.
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Molecular Alterations of KIT Oncogene in Gliomas
Gomes, Ana L.; Reis-Filho, Jorge S.; Lopes, José M.; Martinho, Olga; Lambros, Maryou B. K.; Martins, Albino; Schmitt, Fernando; Pardal, Fernando; Reis, Rui M.
2007-01-01
Gliomas are the most common and devastating primary brain tumours. Despite therapeutic advances, the majority of gliomas do not respond either to chemo or radiotherapy. KIT, a class III receptor tyrosine kinase (RTK), is frequently involved in tumourigenic processes. Currently, KIT constitutes an attractive therapeutic target. In the present study we assessed the frequency of KIT overexpression in gliomas and investigated the genetic mechanisms underlying KIT overexpression. KIT (CD117) immunohistochemistry was performed in a series of 179 gliomas of various grades. KIT activating gene mutations (exons 9, 11, 13 and 17) and gene amplification analysis, as defined by chromogenic in situ hybridization (CISH) and quantitative real-time PCR (qRT-PCR) were performed in CD117 positive cases. Tumour cell immunopositivity was detected in 15.6% (28/179) of cases, namely in 25% (1/4) of pilocytic astrocytomas, 25% (5/20) of diffuse astrocytomas, 20% (1/5) of anaplastic astrocytomas, 19.5% (15/77) of glioblastomas and one third (3/9) of anaplastic oligoastrocytomas. Only 5.7% (2/35) of anaplastic oligodendrogliomas showed CD117 immunoreactivity. No association was found between tumour CD117 overexpression and patient survival. In addition, we also observed CD117 overexpression in endothelial cells, which varied from 0–22.2% of cases, being more frequent in high-grade lesions. No KIT activating mutations were identified. Interestingly, CISH and/or qRT-PCR analysis revealed the presence of KIT gene amplification in 6 glioblastomas and 2 anaplastic oligoastrocytomas, corresponding to 33% (8/24) of CD117 positive cases. In conclusion, our results demonstrate that KIT gene amplification rather than gene mutation is a common genetic mechanism underlying KIT expression in subset of malignant gliomas. Further studies are warranted to determine whether glioma patients exhibiting KIT overexpression and KIT gene amplification may benefit from therapy with anti-KIT RTK inhibitors. PMID:17726262
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nobuhisa, Ikuo, E-mail: nobuhisa.scr@mri.tmd.ac.jp; Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics/Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, 860-0811; Yamasaki, Shoutarou
Long-term reconstituting hematopoietic stem cells first arise from the aorta of the aorta-gonad-mesonephros (AGM) region in a mouse embryo. We have previously reported that in cultures of the dispersed AGM region, CD45{sup low}c-Kit{sup +} cells possess the ability to reconstitute multilineage hematopoietic cells, but investigations are needed to show that this is not a cultured artifact and to clarify when and how this population is present. Based on the expression profile of CD45 and c-Kit in freshly dissociated AGM cells from embryonic day 9.5 (E9.5) to E12.5 and aorta cells in the AGM from E13.5 to E15.5, we defined sixmore » cell populations (CD45{sup -}c-Kit{sup -}, CD45{sup -}c-Kit{sup low}, CD45{sup -}c-Kit{sup high}, CD45{sup low}c-Kit{sup high}, CD45{sup high}c-Kit{sup high}, and CD45{sup high}c-Kit{sup very} {sup low}). Among these six populations, CD45{sup low}c-Kit{sup high} cells were most able to form hematopoietic cell colonies, but their ability decreased after E11.5 and was undetectable at E13.5 and later. The CD45{sup low}c-Kit{sup high} cells showed multipotency in vitro. We demonstrated further enrichment of hematopoietic activity in the Hoechst dye-effluxing side population among the CD45{sup low}c-Kit{sup high} cells. Here, we determined that CD45{sup low}c-Kit{sup high} cells arise from the lateral plate mesoderm using embryonic stem cell-derived differentiation system. In conclusion, CD45{sup low}c-Kit{sup high} cells are the major hematopoietic cells of mouse AGM.« less
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Bone-induced c-kit expression in prostate cancer: a driver of intraosseous tumor growth
Mainetti, Leandro E.; Zhe, Xiaoning; Diedrich, Jonathan; Saliganan, Allen D.; Cho, Won Jin; Cher, Michael L.; Heath, Elisabeth; Fridman, Rafael; Kim, Hyeong-Reh Choi; Bonfil, R. Daniel
2014-01-01
Loss of BRCA2 function stimulates prostate cancer (PCa) cell invasion and is associated with more aggressive and metastatic tumors in PCa patients. Concurrently, the receptor tyrosine kinase c-kit is highly expressed in skeletal metastases of PCa patients and induced in PCa cells placed into the bone microenvironment in experimental models. However, the precise requirement of c-kit for intraosseous growth of PCa and its relation to BRCA2 expression remain unexplored. Here, we show that c-kit expression promotes migration and invasion of PCa cells. Alongside, we found that c-kit expression in PCa cells parallels BRCA2 downregulation. Gene rescue experiments with human BRCA2 transgene in c-kit-transfected PCa cells resulted in reduction of c-kit protein expression and migration and invasion, suggesting a functional significance of BRCA2 downregulation by c-kit. The inverse association between c-kit and BRCA2 gene expressions in PCa cells was confirmed using laser capture microdissection in experimental intraosseous tumors and bone metastases of PCa patients. Inhibition of bone-induced c-kit expression in PCa cells transduced with lentiviral short hairpin RNA reduced intraosseous tumor incidence and growth. Overall, our results provide evidence of a novel pathway that links bone-induced c-kit expression in PCa cells to BRCA2 downregulation and supports bone metastasis. PMID:24798488
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Factors for C-Kit Expression in Cardiac Outgrowth Cells and Human Heart Tissue.
