PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

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Dash Aditya P

2009-07-01

Full Text Available Abstract Background Anopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping. Methods The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR, an Amplification Refractory Mutation System (ARMS and Primer Introduced Restriction Analysis-PCR (PIRA-PCR were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped. Results The genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium. Conclusion The Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

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Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

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Zhiyuan Wu

Full Text Available BACKGROUND: JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. METHODOLOGY/PRINCIPAL FINDINGS: Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. CONCLUSIONS: With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

EGFR Mutation Status in Uighur Lung Adenocarcinoma Patients

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Li SHAN

2013-02-01

Full Text Available Background and objective Epidermal growth factor receptor (EGFR, a transmembrane protein, is a member of the tyrosine kinase family. Gefitinib, an EGFR tyrosine-kinase inhibitors, has shown a high response rate in the treatment of lung cancer in patients with EGFR mutation. However, significant differences in EGFR mutations exist among different ethnic groups. The aim of this study is to investigate the prevalence of EGFR mutations in Uighur lung adenocarcinoma patients by using a rapid and sensitive detection method and to analyze EGFR mutation differences compared with Han lung adenocarcinoma patients. Methods We examined lung adenocarcinoma tissues from 138 patients, including 68 Uighur lung adenocarcinoma patients and 70 Han lung adenocarcinoma patients, for EGFR mutations in exons 18, 19, 20, and 21 by using the amplification refractory mutation system (ARMS PCR method. The mutation differences between Uighur and Han lung adenocarcinoma were compared by using the chi-square test method. Results EGFR mutations were detected in 43 (31.2% of the 138 lung adenocarcinoma patients. EGFR mutations were detected in 11 (16.2% of the 68 Uighur lung adenocarcinoma patients and in 32 (45.7% of the 70 Han lung adenocarcinoma patients. Significant differences were observed in the EGFR mutations between Uighur lung adenocarcinoma patients and Han lung adenocarcinoma patients (P<0.001. Conclusion Our results indicate that the EGFR mutation in Uighur lung adenocarcinoma patients (16.2% is significantly lower than that in Han lung adenocarcinoma patients (45.7%.

Intrachromosomal amplification, locus deletion and point mutation in the aquaglyceroporin AQP1 gene in antimony resistant Leishmania (Viannia guyanensis.

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Rubens Monte-Neto

2015-02-01

Full Text Available Antimony resistance complicates the treatment of infections caused by the parasite Leishmania.Using next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1. Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion.This is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.

The Association of Factor V Leiden Mutation with Recurrent Pregnancy Loss

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Kashif, M.; Saeed, A.

2015-01-01

Objective: To determine the association of factor V Leiden mutation with recurrent pregnancy loss. Methods: The case-control study was conducted at the Department of Haematology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from January to June 2012, and comprised women of 18 to 45 years of age who had a history of recurrent pregnancy loss, and controls with no history of pregnancy loss. All the subjects belonged to Punjabi ethnic group. Three ml blood was taken from cases and controls and deoxyribonucleic acid was extracted. In order to identify Factor V Leiden mutation, polymerase chain reaction method was utilised combined with the amplification refractory mutation system. Data was analysed using SPSS 17. Results: Of the 112 subjects, 56(50 percent) were in each of the two groups. The presence of factor V Leiden mutation among the cases was 3(5.4 percent) while it was absent among the controls. The mutation was significantly associated with recurrent pregnancy loss (p=0.017).Recurrent pregnancy loss was higher in cases than controls (p=0.001). Conclusion: Factor V Leiden mutation was significantly associated with recurrent pregnancy loss. It should be considered one of the causes of recurrent pregnancy loss. (author)

Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.

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Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei

2014-09-15

Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

REFRACTORY THROMBOCYTOPENIA AND NEUTROPENIA: A DIAGNOSTIC CHALLENGE

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Emmanuel Gyan

2015-02-01

Full Text Available Background. The 2008 WHO classification identified refractory cytopenia with unilineage dysplasia (RCUD as a composite entity encompassing refractory anemia, refractory thrombocytopenia (RT, and refractory neutropenia (RN, characterized by 10% or more dysplastic cells in the bone marrow respective lineage. The diagnosis of RT and RN is complicated by several factors.  Diagnosing RT first requires exclusion of familial thrombocytopenia, chronic auto-immune thrombocytopenia, concomitant medications, viral infections, or hypersplenism. Diagnosis of RN should also be made after ruling out differential diagnoses such as ethnic or familial neutropenia, as well as acquired, drug-induced, infection-related or malignancy-related neutropenia. An accurate quantification of dysplasia should be performed in order to distinguish RT or RN from the provisional entity named idiopathic cytopenia of unknown significance (ICUS. Cytogenetic analysis, and possibly in the future somatic mutation analysis (of genes most frequently mutated in MDS, and flow cytometry analysis aberrant antigen expression on myeloid cells may help in this differential diagnosis. Importantly, we and others found that, while isolated neutropenia and thrombocytopenia are not rare in MDS, those patients can generally be classified (according to WHO 2008 classification as refractory cytopenia with multilineage dysplasia or refractory anemia with excess blasts, while RT and RN (according to WHO 2008 are quite rare.These results suggest in particular that identification of RT and RN as distinct entities could be reconsidered in future WHO classification updates.

Mutations and amplification of EPSPS gene confer resistance to glyphosate in goosegrass (Eleusine indica).

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Chen, Jingchao; Huang, Hongjuan; Zhang, Chaoxian; Wei, Shouhui; Huang, Zhaofeng; Chen, Jinyi; Wang, Xu

2015-10-01

Field-evolved resistance of goosegrass to glyphosate is due to double or single mutation in EPSPS , or amplification of EPSPS leads to increased transcription and protein levels. Glyphosate has been used widely in the south of China. The high selection pressure from glyphosate use has led to the evolution of resistance to glyphosate in weeds. We investigated the molecular mechanisms of three recently discovered glyphosate-resistant Eleusine indica populations (R1, R2 and R3). The results showed that R1 and R2 had double Thr102Ile and Pro106Ser mutation and a single mutation of Pro106Leu in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, respectively. Escherichia coli containing the mutated EPSPS genes was tolerant to glyphosate. EPSPS activity in R1 and R2 plants was higher than in the sensitive plants. There was no amino acid substitution in EPSPS gene in R3. However, expression of EPSPS in R3 plants was higher than in glyphosate-susceptible (S) population (13.8-fold) after glyphosate treatment. EPSPS enzyme activity in both R3 and S plants was inhibited by glyphosate, while shikimate accumulation in R3 was significantly lower than for the S population. Further analysis revealed that the genome of R3 contained 28.3-fold more copies of the EPSPS gene than that of susceptible population. EPSPS expression was positively correlated with copy number of EPSPS. In conclusion, mutation of the EPSPS gene and increased EPSPS expression are part of the molecular mechanisms of resistance to glyphosate in Eleusine indica.

Detection of wild-type EGFR amplification and EGFRvIII mutation in CSF-derived extracellular vesicles of glioblastoma patients.

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Figueroa, Javier M; Skog, Johan; Akers, Johnny; Li, Hongying; Komotar, Ricardo; Jensen, Randy; Ringel, Florian; Yang, Isaac; Kalkanis, Steven; Thompson, Reid; LoGuidice, Lori; Berghoff, Emily; Parsa, Andrew; Liau, Linda; Curry, William; Cahill, Daniel; Bettegowda, Chetan; Lang, Frederick F; Chiocca, E Antonio; Henson, John; Kim, Ryan; Breakefield, Xandra; Chen, Clark; Messer, Karen; Hochberg, Fred; Carter, Bob S

2017-10-19

RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR). CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs. EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61% and a specificity of 98% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM. Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

MDM2 Amplification and PI3KCA Mutation in a Case of Sclerosing Rhabdomyosarcoma

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Ken Kikuchi

2013-01-01

Full Text Available A rare sclerosing variant of rhabdomyosarcoma characterized by prominent hyalinization and pseudovascular pattern has recently been described as a subtype biologically distinct from embryonal, alveolar, and pleomorphic forms. We present cytogenetic and molecular findings as well as experimental studies of an unusual case of sclerosing rhabdomyosarcoma. The primary lesion arose within the plantar subcutaneous tissue of the left foot of an otherwise healthy 23-year-old male who eventually developed pulmonary nodules despite systemic chemotherapy. Two genetic abnormalities identified in surgical and/or autopsy samples of the tumor were introduced into 10T1/2 murine fibroblasts to determine whether these genetic changes cooperatively facilitated transformation and growth. Cytogenetic analysis revealed a complex abnormal hyperdiploid clone, and MDM2 gene amplification was confirmed by fluorescence in situ hybridization. Cancer gene mutation screening using a combination of multiplexed PCR and mass spectroscopy revealed a PIK3CA exon 20 H1047R mutation in the primary tumor, lung metastasis, and liver metastasis. However, this mutation was not cooperative with MDM2 overexpression in experimental assays for transformation or growth. Nevertheless, MDM2 and PIK3CA are genes worthy of further investigation in patients with sclerosing rhabdomyosarcoma and might be considered in the enrollment of these patients into clinical trials of targeted therapeutics.

An MPL W515L mutation in refractory anemia with ringed sideroblasts associated with marked thrombocytosis: A case report.

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Hao, Lin; Sen, Sandeep; Sugumar, Dhivya

2015-02-01

The current study presents the case of a 63-year-old patient exhibiting refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), who was positive for the MPL W515L mutation, but negative for the JAK2 V617F mutation. Following diagnosis, the patient remained asymptomatic for over three years, however, in August 2012, the patient relapsed and was administered with supportive treatment in the form of subcutaneous darbepoetin α at a dose of 300 μg/week, which resulted in an increased hemoglobin concentration, allowing the patient to remain transfusion-independent. The MPL W515L mutation has been reported in two previous cases of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) with ringed sideroblasts, however, to the best of our knowledge, the current report is the first to present a case of RARS-T with an MPL W515L mutation. A clinical trial designed to evaluate the efficacy of a targeted agent against the JAK2 V617F mutation is currently ongoing, with the aim of providing a novel therapeutic strategy for treating MDS/MPN patients. As MPL is located upstream of the JAK-STAT signaling pathway, it is a possible therapeutic target in MDS/MPN patients positive for an MPL W515L mutation, but negative for a JAK2 V617F mutation.

Amplification-free liquid biopsy by fluorescence approach

DEFF Research Database (Denmark)

Uhd, Jesper; Okholm, Anders; Kjems, Jargen

2017-01-01

Liquid biopsy is an attractive new paradigm of modern cancer research and clinical oncology. Synergy of fluorescence microscopy with mutation specific molecular probes is a method that we developed for the detection of tumor related circulating DNA, ctDNA. The present detection methods of ctDNA...... samples include amplification-based techniques that have multiple challenges, are often time consuming and rather expensive. In this work, we successfully applied the hybridization assay and advanced microscopy for the reliable amplification-free detection and quantification of cancer associated mutations...... in ctDNA....

Genetic signatures from amplification profiles characterize DNA mutation in somatic and radiation-induced sports of chrysanthemum

International Nuclear Information System (INIS)

Trigiano, R.N.; Scott, M.C.; Caetano-Anolles, G.

1998-01-01

The chrysanthemum (Dendranthema grandiflora Tzvelev.) cultivars 'Dark Charm', 'Salmon Charm', 'Coral Charm' and 'Dark Bronze Charm' are either radiation-induced mutants or spontaneous sports of 'Charm' and constitute a family or series of plants that primarily differ in flower color. These cultivars, which were difficult to differentiate genetically by DNA amplification fingerprinting (DAF), were easily identified by using arbitrary signatures from amplification profiles (ASAP). Genomic DNA was first amplified with three standard octamer arbitrary primers, all of which produced monomorphic profiles. Products from each of these DNA fingerprints were subsequently reamplified using four minihairpin decamer primers. The 12 primer combinations produced signatures containing approximately 37% polymorphic character loci, which were used to estimate genetic relationships between cultivars. Forty-six (32%) unique amplification products were associated with individual cultivars. The number of ASAP polymorphisms detected provided an estimate of the mutation rate in the mutant cultivars, ranging from 0.03% to 1.6% of nucleotide changes within an average of 18 kb of arbitrary amplified DAF sequence. The ASAP technique permits the clear genetic identification of somatic mutants and radiation-induced sports that are genetically highly homogeneous and should facilitate marker assisted breeding and protection of plant breeders rights of varieties or cultivars

RAPID DETECTION OF -THALASSEMIA MUTATIONS IN THAILAND USING MULTIPLEX ARMS

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D. Shimbhu

2017-11-01

Full Text Available The number of mutations underlining b-thalassemia generate a wide variety of different clinical phenotypes. An understanding of the genotype is important for medical personnel in order to provide proper counseling to patients and their families. Characterization of these mutations should aid the planning of a prenatal diagnosis program for bthalassemia. The heterogeneity of the mutations makes it difficult and time consuming to identify the mutation in some individuals. We developed a single-tube multiplex amplification refractory mutation system (multiplex ARMS to identify common ethnic- specific b-thalassemia mutations. Confirmation of multiplex ARMS results was carried out using direct sequencing. Three thousand three hundred twenty two people from Phitsanulok province were screened for the b-thalassemia trait by quantitation of HbA2 with microcolumn chromatography and the genotypes of mutations were characterized using multiplex ARMS and direct sequencing. We found that the deletion at codons 41/42 (-TCTT was the most frequent (48%, codon 17 (A®T (30%, -28 (A®G (6% and IVS-I-1(G®T (6% were the second and third in frequency respectively. A -87 (C®A mutation (4%, IVS II-654 (C®T (2%, codons 71/72 (+A (2% and codon 35 (C®A mutations (2% were also found. These techniques were found to be a valuable tool for analysis of b-thalassemia mutations because they are accurate, simple, and speedy in operation. The application for the diagnosis of severe thalassemia in high-risk pregnancies is promising.