Matsushita, Satoshi; Minematsu, Kazuo; Yamamoto, Taira; Inaba, Hirotaka; Kuwaki, Kenji; Shimada, Akie; Yokoyama, Yasutaka; Amano, Atsushi
2017-12-12
We determined the factors associated with the expression of c-kit in the heart and the proliferation of c-kit-positive (c-kit pos ) cardiac stem cells among the outgrowth cells cultured from human cardiac explants.Samples of the right atrium (RA), left atrium (LA), and left ventricle obtained from patients during open-heart surgery were processed for cell culture of outgrowth cells and tissue analysis. The total number of growing cells and the population of c-kit pos cells were measured and compared with c-kit expression in native tissues and characteristics of the patients according to the region of the heart.We analyzed 452 samples from 334 patients. Atrial fibrillation (AF) in the patients reduced the number of outgrowth cells from the RA and LA, and aging was a co-factor for the LA. The c-kit pos population from the RA was associated with serum brain natriuretic peptide (BNP). C-kit expression in native tissue was also associated with BNP expression. However, we observed no relationship in expression between outgrowth cells and native tissue. In addition, the RA tissue provided the highest number of c-kit pos cells, and the left ventricle provided the lowest.C-kit was weakly expressed in response to damage. In addition, no correlation between outgrowth cells and native tissue was found for c-kit expression.
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Obata, Yuuki; Horikawa, Keita; Shiina, Isamu; Takahashi, Tsuyoshi; Murata, Takatsugu; Tasaki, Yasutaka; Suzuki, Kyohei; Yonekura, Keita; Esumi, Hiroyasu; Nishida, Toshirou; Abe, Ryo
2018-02-28
Most gastrointestinal stromal tumours (GISTs) are caused by constitutively active mutations in Kit tyrosine kinase. The drug imatinib, a specific Kit inhibitor, improves the prognosis of metastatic GIST patients, but these patients become resistant to the drug by acquiring secondary mutations in the Kit kinase domain. We recently reported that a Kit mutant causes oncogenic signals only on the Golgi apparatus in GISTs. In this study, we show that in GIST, 2-methylcoprophilinamide (M-COPA, also known as "AMF-26"), an inhibitor of biosynthetic protein trafficking from the endoplasmic reticulum (ER) to the Golgi, suppresses Kit autophosphorylation at Y703/Y721/Y730/Y936, resulting in blockade of oncogenic signalling. Results of our M-COPA treatment assay show that Kit Y703/Y730/Y936 in the ER are dephosphorylated by protein tyrosine phosphatases (PTPs), thus the ER-retained Kit is unable to activate downstream molecules. ER-localized Kit Y721 is not phosphorylated, but not due to PTPs. Importantly, M-COPA can inhibit the activation of the Kit kinase domain mutant, resulting in suppression of imatinib-resistant GIST proliferation. Our study demonstrates that Kit autophosphorylation is spatio-temporally regulated and may offer a new strategy for treating imatinib-resistant GISTs. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.
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Levitation Kits Demonstrate Superconductivity.
ERIC Educational Resources Information Center
Worthy, Ward
1987-01-01
Describes the "Project 1-2-3" levitation kit used to demonstrate superconductivity. Summarizes the materials included in the kit. Discusses the effect demonstrated and gives details on how to obtain kits. Gives an overview of the documentation that is included. (CW)
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NASA Astrophysics Data System (ADS)
2012-01-01
WE RECOMMEND Air swimmers Helium balloon swims like a fish Their Arrows will Darken the Sun: The Evolution and Science of Ballistics Ballistics book hits the spot Physics Experiments for your Bag Handy experiments for your lessons Quantum Physics for Poets Book shows the economic importance of physics SEP colour wheel kit Wheels investigate colour theory SEP colour mixing kit Cheap colour mixing kit uses red, green and blue LEDs iHandy Level iPhone app superbly measures angles Photonics Explorer kit Free optics kit given to schools WORTH A LOOK DrDAQ DrDAQ software gets an upgrade WEB WATCH Websites show range of physics
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FUNCTIONAL DEREGULATION OF KIT: LINK TO MAST CELL PROLIFERATIVE DISEASES AND OTHER NEOPLASMS
Cruse, Glenn; Metcalfe, Dean D.; Olivera, Ana
2014-01-01
SYNOPSIS Signaling through the receptor tyrosine kinase KIT mediates differentiation, proliferation and survival of hematopoietic precursor cells and mast cells. Constitutive KIT signaling due to somatic point mutations in c-Kit is an important occurrence in the development of mast cell proliferation disorders and other hematological malignancies. In this review, we discuss the common gain-of-function mutations found in these malignancies, particularly in mast cell proliferation disorders, and summarize the current understanding of the molecular mechanisms by which transforming point mutations in KIT may affect KIT structure and function and lead to altered downstream signaling and cellular transformation. Drugs targeting KIT have shown mixed success in the treatment of these diseases. A brief overview of the most common KIT inhibitors currently used, the reasons for the varied clinical results of such inhibitors and a discussion of potential new strategies are provided. PMID:24745671
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Pharmacists' views on and experiences with bowel cancer screening kits in Auckland, New Zealand.
Martini, Nataly; Basdew, Kamlika; Kammona, Ala; Shen, Amy; Taylor, Caragh; McIntosh, Timothy R; Barnes, Joanne
2014-08-01
To explore the views of New Zealand pharmacists on bowel cancer screening, particularly with regards to faecal occult blood testing (FOBT) kits, self-perceived knowledge on FOBT kits and barriers, motivators and experiences with selling and counselling consumers with respect to FOBT kits. Semi-structured interviews were conducted face to face or by telephone with 20 community pharmacists in the Auckland region. Interviews were recorded and transcribed verbatim and data were coded and analysed using NVivo software to identify key themes. Participant pharmacists believed that they were well placed to provide advice on FOBT kits to consumers. Barriers to selling the kits included cost and perceived lack of test sensitivity of the kits, poor consumer demand, pharmacists' lack of training and information, and a belief that selling FOBT kits was outside the pharmacists' scope of practice. Motivators to selling the kits included customer convenience, ease of use, confidence in the kits and embracing new roles for pharmacists. Pharmacists were concerned that use of the kits may increase the burden on the public health system through customer anxiety over test results; however, they agreed that there was a need for bowel cancer screening and awareness and that people concerned about bowel cancer should make visiting their general practitioner a priority. Pharmacists' views were mixed. Pharmacists' training and competence with respect to the provision of bowel cancer kits, and how a bowel cancer screening service can be developed to optimise public health outcomes, need to be addressed. © 2013 Royal Pharmaceutical Society.