MDM2 and CDK4 amplifications are rare events in salivary duct carcinomas.

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Grünewald, Inga; Trautmann, Marcel; Busch, Alina; Bauer, Larissa; Huss, Sebastian; Schweinshaupt, Petra; Vollbrecht, Claudia; Odenthal, Margarete; Quaas, Alexander; Büttner, Reinhard; Meyer, Moritz F; Beutner, Dirk; Hüttenbrink, Karl-Bernd; Wardelmann, Eva; Stenner, Markus; Hartmann, Wolfgang

2016-11-15

Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands associated with poor clinical outcome. SDCs are known to carry TP53 mutations in about 50%, however, only little is known about alternative pathogenic mechanisms within the p53 regulatory network. Particularly, data on alterations of the oncogenes MDM2 and CDK4 located in the chromosomal region 12q13-15 are limited in SDC, while genomic rearrangements of the adjacent HMGA2 gene locus are well documented in subsets of SDCs. We here analyzed the mutational status of the TP53 gene, genomic amplification of MDM2, CDK4 and HMGA2 rearrangement/amplification as well as protein expression of TP53 (p53), MDM2 and CDK4 in 51 de novo and ex pleomorphic adenoma SDCs.25 of 51 cases were found to carry TP53 mutations, associated with extreme positive immunohistochemical p53 staining levels in 13 cases. Three out of 51 tumors had an MDM2 amplification, one of them coinciding with a CDK4 amplification and two with a HMGA2 rearrangement/amplification. Two of the MDM2 amplifications occurred in the setting of a TP53 mutation. Two out of 51 cases showed a CDK4 amplification, one synchronously being MDM2 amplified and the other one displaying concurrent low copy number increases of both, MDM2 and HMGA2.In summary, we here show that subgroups of SDCs display genomic amplifications of MDM2 and/or CDK4, partly in association with TP53 mutations and rearrangement/amplification of HMGA2. Further research is necessary to clarify the role of chromosomal region 12q13-15 alterations in SDC tumorigenesis and their potential prognostic and therapeutic relevance.

Competitive amplification of differentially melting amplicons (CADMA improves KRAS hotspot mutation testing in colorectal cancer

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Kristensen Lasse

2012-11-01

Full Text Available Abstract Background Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR. Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples. Methods We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA. The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit. Results The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing. Conclusions The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification

International Nuclear Information System (INIS)

Marcinkowska-Swojak, Malgorzata; Handschuh, Luiza; Wojciechowski, Pawel; Goralski, Michal; Tomaszewski, Kamil; Kazmierczak, Maciej; Lewandowski, Krzysztof; Komarnicki, Mieczyslaw

2016-01-01

Highlights: • The NPM1 mutations were detected exclusively in AML accounting for 25% of cases. • The NPM1 gene did not reveal any copy number alterations. • The NPM1mut+ assay is a reliable test for the analysis of mutations and CNA in NPM1. - Abstract: The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The

Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification

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Marcinkowska-Swojak, Malgorzata, E-mail: m-marcinkowska@o2.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Handschuh, Luiza, E-mail: luizahan@ibch.poznan.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); Wojciechowski, Pawel, E-mail: Pawel.Wojciechowski@cs.put.poznan.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Institute of Computing Science, Poznan University of Technology, Piotrowo 2, 60-965 Poznan (Poland); Goralski, Michal, E-mail: mgoralsk@ibch.poznan.pl [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Tomaszewski, Kamil, E-mail: kamil.tomaszewsky@gmail.com [European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan (Poland); Kazmierczak, Maciej, E-mail: maciej.kazmierczak@onet.eu [Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); Lewandowski, Krzysztof, E-mail: krzysztof.lewandowski@skpp.edu.pl [Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); Komarnicki, Mieczyslaw, E-mail: mak7@pro.onet.pl [Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan (Poland); and others

2016-04-15

Highlights: • The NPM1 mutations were detected exclusively in AML accounting for 25% of cases. • The NPM1 gene did not reveal any copy number alterations. • The NPM1mut+ assay is a reliable test for the analysis of mutations and CNA in NPM1. - Abstract: The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The

High rate of mutation K103N causing resistance to nevirapine in Indian children with acquired immunodeficiency syndrome

Directory of Open Access Journals (Sweden)

Sehgal S

2008-01-01

Full Text Available In north India the number of paediatric cases with acquired immunodeficiency syndrome (AIDS is on the rise. Most drug combinations used for treatment of AIDS incorporate nevirapine, resistance to which develops very fast if given singly or because of unplanned interruptions. This paper investigates presence of mutations at codon 103 and codon 215 of the HIV pol gene causing resistance to nevirapine and zidovudine (AZT respectively in 25 children with AIDS. Mutations T215Y and K103N were detected by a nested cum amplification refractory mutation system polymerase chain reaction (ARMS PCR and the results were confirmed by direct sequencing in five randomly selected cases. Nineteen patients had received nevirapine containing regimen and six were drug naive. Mutation K103N was observed in 56% (14/25 of the children while mutation T215Y was found in none. Two of the six drug naοve children also showed K103N mutation. Thus, Indian children drug naοve or treated with nevirapine containing regimens show a high rate of mutation conferring resistance to nevirapine which calls for a judicious use of nevirapine both in antenatal and postnatal setting.

Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

OpenAIRE

Wang, Huiping; Kong, Fanrong; Sorrell, Tania C; Wang, Bin; McNicholas, Paul; Pantarat, Namfon; Ellis, David; Xiao, Meng; Widmer, Fred; Chen, Sharon CA

2009-01-01

Abstract Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in th...

The association of JAK2V617F mutation and leukocytosis with thrombotic events in essential thrombocythemia.

Science.gov (United States)

Hsiao, Hui-Hua; Yang, Ming-Yu; Liu, Yi-Chang; Lee, Ching-Ping; Yang, Wen-Chi; Liu, Ta-Chih; Chang, Chao-Sung; Lin, Sheng-Fung

2007-11-01

The Janus kinase 2 mutation, JAK2 (V617F), and megakaryocytic mutations, MPL (W515L/K), have been identified and correlated with a subtype of essential thrombocythemia (ET) patients. We investigated the frequency of mutations in ET patients and analyzed the relationship with their clinical features. Fifty-three ET patients were enrolled in the study. The amplification refractory mutation system was applied for the mutation survey of the JAK2V617F, while the polymerase chain reaction with sequencing was used for the mutation survey of MPLW515L/K. Thirty-five (66%) patients harboring the JAK2 (V617F) mutation, including 3 homozygous and 32 heterozygous changes, but no MPLW515L/K mutation, were found. During follow-up, 17 (32.1%) patients suffered from documented thrombotic events, with 15 having JAK2V617F mutations. Statistical analysis showed that patients with the JAK2 mutation had significantly higher leukocytes, hemoglobin level, and thrombotic event (p = 0.043, p = 0.001, and p = 0.029, respectively). Thrombotic events were also significantly correlated with leukocytosis and older age. The JAK2V617F mutation was noted in a certain population of ET patients and correlated with leukocytosis, high hemoglobin level, and thrombosis. Therefore, detection of the JAK2V617F mutation can affect not only the diagnosis, but also the management of ET patients.

Low frequency of c-MPL gene mutations in Iranian patients with Philadelphia-negative myeloproliferative disorders.

Science.gov (United States)

Ghotaslou, A; Nadali, F; Chahardouli, B; Alizad Ghandforosh, N; Rostami, S H; Alimoghaddam, K; Ghavamzadeh, A

2015-01-01

Myeloproliferative disorders are a group of diseases characterized by increased proliferation of myeloid lineage. In addition to JAK2V617F mutation, several mutations in the c-MPL gene have been reported in patients with philadelphia-negative chronic myeloproliferative disorders that could be important in the pathogenesis of diseases. The aim of the present study was to investigate the frequency of c-MPL and JAK2V617F mutations in Iranian patients with Philadelphia-negativemyeloproliferative disorders. Peripheral blood samples were collected from 60 patients with Philadelphia-negative MPD) Subgroups ET and PMF) and 25 healthy subjects as control group. The mutation status of c-MPL and Jak2V617F were investigated by using Amplification-refractory mutation system (ARMS) and Allele-Specific PCR (AS-PCR), respectively. The results were confirmed by sequencing. Among 60 patients, 34 (56.6%) and 1(1.7%) had Jak2V617F and c-MPL mutation, respectively. Patients with Jak2V617F mutation had higher WBC counts and hemoglobin concentration than those without the mutation (p= 0.005, p=0.003). In addition, for all healthy subjects in control group, mutations were negative. The present study revealed that the c-MPL mutations unlike the Jak2V617F mutations are rare in Iranian patients with Ph-negative MPNs and the low mutation rate should be considered in the design of screening strategies of MPD patients.

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis

DEFF Research Database (Denmark)

De Roock, Wendy; Claes, Bart; Bernasconi, David

2010-01-01

with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre...

Unique TTC repeat base pair loss mutation in cases of pure neural leprosy: A survival strategy of Mycobacterium leprae?

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Abhishek De

2015-01-01

Full Text Available Background: Genomic reduction helps obligate intracellular microbes to survive difficult host niches. Adaptation of Mycobacterium leprae in cases of pure neural leprosy (PNL in the intracellular niche of peripheral nerves can be associated with some gene loss. Recently, a stable but variable number of tandem repefzats (TTC have been reported in strains of M. leprae. FolP and rpoB genes are the two common mutation sites which deal with the susceptibility of the bacteria to drugs. Aim: We attempted to find if genomic reduction of M. leprae in context of these TTC repeats or mutations in folP1 and rpoB can be the reason for the restriction of M. leprae in the nerves in PNL. Materials and Methods: DNA extracts taken from fine needle aspiration of affected nerves of 24 PNL cases were studied for tandem repeats with 21TTC primer in multiplex-PCR. Mutations were also studied by PCR Amplification of SRDR (Sulphone Resistance Determining Region of the folP1 and multiple primer PCR amplification refractory mutation system (MARS of the rpoB. Results: Of the 24 PNL, only 1 patient showed mutation in the rpoB gene and none in the folp1 gene. Studying the mutation in TTC region of the M. leprae gene we found that all the cases have a loss of a few bases in the sequence. Conclusion: We can conclude that there is consistent loss in the bases in the TTC region in all cases of pure neural Hansen and we postulate that it may be an adaptive response of the bacteria to survive host niche resulting in its restriction to peripheral nerves.

Iron Refractory Iron Deficiency Anaemia: A Rare Cause of Iron Deficiency Anaemia

LENUS (Irish Health Repository)

McGrath, T

2018-01-01

We describe the case of a 17-month-old boy with a hypochromic microcytic anaemia, refractory to oral iron treatment. After exclusion of dietary and gastrointestinal causes of iron deficiency, a genetic cause for iron deficiency was confirmed by finding two mutations in the TMPRSS6 gene, consistent with a diagnosis of iron-refractory iron deficiency anaemia (IRIDA).

Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

Science.gov (United States)

Takei, Hiraku; Morishita, Soji; Araki, Marito; Edahiro, Yoko; Sunami, Yoshitaka; Hironaka, Yumi; Noda, Naohiro; Sekiguchi, Yuji; Tsuneda, Satoshi; Ohsaka, Akimichi; Komatsu, Norio

2014-01-01

A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

Directory of Open Access Journals (Sweden)

Hiraku Takei

Full Text Available A gain-of-function mutation in the myeloproliferative leukemia virus (MPL gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs. The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5% of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

Anaesthesia for left thoracoscopic sympathectomy for refractory long ...

African Journals Online (AJOL)

... mutations including life-threatening cardiac arrhythmias and sudden death. Left thoracoscopic sympathectomy is an effective treatment for patients who are refractory to medical therapy or who need frequent epicardial internal cardio defibrillator intervention. The authors report three cases, one adult and two children, who ...

GAB2 amplifications refine molecular classification of melanoma.

Science.gov (United States)

Chernoff, Karen A; Bordone, Lindsey; Horst, Basil; Simon, Katherine; Twadell, William; Lee, Keagan; Cohen, Jason A; Wang, Shuang; Silvers, David N; Brunner, Georg; Celebi, Julide Tok

2009-07-01

Gain-of-function mutations in BRAF, NRAS, or KIT are associated with distinct melanoma subtypes with KIT mutations and/or copy number changes frequently observed among melanomas arising from sun-protected sites, such as acral skin (palms, soles, and nail bed) and mucous membranes. GAB2 has recently been implicated in melanoma pathogenesis, and increased copy numbers are found in a subset of melanomas. We sought to determine the association of increased copy numbers of GAB2 among melanoma subtypes in the context of genetic alterations in BRAF, NRAS, and KIT. A total of 85 melanomas arising from sun-protected (n = 23) and sun-exposed sites (n = 62) were analyzed for copy number changes using array-based comparative genomic hybridization and for gain-of-function mutations in BRAF, NRAS, and KIT. GAB2 amplifications were found in 9% of the cases and were associated with melanomas arising from acral and mucosal sites (P = 0.005). Increased copy numbers of the KIT locus were observed in 6% of the cases. The overall mutation frequencies for BRAF and NRAS were 43.5% and 14%, respectively, and were mutually exclusive. Among the acral and mucosal melanomas studied, the genetic alteration frequency was 26% for GAB2, 13% for KIT, 30% for BRAF, and 4% for NRAS. Importantly, the majority of GAB2 amplifications occurred independent from genetic events in BRAF, NRAS, and KIT. GAB2 amplification is critical for melanomas arising from sun-protected sites. Genetic alterations in GAB2 will help refine the molecular classification of melanomas.