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Repeatability and validity of a field kit for estimation of cholinesterase in whole blood.
London, L; Thompson, M L; Sacks, S; Fuller, B; Bachmann, O M; Myers, J E
1995-01-01
OBJECTIVES--To evaluate a spectrophotometric field kit (Test-Mate-OP) for repeatability and validity in comparison with reference laboratory methods and to model its anticipated sensitivity and specificity based on these findings. METHODS--76 farm workers between the age of 20 and 55, of whom 30 were pesticide applicators exposed to a range of organophosphates in the preceding 10 days, had blood taken for plasma cholinesterase (PCE) and erythrocyte cholinesterase (ECE) measurement by field kit or laboratory methods. Paired blinded duplicate samples were taken from subgroups in the sample to assess repeatability of laboratory and field kit methods. Field kits were also used to test venous blood in one subgroup. The variance obtained for the field kit tests was then applied to two hypothetical scenarios that used published action guidelines to model the kit's sensitivity and specificity. RESULTS--Repeatability for PCE was much poorer and for ECE slightly poorer than that of laboratory measures. A substantial upward bias for field kit ECE relative to laboratory measurements was found. Sensitivity of the kit to a 40% drop in PCE was 67%, whereas that for ECE was 89%. Specificity of the kit with no change in mean of the population was 100% for ECE and 91% for PCE. CONCLUSION--Field kit ECE estimation seems to be sufficiently repeatable for surveillance activities, whereas PCE does not. Repeatability of both tests seems to be too low for use in epidemiological dose-response investigations. Further research is indicated to characterise the upward bias in ECE estimation on the kit. PMID:7697143
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Evaluation of a Commercial Latex Agglutination Test Kit for Cryptococcal Antigen
Kaufman, Leo; Cowart, Glenda; Blumer, Sharon; Stine, Amy; Wood, Ross
1974-01-01
Two dozen Crypto-LA kits for detecting Cryptococcus neoformans capsular polysaccharide antigens were evaluated. Ten kits proved reliable for detecting and titering antigen in clinical materials. Fourteen kits were found to be inadequate. PMID:4596394
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Chen, Evan C; Karl, Taylor A; Kalisky, Tomer; Gupta, Santosh K; O'Brien, Catherine A; Longacre, Teri A; van de Rijn, Matt; Quake, Stephen R; Clarke, Michael F; Rothenberg, Michael E
2015-09-01
Receptor tyrosine kinase (RTK) inhibitors have advanced colon cancer treatment. We investigated the role of the RTK KIT in development of human colon cancer. An array of 137 patient-derived colon tumors and their associated xenografts were analyzed by immunohistochemistry to measure levels of KIT and its ligand KITLG. KIT and/or KITLG was stably knocked down by expression of small hairpin RNAs from lentiviral vectors in DLD1, HT29, LS174T, and COLO320 DM colon cancer cell lines, and in UM-COLON#8 and POP77 xenografts; cells transduced with only vector were used as controls. Cells were analyzed by real-time quantitative reverse transcription polymerase chain reaction, single-cell gene expression analysis, flow cytometry, and immunohistochemical, immunoblot, and functional assays. Xenograft tumors were grown from control and KIT-knockdown DLD1 and UM-COLON#8 cells in immunocompromised mice and compared. Some mice were given the RTK inhibitor imatinib after injection of cancer cells; tumor growth was measured based on bioluminescence. We assessed tumorigenicity using limiting dilution analysis. KIT and KITLG were expressed heterogeneously by a subset of human colon tumors. Knockdown of KIT decreased proliferation of colon cancer cell lines and growth of xenograft tumors in mice compared with control cells. KIT knockdown cells had increased expression of enterocyte markers, decreased expression of cycling genes, and, unexpectedly, increased expression of LGR5 associated genes. No activating mutations in KIT were detected in DLD1, POP77, or UM-COLON#8 cells. However, KITLG-knockdown DLD1 cells formed smaller xenograft tumors than control cells. Gene expression analysis of single CD44(+) cells indicated that KIT can promote growth via KITLG autocrine and/or paracrine signaling. Imatinib inhibited growth of KIT(+) colon cancer organoids in culture and growth of xenograft tumors in mice. Cancer cells with endogenous KIT expression were more tumorigenic in mice. KIT and KITLG are expressed by a subset of human colon tumors. KIT signaling promotes growth of colon cancer cells and organoids in culture and xenograft tumors in mice via its ligand, KITLG, in an autocrine or paracrine manner. Patients with KIT-expressing colon tumors can benefit from KIT RTK inhibitors. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.
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Chen, Evan C.; Karl, Taylor A.; Kalisky, Tomer; Gupta, Santosh K.; O’Brien, Catherine A.; Longacre, Teri A.; van de Rijn, Matt; Quake, Stephen R.; Clarke, Michael F.; Rothenberg, Michael E.