[Frequency of the most common mutations of the CFTR gene in peruvian patients with cystic fibrosis using the ARMS-PCR technique].

Science.gov (United States)

Aquino, Ruth; Protzel, Ana; Rivera, Juan; Abarca, Hugo; Dueñas, Milagros; Nestarez, Cecilia; Purizaga, Nestor; Diringer, Benoit

2017-01-01

To determine the frequency of the ten most common mutations of the CFTR gene reported in Latin Americausing amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) in patients with cystic fibrosis (CF) in two referral hospitals in Peru during the year 2014. The frequency of the ten most common mutations of the CFTR gene was assessed in patients of the Hospital Nacional Edgardo Rebagliati Martins and the Instituto Nacional de Salud del Niño, both located in Lima, Peru. Blood samples were collected from 36 patients with CF, and the ARMS-PCR technique was used to determine the presence of these mutations. The study group included 73.5% of patients with a known diagnosis of CF in the country when the study was carried out. ARMS-PCR allowed three of the mutations to be identified in a combined 30.6% of the alleles from patients with CF, and 64.9% of the mutated alleles were not identified. The mutations found were p.Phe508del (22,2%), p.Gly542* (6,9%), and p.Arg1162* (1,4%). There is significant variability in both the frequency and type of mutations present in our study population and in what has been reported in other Latin American countries. It is necessary to perform studies that use complete sequencing technology for the CFTR gene to identify other mutations present in our population.

A novel FKRP-related muscular dystrophy founder mutation in South African Afrikaner patients with a phenotype suggestive of a dystrophinopathy

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M M Mudau

2017-01-01

Full Text Available Background. Fukutin-related protein (FKRP muscular dystrophy is an autosomal recessive disorder caused by mutations in the FKRP gene. The condition is often misdiagnosed as a dystrophinopathy. A previously unreported mutation, c.1100T>C in exon 4 of FKRP, had been identified in homozygous form in two white South African (SA Afrikaner patients clinically diagnosed with a dystrophinopathy. Objectives. To investigate whether the c.1100T>C mutation and the common European FKRP mutation c.826C>A are present in other patients of Afrikaner origin with suspected dystrophinopathy, and whether a founder haplotype exists. Methods. The c.1100T>C mutation was initially tested for using an amplification refractory mutation system technique in 45 white SA Afrikaner patients who had tested negative using multiplex ligation probe amplification screening for exonic deletions/duplications in the dystrophin gene. Sequencing analysis was used to confirm the c.1100T>C mutation and screen for the c.826C>A mutation. Two cohorts (each numbering 100 of Afrikaans and other white controls were screened for the c.1100T>C and c.826C>A mutations, respectively. Results. Of the 45 patients, 8 patients (17.8% were homozygous for c.1100T>C, 2 (4.4% were compound heterozygotes for c.1100T>C and c.826C>A, and 1 (2.2% was heterozygous for c.1100T>C with a second unidentified mutation. The c.1100T>C mutation was found in 1/100 controls, but no heterozygotes for the c.826C>A mutation were identified. Linked marker analysis for c.1100T>C showed a common haplotype, suggesting a probable founder mutation in the SA Afrikaner population. Conclusion. FKRP mutations may be relatively common in Afrikaners, and screening should be considered in patients who have a suggestive phenotype and test negative for a dystrophinopathy. This test will be useful for offering diagnostic, carrier and prenatal testing for affected individuals and their families. As FKRP muscular dystrophy is autosomal

Geographical distribution of β-globin gene mutations in Syria.

Science.gov (United States)

Murad, Hossam; Moasses, Faten; Dabboul, Amir; Mukhalalaty, Yasser; Bakoor, Ahmad Omar; Al-Achkar, Walid; Jarjour, Rami A

2018-04-11

Objectives β-Thalassemia disease is caused by mutations in the β-globin gene. This is considered as one of the common genetic disorders in Syria. The aim of this study was to identify the geographical distribution of the β-thalassemia mutations in Syria. Methods β-Globin gene mutations were characterized in 636 affected patients and 94 unrelated carriers using the amplification refractory mutations system-polymerase chain reaction technique and DNA sequencing. Results The study has revealed the presence of 38 β-globin gene mutations responsible for β-thalassemia in Syria. Important differences in regional distribution were observed. IVS-I.110 [G > A] (22.2%), IVS-I.1 [G > A] (17.8%), Cd 39 [C > T] (8.2%), IVS-II.1 [G > A] (7.6%), IVS-I.6 [T > C] (7.1%), Cd 8 [-AA] (6%), Cd 5 [-CT] (5.6%) and IVS-I.5 [G > C] (4.1%) were the eight predominant mutations found in our study. The coastal region had higher relative frequencies (37.9 and 22%) than other regions. A clear drift in the distribution of the third common Cd 39 [C > T] mutation in the northeast region (34.8%) to the northwest region (2.5%) was noted, while the IVS-I.5 [G > C] mutation has the highest prevalence in north regions. The IVS-I.6 [T > C] mutation had a distinct frequency in the middle region. Ten mutations -86 [C > G], -31 [A > G], -29 [A > G], 5'UTR; +22 [G > A], CAP + 1 [A > C], Codon 5/6 [-TG], IVS-I (-3) or codon 29 [C > T], IVS-I.2 [T > A], IVS-I.128 [T > G] and IVS-II.705 [T > G] were found in Syria for the first time. Conclusions These data will significantly facilitate the population screening, genetic counseling and prenatal diagnosis in Syrian population.

Frequency and Clinical Implication of the R450H Mutation in the Thyrotropin Receptor Gene in the Japanese Population Detected by Smart Amplification Process 2

Science.gov (United States)

Yanagawa, Yoshimaro; Aoki, Tomoyuki; Morimura, Tadashi; Araki, Osamu; Kimura, Takao; Ogiwara, Takayuki; Kotajima, Nobuo; Yanagawa, Masumi; Murakami, Masami

2014-01-01

In Japanese pediatric patients with thyrotropin (TSH) resistance, the R450H mutation in TSH receptor gene (TSHR) is occasionally observed. We studied the frequency and clinical implication of the R450H mutation in TSHR in the general population of Japanese adults using smart amplification process 2 (SmartAmp2). We designed SmartAmp2 primer sets to detect this mutation using a drop of whole blood. We analyzed thyroid function, antithyroid antibodies, and this mutation in 429 Japanese participants who had not been found to have thyroid disease. Two cases without antithyroid antibodies were heterozygous for the R450H mutation in TSHR. Thus, the prevalence of this mutation was 0.47% in the general population and 0.63% among those without antithyroid antibodies. Their serum TSH concentrations were higher than the average TSH concentration not only in subjects without antithyroid antibodies but also in those with antithyroid antibodies. The R450H mutation in TSHR is relatively common in the Japanese population and potentially affects thyroid function. The present study demonstrates that the SmartAmp2 method is useful to detect the R450H mutation in TSHR, which is one of the common causes of TSH resistance in the Japanese population. PMID:24895636

Use of human tissue to assess the oncogenic activity of melanoma-associated mutations.

Science.gov (United States)

Chudnovsky, Yakov; Adams, Amy E; Robbins, Paul B; Lin, Qun; Khavari, Paul A

2005-07-01

Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification. Melanomas also commonly show impairment of the p16(INK4A)-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16(INK4A) and ARF protein loss. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations. TERT amplification also occurs in melanoma. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

Characterisation and confirmation of rare beta-thalassaemia mutations in the Malay, Chinese and Indian ethnic groups in Malaysia.

Science.gov (United States)

Tan, Jin Ai Mary Anne; Chin, Pui See; Wong, Yean Ching; Tan, Kim Lian; Chan, Lee Lee; George, Elizabeth

2006-10-01

In Malaysia, about 4.5% of the Malay and Chinese populations are heterozygous carriers of beta-thalassaemia. The initial identification of rare beta-globin gene mutations by genomic sequencing will allow the development of simpler and cost-effective PCR-based techniques to complement the existing amplification refractory mutation system (ARMS) and gap-PCR used for the identification of beta-thalassaemia mutations. DNA from 173 beta-thalassaemia carriers and five beta-thalassaemia major patients from the Malay, Chinese and Indian ethnic groups were first analysed by ARMS and gap-PCR. Ninety-five per cent (174/183) of the 183 beta-globin genes studied were characterised using these two techiques. The remaining nine uncharacterised beta-globin genes (4.9%) were analysed using genomic sequencing of a 904 bp amplified PCR product consisting of the promoter region, exon 1, intervening sequence (IVS) 1, exon 2 and the 5' IVS2 regions of the beta-globin gene. The rare beta-globin mutations detected in the Chinese patients were CD27/28 (+C) and CD43 (GAG-TAG), and -88 (C-T) in an Indian patient. Beta-globin mutations at CD16 (-C), IVS1-1 (G-A), IVS2-1 (G-A), -86 (C-G) and Haemoglobin South Florida (CD1, GTG-ATG) were confirmed in the Malay patients. The seven rare beta-globin mutations and a rare haemoglobin variant confirmed in this study have been described in other populations but have not been previously described in Malaysian beta-thalassemia patients.

Targeting MET Amplification as a New Oncogenic Driver

International Nuclear Information System (INIS)

Kawakami, Hisato; Okamoto, Isamu; Okamoto, Wataru; Tanizaki, Junko; Nakagawa, Kazuhiko; Nishio, Kazuto

2014-01-01

Certain genetically defined cancers are dependent on a single overactive oncogene for their proliferation and survival, a phenomenon known as “oncogene addiction”. A new generation of drugs that selectively target such “driver oncogenes” manifests a clinical efficacy greater than that of conventional chemotherapy in appropriate genetically defined patients. MET is a proto-oncogene that encodes a receptor tyrosine kinase, and aberrant activation of MET signaling occurs in a subset of advanced cancers as result of various genetic alterations including gene amplification, polysomy, and gene mutation. Our preclinical studies have shown that inhibition of MET signaling either with the small-molecule MET inhibitor crizotinib or by RNA interference targeted to MET mRNA resulted in marked antitumor effects in cancer cell lines with MET amplification both in vitro and in vivo. Furthermore, patients with non-small cell lung cancer or gastric cancer positive for MET amplification have shown a pronounced clinical response to crizotinib. Accumulating preclinical and clinical evidence thus suggests that MET amplification is an “oncogenic driver” and therefore a valid target for treatment. However, the prevalence of MET amplification has not been fully determined, possibly in part because of the difficulty in evaluating gene amplification. In this review, we provide a rationale for targeting this genetic alteration in cancer therapy

Targeting MET Amplification as a New Oncogenic Driver

Energy Technology Data Exchange (ETDEWEB)

Kawakami, Hisato [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Okamoto, Isamu, E-mail: okamotoi@kokyu.med.kyushu-u.ac.jp [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Center for Clinical and Translational Research, Kyushu University Hospital, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582 (Japan); Okamoto, Wataru [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Division of Transrlational Research, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577 (Japan); Tanizaki, Junko [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, HIM223, 450 Brookline Avenue, Boston, MA 02215 (United States); Nakagawa, Kazuhiko [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Nishio, Kazuto [Department of Genome Biology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan)

2014-07-22

Certain genetically defined cancers are dependent on a single overactive oncogene for their proliferation and survival, a phenomenon known as “oncogene addiction”. A new generation of drugs that selectively target such “driver oncogenes” manifests a clinical efficacy greater than that of conventional chemotherapy in appropriate genetically defined patients. MET is a proto-oncogene that encodes a receptor tyrosine kinase, and aberrant activation of MET signaling occurs in a subset of advanced cancers as result of various genetic alterations including gene amplification, polysomy, and gene mutation. Our preclinical studies have shown that inhibition of MET signaling either with the small-molecule MET inhibitor crizotinib or by RNA interference targeted to MET mRNA resulted in marked antitumor effects in cancer cell lines with MET amplification both in vitro and in vivo. Furthermore, patients with non-small cell lung cancer or gastric cancer positive for MET amplification have shown a pronounced clinical response to crizotinib. Accumulating preclinical and clinical evidence thus suggests that MET amplification is an “oncogenic driver” and therefore a valid target for treatment. However, the prevalence of MET amplification has not been fully determined, possibly in part because of the difficulty in evaluating gene amplification. In this review, we provide a rationale for targeting this genetic alteration in cancer therapy.

Quantum dots immunofluorescence histochemical detection of EGFR gene mutations in the non-small cell lung cancers using mutation-specific antibodies

Directory of Open Access Journals (Sweden)