2015-01-01
Background & Aims Receptor tyrosine kinase (RTK) inhibitors have advanced colon cancer treatment. We investigated the role of the RTK KIT in development of human colon cancer. Methods An array of 137 patient-derived colon tumors and their associated xenografts were analyzed by immunohistochemistry to measure levels of KIT and its ligand KITLG. KIT and/or KITLG was stably knocked down by expression of small hairpin RNAs from lentiviral vectors in DLD1, HT29, LS174T, and COLO320 colon cancer cell lines, and in UM-COLON#8 and POP77 xenografts; cells transduced with only vector were used as controls. Cells were analyzed by real-time quantitative reverse transcription PCR, single-cell gene expression analysis, flow cytometry, and immunohistochemical, immunoblot, and functional assays. Xenograft tumors were grown from control and KIT-knockdown DLD1 and UM-COLON#8 cells in immunocompromised mice and compared. Some mice were given the RTK inhibitor imatinib following injection of cancer cells; tumor growth was measured based on bioluminescence. We assessed tumorigenicity using limiting dilution analysis. Results KIT and KITLG were expressed heterogeneously by a subset of human colon tumors. Knockdown of KIT decreased proliferation of colon cancer cell lines and growth of xenograft tumors in mice, compared with control cells. KIT knockdown cells had increased expression of enterocyte markers, decreased expression of cycling genes, and, unexpectedly, increased expression of LGR5-associated genes. No activating mutations in KIT were detected in DLD1, POP77, or UM-COLON#8 cell lines. However, KITLG-knockdown DLD1 cells formed smaller xenograft tumors than control cells. Gene expression analysis of single CD44+ cells indicated that KIT may promote growth via KITLG autocrine and/or paracrine signaling. Imatinib inhibited growth of KIT+ colon cancer organoids in culture and growth of xenograft tumors in mice. Cancer cells with endogenous KIT expression were more tumorigenic in mice. Conclusions KIT and KITLG are expressed by a subset of human colon tumors. KIT signaling promotes growth of colon cancer cells and organoids in culture and xenograft tumors in mice via its ligand, KITLG, in an autocrine or paracrine manner. Patients with KIT-expressing colon tumors may benefit from KIT RTK inhibitors. PMID:26026391
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Combined KIT and FGFR2b Signaling Regulates Epithelial Progenitor Expansion during Organogenesis
Lombaert, Isabelle M.A.; Abrams, Shaun R.; Li, Li; Eswarakumar, Veraragavan P.; Sethi, Aditya J.; Witt, Robert L.; Hoffman, Matthew P.
2013-01-01
Summary Organ formation and regeneration require epithelial progenitor expansion to engineer, maintain, and repair the branched tissue architecture. Identifying the mechanisms that control progenitor expansion will inform therapeutic organ (re)generation. Here, we discover that combined KIT and fibroblast growth factor receptor 2b (FGFR2b) signaling specifically increases distal progenitor expansion during salivary gland organogenesis. FGFR2b signaling upregulates the epithelial KIT pathway so that combined KIT/FGFR2b signaling, via separate AKT and mitogen-activated protein kinase (MAPK) pathways, amplifies FGFR2b-dependent transcription. Combined KIT/FGFR2b signaling selectively expands the number of KIT+K14+SOX10+ distal progenitors, and a genetic loss of KIT signaling depletes the distal progenitors but also unexpectedly depletes the K5+ proximal progenitors. This occurs because the distal progenitors produce neurotrophic factors that support gland innervation, which maintains the proximal progenitors. Furthermore, a rare population of KIT+FGFR2b+ cells is present in adult glands, in which KIT signaling also regulates epithelial-neuronal communication during homeostasis. Our findings provide a framework to direct regeneration of branched epithelial organs. PMID:24371813
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Exosome-delivered microRNAs promote IFN-α secretion by human plasmacytoid DCs via TLR7.
Salvi, Valentina; Gianello, Veronica; Busatto, Sara; Bergese, Paolo; Andreoli, Laura; D'Oro, Ugo; Zingoni, Alessandra; Tincani, Angela; Sozzani, Silvano; Bosisio, Daniela
2018-05-17
The excessive production of type I IFNs is a hallmark and a main pathogenic mechanism of many autoimmune diseases, including systemic lupus erythematosus (SLE). In these pathologies, the sustained secretion of type I IFNs is dependent on the improper activation of plasmacytoid DCs (pDCs) by self-nucleic acids. However, the nature and origin of pDC-activating self-nucleic acids is still incompletely characterized. Here, we report that exosomes isolated from the plasma of SLE patients can activate the secretion of IFN-α by human blood pDCs in vitro. This activation requires endosomal acidification and is recapitulated by microRNAs isolated from exosomes, suggesting that exosome-delivered microRNAs act as self-ligands of innate single-stranded endosomal RNA sensors. By using synthetic microRNAs, we identified an IFN induction motif that is responsible for the TLR7-dependent activation, maturation, and survival of human pDCs. These findings identify exosome-delivered microRNAs as potentially novel TLR7 endogenous ligands able to induce pDC activation in SLE patients. Therefore, microRNAs may represent novel pathogenic mediators in the onset of autoimmune reactions and potential therapeutic targets in the treatment of type I IFN-mediated diseases.
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NASA Astrophysics Data System (ADS)
Kim, Hye Jin; Kang, Dong-Hoon; Lee, Eunji; Hwang, Kyo Seon; Shin, Hyun-Joon; Kim, Jinsik
2018-02-01
We propose a simple fluorescent bio-chip based on two types of alternative current-dielectrophoretic (AC-DEP) force, attractive (positive DEP) and repulsive (negative DEP) force, for simultaneous nano-molecules analysis. Various radius of micro-holes on the bio-chip are designed to apply the different AC-DEP forces, and the nano-molecules are concentrated inside the micro-hole arrays according to the intensity of the DEP force. The bio-chip was fabricated by Micro Electro Mechanical system (MEMS) technique, and was composed of two layers; a SiO2 layer and Ta/Pt layer were accomplished for an insulation layer and a top electrode with micro-hole arrays to apply electric fields for DEP force, respectively. Each SiO2 and Ta/Pt layers were deposited by thermal oxidation and sputtering, and micro-hole arrays were fabricated with Inductively Coupled Plasma (ICP) etching process. For generation of each positive and negative DEP at micro-holes, we applied two types of sine-wave AC voltage with different frequency range alternately. The intensity of the DEP force was controlled by the radius of the micro-hole and size of nano-molecule, and calculated with COMSOL multi-physics. Three types of nano-molecules labelled with different fluorescent dye were used and the intensity of nano-molecules was examined by the fluorescent optical analysis after applying the DEP force. By analyzing the fluorescent intensities of the nano-molecules, we verify the various nano-molecules in analyte are located successfully inside corresponding micro-holes with different radius according to their size.
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Hannemann, Frank; Bernhardt, Rita; Jose, Joachim
2007-10-01
Biocatalysis, the conversion of substrates into valuable products by the use of enzymes, has some striking advantages in comparison to standard organic chemistry for drug synthesis. By biocatalysis, substrates that contain several identical reactive groups at different positions can be converted with high regio-selectivity and enantio-selectivity. In this study, an E. coli isolate (E132) was identified which was able to convert the steroid desoxycorticosterone into the product 4-pregnen-20,21-diol-3-one in real terms. The product was purified from the cell culture supernatant by HPLC and its structure was demonstrated by mass spectrometry and NMR spectroscopy. It was tested on inhibition of human 5alpha-reductases type I and type II. At a concentration of 10 microM, inhibition was 49.0% for type I and 81.8% for type II, whereas there was no inhibition of human aromatase (CYP19) at 20 microM and human 17alpha-hydroxylase-C17,20-lyase (CYP17) at 2.5 microM detectable. The IC50 value of 4-pregnen-20,21-diol-3-one for human 5alpha-reductase type II was determined to be 1.56 microM.
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Dewetting Based Fabrication of Fibrous Micro-Scaffolds as Potential Injectable Cell Carriers
Song, Hokyung; Yin, Liya; Chilian, William M.; Newby, Bi-min Zhang
2014-01-01
Although regenerative medicine utilizing tissue scaffolds has made enormous strides in recent years, many constraints still hamper their effectiveness. A limitation of many scaffolds is that they form surface patches, which are not particularly effective for some types of “wounds” that are deep within tissues, e.g., stroke, myocardial infarction. In this study, we reported the generation of fibrous micro-scaffolds feasible for delivering cells by injection into the tissue parenchyma. The micro-scaffolds (widths < 100 μm) were made by dewetting of poly (lactic-coglycolic acid) thin films containing parallel strips, and cells were seeded to form cell/polymer micro-constructs during or post the micro-scaffold fabrication process. Five types of cells including rat induced vascular progenitor cells were assessed for the formation of the micro-constructs. Critical factors in forming fibrous micro-scaffolds via dewetting of polymer thin films were found to be properties of polymers and supporting substrates, temperature, and proteins in the culture medium. Also, the ability of cells to attach to the micro-scaffolds was essential for forming cell/polymer micro-constructs. Both in vitro and in vivo assessments of injecting these micro-scaffolding constructs showed, as compared to free cells, enhanced cell retention at the injected site, which could lead to improved tissue engineering and regeneration. PMID:25579969
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LogiKit - assisting complex logic specification and implementation for embedded control systems
NASA Astrophysics Data System (ADS)
Diglio, A.; Nicolodi, B.
2002-07-01
LogiKit provides an overall lifecycle solution. LogiKit is a powerful software engineering case toolkit for requirements specification, simulation and documentation. LogiKit also provides an automatic ADA software design, code and unit test generator.
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Psychometric Properties of the Concept Assessment Kit-Conservation.
ERIC Educational Resources Information Center
Lehnert, Linda; And Others
1986-01-01
This study investigated the psychometric properties of the Educational and Industrial Testing Service Concept Assessment Kit-Conservation (EITS Kit). Presented are an overview of the concept of conservation, a description of the EITS Kit, and results of the study. (MT)
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Rastawicki, Waldemar; Rokosz, Natalia; Jagielski, Marek
2011-01-01
The Binax and the Biotest urinary antigen kits for detection of L. pneumophila antigen were compared by testing of selected 67 urine samples obtained from EWGLI as reference samples in External Quality Assessment Scheme. Thirty nine were positive with the Binax kit (100% of sensitivity), and 33 were positive with the Biotest (84.6% of sensitivity). The test specificities were 100% for the both kits. It was concluded that the Binax kit was more suitable for the routine diagnosis of Legionella infections than the Biotest kit.
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Retinoic acid induction of calcium channel expression in human NT2N neurons.
Gao, Z Y; Xu, G; Stwora-Wojczyk, M M; Matschinsky, F M; Lee, V M; Wolf, B A
1998-06-18
Ca2+ channel expression and regulation of intracellular Ca2+ homeostasis were studied during retinoic acid (RA)-induced differentiation of the human teratocarcinoma cell line Ntera 2/C1.D1 (NT2- cells) into NT2N neurons, a unique model of human neurons in culture. The cytosolic Ca2+ level of undifferentiated NT2- cells was low (75 +/- 5 nM) and stable under basal conditions, and it was only marginally decreased (by 9%) upon removal of extracellular Ca2+. After 10 microM RA treatment, NT2- cells were irreversibly differentiated into a phenotype of neuron-like NT2N cells. Cytosolic Ca2+ level of NT2N neurons was higher (106 +/- 14 nM) than that of NT2- cells and spontaneously fluctuated (0.208 +/- 0.038 transients/min) under basal conditions. Although K+ increased 86Rb fluxes in both NT2- cells and NT2N neurons, it only increased cytosolic Ca2+ level in NT2N neurons. The K+-induced increase in cytosolic Ca2+ in NT2N neurons was antagonized by 0.1-10 microM nifedipine or verapamil, 5 microM omega-CgTx GVIA, but not by 1 microM omega-agatoxin IVA, 1 microM omega-agatoxin TK, 1 microM FTX-3.3, or 100 microM Ni+ implicating L- and N-type voltage-dependent Ca2+ channels. In L- and N-type channels, but not in P- and Q-types, mRNAs were expressed in NT2N neurons as well as NT2- cells. Quantitative analysis of L- and N-type Ca2+ protein levels showed major differences between NT2- cells and NT2N neurons. In NT2- cells, N-type Ca2+ channels were undetectable while L-type channels levels were fivefold lower compared to NT2N neurons. Our findings show that L- and N-type channels are expressed during differentiation of NT2- cells into neurons, and that these voltage-dependent Ca2+ channels have a major role in regulating intracellular Ca2+ homeostasis and neuronal excitability. Copyright 1998 Academic Press.