Qu YG

2014-12-01

Full Text Available Yan-Gang Qu,1 Qian Zhang,2 Qi Pan,3 Xian-Da Zhao,4 Yan-Hua Huang,2 Fu-Chun Chen,3 Hong-Lei Chen41Department of Pathology, The Central Hospital of Enshi Autonomous Prefecture, Enshi, 2Department of Molecular Pathology, Wuhan Nano Tumor Diagnosis Engineering Research Center, Wuhan, Hubei, People’s Republic of China; 3Department of Thoracosurgery, Traditional Chinese Medical Hospital of Wenling, Wenling, Zhejiang, People’s Republic of China; 4Department of Pathology, School of Basic Medical Science, Wuhan University, Wuhan, Hubei, People’s Republic of ChinaBackground: Epidermal growth factor receptor (EGFR mutation status plays an important role in therapeutic decision making for non-small cell lung cancer (NSCLC patients. Since EGFR mutation-specific antibodies (E746-A750del and L858R have been developed, EGFR mutation detection by immunohistochemistry (IHC is a suitable screening test. On this basis, we want to establish a new screening test, quantum dots immunofluorescence histochemistry (QDs-IHC, to assess EGFR gene mutation in NSCLC tissues, and we compared it to traditional IHC and amplification refractory mutation system (ARMS.Materials and methods: EGFR gene mutations were detected by QDs-IHC, IHC, and ADx-ARMS in 65 cases of NSCLC composed of 55 formalin-fixed, paraffin-embedded specimens and ten pleural effusion cell blocks, including 13 squamous cell carcinomas, two adenosquamous carcinomas, and 50 adenocarcinomas.Results: Positive rates of EGFR gene mutations detected by QDs-IHC, IHC, and ADx-ARMS were 40.0%, 36.9%, and 46.2%, respectively, in 65 cases of NSCLC patients. The sensitivity of QDs-IHC when detecting EGFR mutations, as compared to ADx-ARMS, was 86.7% (26/30; the specificity for both antibodies was 100.0% (26/26. IHC sensitivity was 80.0% (24/30 and the specificity was 92.31% (24/26. When detecting EGFR mutations, QDs-IHC and ADx-ARMS had perfect consistency (κ=0.882; P<0.01. Excellent agreement was observed

Digital Microfluidics for Nucleic Acid Amplification

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Beatriz Coelho

2017-06-01

Full Text Available Digital Microfluidics (DMF has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

Presence of c.3956delC mutation in familial adenomatous polyposis patients from Brazil.

Science.gov (United States)

Moreira-Nunes, Caroline Aquino; Alcântara, Diego di Felipe Ávila; Lima-Júnior, Sérgio Figueiredo; Cavalléro, Sandro Roberto de Araújo; Rey, Juan Antonio; Pinto, Giovanny Rebouças; de Assumpção, Paulo Pimentel; Burbano, Rommel Rodriguez

2015-08-21

To characterize APC gene mutations and correlate them with patient phenotypes in individuals diagnosed with familial adenomatous polyposis (FAP) in northern Brazil. A total of 15 individuals diagnosed with FAP from 5 different families from the north of Brazil were analyzed in this study. In addition to patients with histopathological diagnosis of FAP, family members who had not developed the disease were also tested in order to identify mutations and for possible genetic counseling. All analyzed patients or their guardians signed a consent form approved by the Research Ethics Committee of the João de Barros Barreto University Hospital (Belem, Brazil). DNA extracted from the peripheral blood of a member of each of the affected families was subjected to direct sequencing. The proband of each family was sequenced to identify germline mutations using the Ion Torrent platform. To validate the detected mutations, Sanger sequencing was also performed. The samples from all patients were also tested for the identification of mutations by real-time quantitative polymerase chain reaction using the amplification refractory mutation system. Through interviews with relatives and a search of medical records, it was possible to construct genograms for three of the five families included in the study. All 15 patients from the five families with FAP exhibited mutations in the APC gene, and all mutations were detected in exon 15 of the APC gene. In addition to the patients with a histological diagnosis of FAP, family members without disease symptoms showed the mutation in the APC gene. In the present study, we detected two of the three most frequent germline mutations in the literature: the mutation at codon 1309 and the mutation at codon 1061. The presence of c.3956delC mutation was found in all families from this study, and suggests that this mutation was introduced in the population of the State of Pará through ancestor immigration (i.e., a de novo mutation that arose in one

Severe neonatal epileptic encephalopathy and KCNQ2 mutation: neuropathological substrate?

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Charlotte eDalen Meurs-Van Der Schoor

2014-12-01

Full Text Available Background:Neonatal convulsions are clinical manifestations in a heterogeneous group of disorders with different etiology and outcome. They are attributed to several genetic causes. Methods:We describe a patient with intractable neonatal seizures who died from respiratory compromise during a status epilepticus. Results:This case report provides EEG, MRI, genetic analysis and neuropathological data. Genetic analysis revealed a de novo heterozygous missense mutation in the KCNQ2 gene, which encodes a subunit of a voltage-gated potassium channel. KCNQ2 gene mutation is associated with intractable neonatal seizures. EEG, MRI data as well as mutation analysis have been described in other KCNQ2 cases. Postmortem neuropathologic investigation revealed mild malformation of cortical development with increased heterotopic neurons in the deep white matter compared to an age-matched control subject. The new finding of this study is the combination of a KCNQ2 mutation and the cortical abnormalities. Conclusions:KCNQ2 mutations should be considered in neonates with refractory epilepsy of unknown cause. The mild cortical malformation is an important new finding, though it remains unknown whether these cortical abnormalities are due to the KCNQ2 mutation or are secondary to the refractory seizures.

Spectrum of mutations in RARS-T patients includes TET2 and ASXL1 mutations

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Szpurka, Hadrian; Jankowska, Anna M.; Makishima, Hideki; Bodo, Juraj; Bejanyan, Nelli; Hsi, Eric D.; Sekeres, Mikkael A.; Maciejewski, Jaroslaw P.

2010-01-01

While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation ...

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

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Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo

2012-01-01

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397

EGFR Mutations in Surgically Resected Fresh Specimens from 697 Consecutive Chinese Patients with Non-Small Cell Lung Cancer and Their Relationships with Clinical Features

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Yuanyang Lai

2013-12-01

Full Text Available We aimed to reveal the true status of epidermal growth factor receptor (EGFR mutations in Chinese patients with non-small cell lung cancer (NSCLC after lung resections. EGFR mutations of surgically resected fresh tumor samples from 697 Chinese NSCLC patients were analyzed by Amplification Refractory Mutation System (ARMS. Correlations between EGFR mutation hotspots and clinical features were also explored. Of the 697 NSCLC patients, 235 (33.7% patients had tyrosine kinase inhibitor (TKIs sensitive EGFR mutations in 41 (14.5% of the 282 squamous carcinomas, 155 (52.9% of the 293 adenocarcinomas, 34 (39.5% of the 86 adenosquamous carcinomas, one (9.1% of the 11 large-cell carcinomas, 2 (11.1% of the 18 sarcomatoid carcinomas, and 2 (28.6% of the 7 mucoepidermoid carcinomas. TKIs sensitive EGFR mutations were more frequently found in female patients (p < 0.001, non-smokers (p = 0.047 and adenocarcinomas (p < 0.001. The rates of exon 19 deletion mutation (19-del, exon 21 L858R point mutation (L858R, exon 21 L861Q point mutation (L861Q, exon 18 G719X point mutations (G719X, including G719C, G719S, G719A were 43.4%, 48.1%, 1.7% and 6.8%, respectively. Exon 20 T790M point mutation (T790M was detected in 3 squamous carcinomas and 3 adenocarcinomas and exon 20 insertion mutation (20-ins was detected in 2 patients with adenocarcinoma. Our results show the rates of EGFR mutations are higher in all types of NSCLC in Chinese patients. 19-del and L858R are two of the more frequent mutations. EGFR mutation detection should be performed as a routine postoperative examination in Chinese NSCLC patients.