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Nagai, Toshiya; Sakurai, Saki; Natori, Naoki; Hataoka, Manaka; Kinoshita, Takako; Inoue, Hiroyoshi; Hanaya, Kengo; Shoji, Mitsuru; Sugai, Takeshi
2018-04-01
Commercially available "Chiralscreen® OH" starter kit containing five types of carbonyl reductases (E001, E007, E031, E039, and E078) was used for the reduction of several aromatic and aliphatic ketones to obtain enantiomerically enriched drug precursors and an insect pheromone. Almost stereochemically pure secondary alcohols, used in the synthesis of drugs such as (R)-rasagiline mesylate, (S)-rivastigmine, (R)-chlorphenesin carbamate, and (R)-mexiletine, and the insect pheromone (4S,5R)-sitophilure, were conveniently obtained. The enzymes worked well with ketones containing at least one non-bulky substituent at the carbonyl group. The diverse stereochemical preference of the above five carbonyl reductases was clarified. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Devesse, Laurence; Ballard, David; Davenport, Lucinda; Riethorst, Immy; Mason-Buck, Gabriella; Syndercombe Court, Denise
2018-05-01
By using sequencing technology to genotype loci of forensic interest it is possible to simultaneously target autosomal, X and Y STRs as well as identity, ancestry and phenotypic informative SNPs, resulting in a breadth of data obtained from a single run that is considerable when compared to that generated with standard technologies. It is important however that this information aligns with the genotype data currently obtained using commercially available kits for CE-based investigations such that results are compatible with existing databases and hence can be of use to the forensic community. In this work, 400 samples were typed using commercially available STR kits and CE, as well as using the Ilumina ForenSeq™ DNA Signature Prep Kit and MiSeq ® FGx to assess concordance of autosomal STRs and population variability. Results show a concordance rate between the two technologies exceeding 99.98% while numerous novel sequence based alleles are described. In order to make use of the sequence variation observed, sequence specific allele frequencies were generated for White British and British Chinese populations. Copyright © 2017 Elsevier B.V. All rights reserved.
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Peter, Harald; Berggrav, Kathrine; Thomas, Peter; Pfeifer, Yvonne; Witte, Wolfgang; Templeton, Kate
2012-01-01
Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to antibiotic therapy, as they confer resistance to carbapenems, which are used to treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we describe the development and evaluation of a DNA microarray for the detection and genotyping of KPC genes (blaKPC) within a 5-h period. To test the whole assay procedure (DNA extraction plus a DNA microarray assay) directly from clinical specimens, we compared two commercial DNA extraction kits (the QIAprep Spin miniprep kit [Qiagen] and the urine bacterial DNA isolation kit [Norgen]) for the direct DNA extraction from urine samples (dilution series spiked in human urine). Reliable single nucleotide polymorphism (SNP) typing was demonstrated using 1 × 105 CFU/ml urine for Escherichia coli (Qiagen and Norgen) and 80 CFU/ml urine, on average, for K. pneumoniae (Norgen). This study presents, for the first time, the combination of a new KPC microarray with commercial sample preparation for detecting and genotyping microbial pathogens directly from clinical specimens; this paves the way toward tests providing epidemiological and diagnostic data, enabling better antimicrobial stewardship. PMID:23035190
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Detection of human papillomavirus DNA in patients referred to a family practice colposcopy clinic.
Holman, J R
1996-01-01
Human papillomavirus (HPV) is strongly implicated in the pathogenesis of cervical neoplasia. The ability of a commercially available kit (Virapap/Viratype) to detect evidence of HPV is compared with cervical cytology, colposcopy, and directed biopsies. During a period of 16 months, cervical samples from 241 consecutive new patients referred for a colposcopy examination were obtained for HPV-DNA hybridization typing according to the kit instructions. Samples were sent to a reference laboratory for testing. The results were compared with results of the colposcopy examination, cervical cytology, and directed cervical biopsy samples processed and evaluated by our hospital laboratory. HPV DNA was detected in 27 of 107 patients who had abnormal colposcopy findings for a sensitivity of 25 +/- 7.5 percent at the 90 percent confidence interval. One of 134 patients with normal findings was positive for a specificity of 99 +/- 5 percent at the 95 percent confidence interval. Based on a 75 percent probability of HPV in the population, the positive predictive value was 99 percent and the negative predictive value 30 percent. With the low negative predictive value and sensitivity, HPV-DNA testing by this commercial kit is not an adequate tool for screening HPV in this population.
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Hatfield, Julianne K; Brown, Melissa A
2015-10-01
Innate lymphoid cells are immune cells that reside in tissues that interface with the external environment and contribute to the first line defense against pathogens. However, they also have roles in promoting chronic inflammation. Here we demonstrate that group 3 ILCs, (ILC3s - CD45+Lin-IL-7Rα+RORγt+), are normal residents of the meninges and exhibit disease-induced accumulation and activation in EAE. In addition to production of the pro-inflammatory cytokines IL-17 and GM-CSF, ILC3s constitutively express CD30L and OX40L, molecules required for memory T cell survival. We show that disease-induced trafficking of transferred wild type T cells to the meninges is impaired in ILC3-deficient Rorc-/- mice. Furthermore, lymphoid tissue inducer cells, a c-kit+ ILC3 subset that promotes ectopic lymphoid follicle development, a hallmark of many autoimmune diseases, are reduced in the meninges of EAE-resistant c-kit mutant Kit(W/Wv) mice. We propose that ILC3s sustain neuroinflammation by supporting T cell survival and reactivation in the meninges. Copyright © 2015 Elsevier Inc. All rights reserved.
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[Analysis of the dilution deviation in CA19-9 measurement].
Hanada, Hiroyuki; Takeoka, Keiko; Nomura, Tomoko; Moriyama, Takanori; Kanakura, Yuzuru
2005-04-01
CA19-9 widely used as a tumor marker of the pancreas and a bile duct. There are a number of reports which describes the measured value discrepancies between RIA and non-RIA kits. RIA results also have shown lack of the linearity over 70 U/ml when the samples are diluted. The pH condition at assay reaction for RIA had been suggested as the major reason, it has been denied by the results from the same pH condition at assay reaction used by COBAS CORE CA19-9 EIA II. On the other hand, the lack of RIA antibody titer is indicated for the discordant results by changing the sample volume to reagent volume ratio in the reaction. Our further investigation also indicates that the specific Lewis blood type, i.e. Le (a-b+), shows the linearity issues by RIA. The discrepancies are not caused by the reaction pH, but the amount of the antibody used in the RIA kit is closely associated. Considering the CA19-9 antibody nature used in RIA kit, which covers broad molecular range, users need to pay more attention to setting up each laboratory's measuring range.
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Fukuishi, Nobuyuki; Igawa, Yuusuke; Kunimi, Tomoyo; Hamano, Hirofumi; Toyota, Masao; Takahashi, Hironobu; Kenmoku, Hiromichi; Yagi, Yasuyuki; Matsui, Nobuaki; Akagi, Masaaki
2013-01-01
While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy. PMID:23573287
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[Forensic Application of HuaxiaTM Platinum Kit].
Wang, Y L; Sheng, X; Li, M; Chen, Y L; Lin, Y; Chen, L Q
2017-04-01
To investigate the genetic polymorphism of 23 autosomal STR loci of Huaxia™ Platinum kit in Chinese Han population, and to evaluate the forensic efficiency of Huaxia™ Platinum kit. A total of 500 unrelated healthy individuals from Han population were genotyped with Huaxia™ Platinum kit. The frequency distribution and the parameter of population genetics of STR loci were analysed statistically. Huaxia™ Platinum kit was compared with other 7 commercial STR kits commonly seen at home and abroad in the number of STR loci, interior label, fluorescent mark, total number of alleles in Ladder and system effectiveness. All the 23 autosomal STR loci were consistent with Hardy-Weinberg equilibrium （ P >0.05）. The discrimination power was 0.791 5-0.986 2. The polymorphism information content （PIC） was 0.559 0-0.914 0. The combined discrimination power （CDP） was 1-4.1×10⁻²⁸, while combined probability of paternity exclusion in trio （CPET） and in duo （CPED） were 1-4.1×10⁻¹⁰ and 1-8.4×10⁻⁷, respectively. Compared with other 7 kits, Huaxia™ Platinum kit contained the most number of alleles within the Ladder. All the 23 autosomal STR loci of Huaxia™ Platinum kit with highly polymorphic in Han population can be used for paternity testing and individual identification. Compared with other 7 kits, it appears that Huaxia™ Platinum kit can provide more genetic information. Copyright© by the Editorial Department of Journal of Forensic Medicine
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Studying the loading effect of acidic type antioxidant on amorphous silica nanoparticle carriers
NASA Astrophysics Data System (ADS)
Ravinayagam, Vijaya; Rabindran Jermy, B.
2017-06-01
The study investigates the suitable nanosilica carriers to transport acidic type cargo molecules for potential targeted drug delivery application. Using phenolic acidic type antioxidant gallic acid (GA) as model compound, the present study investigates the loading effect of GA (0.3-15.9 mmol GA g-1 support) on textural characteristics of amorphous silica nanoparticles such as Q10 silica (1D), structured two-dimensional Si-MCM-41 (2D), and three-dimensional Si-SBA-16 (3D). The variation in the nature of textures after GA loading was analyzed using X-ray diffraction, N2 adsorption, FT-IR, scanning electron microscopy with energy dispersive X-ray spectroscopy, and high-resolution transmission electron microscopy. Among the nanocarriers, high adsorption of GA was found in the following order: Si-SBA-16 (3D)˜Si-KIT-6 (3D) > Si-MCM-41 (2D) > ultralarge pore FDU-12 (ULPFDU-12; 3D) > Q10 (1D)˜mesostructured cellular silica foam (MSU-F). 3D-type silicas Si-SBA-16 and KIT-6 were shown to maintain structural integrity at acidic condition (pH ˜3) and accommodate GA in non-crystalline form. In the case of ULPFDU-12 and MSU-F cellular foam, only crystalline deposition of GA occurs with a significant variation in the surface area and pore volume. [Figure not available: see fulltext.
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Investigator® HDplex (Qiagen) reference population database for forensic use in Argentina.
Martínez, Gustavo; Borosky, Alicia; Corach, Daniel; Llull, Cintia; Locarno, Laura; Lojo, Mercedes; Marino, Miguel; Miozzo, María Cecilia; Modesti, Nidia; Pacharoni, Carla; Pilili, Juan Pablo; Ramella, María Isabel; Sala, Andrea; Schaller, Cecilia; Vullo, Carlos; Toscanini, Ulises
2017-01-01
Currently, autosomal Short Tandem Repeat (STR) markers represent the method of election in forensic human identification. Commercial kits of most common use nowadays -e.g. PowerPlex ® Fusion, Promega Corp.; AmpFlSTR GlobalFiler, Thermofisher scientific; Investigator 24Plex QS,Qiagen-, allow the co-amplification of 23 highly polymorphic STR loci providing a high discrimination power in human identity testing. However, in complex kinship analysis and familial database searches involving distant relationships, additional DNA typing is often required in order to achieve well-founded conclusions. The recently developed kit Investigator ® HDplex (Qiagen) co-amplify twelve autosomal STRs markers (D7S1517, D3S1744, D12S391, D2S1360, D6S474, D4S2366, D8S1132, D5S2500, D18S51, D21S2055, D10S2325, SE33), nine of which are not present in the above mentioned kits, providing a set of efficient supplementary markers for human identification purposes. In this study we genotyped a sample of 980 individuals from urban areas of ten Argentinean provinces using the Investigator ® HDplex kit, aiming to provide forensic estimates for use in forensic casework and parentage testing in Argentina. We report reference allelic frequency databases for each of the provinces studied as well as for the combined samples. No deviation of Hardy-Weinberg equilibrium was observed. A reasonable discrimination capacity and power of exclusion was estimated which allowed predicting an acceptable forensic behavior of this kit, either to be used as the main STR panel for simple cases or as an auxiliary tool in complex cases. Additionally, population comparison tests showed that the studied samples are relatively homogeneous across the country for these STR set. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
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Low-cost field test kits for arsenic detection in water.
Das, Joyati; Sarkar, Priyabrata; Panda, Jigisha; Pal, Priyabrata
2014-01-01
Arsenic, a common contaminant of groundwater, affects human health adversely. According to the World Health Organization (WHO), the maximum recommended contamination level of arsenic in drinking water is 10 μg/L. The purpose of this research was to develop user-friendly kits for detection of arsenic to measure at least up to 10 μg/L in drinking water, so that a preventive measure could be taken. Two different kits for detection of total arsenic in water are reported here. First, the arsenic in drinking water was converted to arsine gas by a strong reducing agent. The arsine produced was then detected by paper strips via generation of color due to reaction with either mercuric bromide (KIT-1) or silver nitrate (KIT-2). These were previously immobilized on the detector strip. The first one gave a yellow color and the second one grey. Both of these kits could detect arsenic contamination within a range of 10 μg/L-250 μg/L. The detection time for both the kits was only 7 min. The kits exhibited excellent performance compared to other kits available in the market with respect to detection time, ease of operation, cost and could be easily handled by a layman. The field trials with these kits gave very satisfactory results. A study on interference revealed that these kits could be used in the presence of 24 common ions present in the arsenic contaminated water. Though the kits were meant for qualitative assay, the results with unknown concentrations of real samples, when compared with atomic absorption spectrophotometer (AAS) were in good agreement as revealed by the t-test.
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Immunomagnetic Separation of Cryptosporidium parvum from Source Water Samples of Various Turbidities
Bukhari, Z.; McCuin, R. M.; Fricker, C. R.; Clancy, J. L.
1998-01-01
Immunomagnetic separation (IMS) procedures which specifically capture Cryptosporidium oocysts and have the potential to isolate oocysts from debris have become commercially available. We compared two IMS kits (kit DB [Dynabeads anti-Cryptosporidium; product no. 730.01; Dynal A.S., Oslo, Norway] and kit IC1 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC, Portland, Maine]) and a modification of kit IC1 (kit IC2 [Crypto Scan IMS; product no. R10; Clearwater Diagnostics Company, LLC]) at three turbidity levels (50, 500, and 5,000 nephelometric turbidity units [ntu]) by using water matrices obtained from different geographical locations. In deionized water, kit DB yielded recoveries between 68 and 83%, whereas the recoveries obtained with kits IC1 and IC2 were more variable and ranged from 0.2 to 74.5%. In water matrices with turbidity levels up to 500 ntu, the oocyst recoveries were more variable with kit DB; however, the recoveries were similar to those obtained in deionized water. In contrast, there were notable reductions in oocyst recoveries in the turbid matrices with kits IC1 and IC2, and the highest recovery (8.3%) was obtained with a 50-ntu sample. An examination of the effects of age on oocyst recovery with kit DB revealed that oocysts up to 16 weeks old yielded recoveries similar to the recoveries observed with fresh oocysts. These data indicate that all IMS kits do not perform equally well, and it is important to conduct in-house quality assurance work before a commercially available IMS kit is selected to replace flotation procedures for recovery of Cryptosporidium oocysts. PMID:9797313
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Elwick, Kyleen; Mayes, Carrie; Hughes-Stamm, Sheree
2018-05-01
In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, been buried, decomposed, and/or contain inhibitory substances. This study compares the performance of a relatively new STR kit in the US market (Investigator® 24plex QS kit; Qiagen) with the GlobalFiler® PCR Amplification kit (Thermo Fisher Scientific) when genotyping highly inhibited and low level DNA samples. In this study, DNA samples ranging from 1 ng to 7.8 pg were amplified to define the sensitivity of two systems. In addition, DNA (1 ng and 0.1 ng input amounts) was spiked with various concentrations of five inhibitors common to human remains (humic acid, melanin, hematin, collagen, calcium). Furthermore, bone (N = 5) and tissue samples from decomposed human remains (N = 6) were used as mock casework samples for comparative analysis with both STR kits. The data suggest that the GlobalFiler® kit may be slightly more sensitive than the Investigator® kit. On average STR profiles appeared to be more balanced and average peak heights were higher when using the GlobalFiler® kit. However, the data also show that the Investigator® kit may be more tolerant to common PCR inhibitors. While both STR kits showed a decrease in alleles as the inhibitor concentration increased, more complete profiles were obtained when the Investigator® kit was used. Of the 11 bone and decomposed tissue samples tested, 8 resulted in more complete and balanced STR profiles when amplified with the GlobalFiler® kit. Copyright © 2018 Elsevier B.V. All rights reserved.
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c-kit expression profile and regulatory factors during spermatogonial stem cell differentiation
2013-01-01
Background It has been proven that c-kit is crucial for proliferation, migration, survival and maturation of spermatogenic cells. A periodic expression of c-kit is observed from primordial germ cells (PGCs) to spermatogenetic stem cells (SSCs), However, the expression profile of c-kit during the entire spermatogenesis process is still unclear. This study aims to reveal and compare c-kit expression profiles in the SSCs before and after the anticipated differentiation, as well as to examine its relationship with retinoic acid (RA) stimulation. Results We have found that there are more than 4 transcripts of c-kit expressed in the cell lines and in the testes. The transcripts can be divided into short and long categories. The long transcripts include the full-length canonical c-kit transcript and the 3′ end short transcript. Short transcripts include the 3.4 kb short transcript and several truncated transcripts (1.9-3.2 kb). In addition, the 3.4 kb transcript (starting from intron 9 and covering exons 10 ~ 21) is discovered to be specifically expressed in the spermatogonia. The extracellular domain of Kit is obtained in the spermatogonia stage, but the intracellular domain (50 kDa) is constantly expressed in both SSCs and spermatogonia. The c-kit expression profiles in the testis and the spermatogonial stem cell lines vary after RA stimulation. The wave-like changes of the quantitative expression pattern of c-kit (increase initially and decrease afterwards) during the induction process are similar to that of the in vivo male germ cell development process. Conclusions There are dynamic transcription and translation changes of c-kit before and after SSCs’ anticipated differentiation and most importantly, RA is a significant upstream regulatory factor for c-kit expression. PMID:24161026