Regional and ethnic distribution of beta thalassemia mutations and effect of consanguinity in patients referred for prenatal diagnosis

International Nuclear Information System (INIS)

Hafeez, M.

2007-01-01

To determine the regional and ethnic distribution of beta thalassemia mutation and the effect of consanguinity in patients referred for prenatal diagnosis of beta b-thalassemia and to target the high risk population for screening. A total of 499 couples were referred to Gentec Lab., Lahore, from all over Pakistan for prenatal diagnosis of b-thalassemia. After counseling, chorionic villus sampling was done between 10-16 weeks of gestation. DNA analysis was done by Amplification Refractory Mutation System (ARMS) for type of mutation in the Armed Forces Institute of Pathology, Rawalpindi. Ethnicity, race and consanguineous relationship of parents was determined.b-thalassemia was prevalent in Punjabis (60.7%) followed by Saraikees (25.5%). Castewise it was most frequent in Rajputs followed by Jatts, Arain, Sheikhs and Pathans. 56.7% of the couples were first cousins and 19.8% were relatives. The commonest mutations were Frameshift 8-9 (Fr8-9) 33.5%, Intervening Sequence 1-5 (IVS 1-5) 17.2%, Fr4142 - 8%, IVS 1-1 - 5.2%, Deletion 619 (Del 619) 4.2% and Codon 5 (Cd 5) - 4.2%. In samples sent for analysis, 53.1% turned out to be carriers (trait), 25.3% were diseased (thalassemia major) and 21.6% were normal. P-value of all results was less than 0.001. In this series, the highest frequency was found in Punjabi Rajputs. The commonest mutation was Fr 8-9. Most parents were first cousins. Premarital thalassemia carrier testing can effectively reduce the disease. (author)

Deletion/duplication mutation screening of TP53 gene in patients with transitional cell carcinoma of urinary bladder using multiplex ligation-dependent probe amplification.

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Bazrafshani, Mohammad Reza R; Nowshadi, Pouriaali A; Shirian, Sadegh; Daneshbod, Yahya; Nabipour, Fatemeh; Mokhtari, Maral; Hosseini, Fatemehsadat; Dehghan, Somayeh; Saeedzadeh, Abolfazl; Mosayebi, Ziba

2016-02-01

Bladder cancer is a molecular disease driven by the accumulation of genetic, epigenetic, and environmental factors. The aim of this study was to detect the deletions/duplication mutations in TP53 gene exons using multiplex ligation-dependent probe amplification (MLPA) method in the patients with transitional cell carcinoma (TCC). The achieved formalin-fixed paraffin-embedded tissues from 60 patients with TCC of bladder were screened for exonal deletions or duplications of every 12 TP53 gene exons using MLPA. The pathological sections were examined by three pathologists and categorized according to the WHO scoring guideline as 18 (30%) grade I, 22 (37%) grade II, 13 (22%) grade III, and 7 (11%) grade IV cases of TCC. None mutation changes of TP53 gene were detected in 24 (40%) of the patients. Furthermore, mutation changes including, 15 (25%) deletion, 17 (28%) duplication, and 4 (7%) both deletion and duplication cases were observed among 60 samples. From 12 exons of TP53 gene, exon 1 was more subjected to exonal deletion. Deletion of exon 1 of TP53 gene has occurred in 11 (35.4%) patients with TCC. In general, most mutations of TP53, either deletion or duplication, were found in exon 1, which was statistically significant. In addition, no relation between the TCC tumor grade and any type of mutation were observed in this research. MLPA is a simple and efficient method to analyze genomic deletions and duplications of all 12 exons of TP53 gene. The finding of this report that most of the mutations of TP53 occur in exon 1 is in contrast to that of the other reports suggesting that exons 5-8 are the most (frequently) mutated exons of TP53 gene. The mutations of exon 1 of TP53 gene may play an important role in the tumorogenesis of TCC. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Spectrum of mutations in RARS-T patients includes TET2 and ASXL1 mutations.

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Szpurka, Hadrian; Jankowska, Anna M; Makishima, Hideki; Bodo, Juraj; Bejanyan, Nelli; Hsi, Eric D; Sekeres, Mikkael A; Maciejewski, Jaroslaw P

2010-08-01

While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation was present in one mutated TET2 and ASXL1 case. JAK2V617F/MPLW515L mutations were absent in TET2/ASXL1 mutants, indicating that similar clinical phenotype can be produced by various MPN-associated mutations and that additional unifying lesions may be present in RARS-T. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Novel Tetra-Primer ARMS-PCR Assays for Thiopurine Intolerance Susceptibility Mutations NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C in East Asians

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Chi-Chun Ho

2017-10-01

Full Text Available Thiopurines are clinically useful in the management of diverse immunological and malignant conditions. Nevertheless, these purine analogues can cause lethal myelosuppression, which may be prevented by prospective testing for variants in the thiopurine S-methyltransferase (TPMT and, in East Asians, Nudix hydrolase 15 (NUDT15 genes. Two single-tube, tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR assays were developed to genotype the common loss-of-function variants NUDT15 c.415C>T (rs116855232 and TPMT*3C c.719A>G (rs1142345. In a group of 60 unselected patients, one and seven were found to be homozygous and heterozygous, respectively, for NUDT15 c.415C>T; one was found to be heterozygous for TPMT*3C c.719A>G. There was no non-specific amplification, and the genotypes were 100% concordant with Sanger sequencing. Limit-of-detection for both assays was below 1 ng of heterozygous template per reaction. Time- and cost-effective ARMS-PCR assays, suitable for genotyping East-Asian patients for thiopurine intolerance, were successfully developed and validated.

The dynamics of diverse segmental amplifications in populations of Saccharomyces cerevisiae adapting to strong selection.

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Payen, Celia; Di Rienzi, Sara C; Ong, Giang T; Pogachar, Jamie L; Sanchez, Joseph C; Sunshine, Anna B; Raghuraman, M K; Brewer, Bonita J; Dunham, Maitreya J

2014-03-20

Population adaptation to strong selection can occur through the sequential or parallel accumulation of competing beneficial mutations. The dynamics, diversity, and rate of fixation of beneficial mutations within and between populations are still poorly understood. To study how the mutational landscape varies across populations during adaptation, we performed experimental evolution on seven parallel populations of Saccharomyces cerevisiae continuously cultured in limiting sulfate medium. By combining quantitative polymerase chain reaction, array comparative genomic hybridization, restriction digestion and contour-clamped homogeneous electric field gel electrophoresis, and whole-genome sequencing, we followed the trajectory of evolution to determine the identity and fate of beneficial mutations. During a period of 200 generations, the yeast populations displayed parallel evolutionary dynamics that were driven by the coexistence of independent beneficial mutations. Selective amplifications rapidly evolved under this selection pressure, in particular common inverted amplifications containing the sulfate transporter gene SUL1. Compared with single clones, detailed analysis of the populations uncovers a greater complexity whereby multiple subpopulations arise and compete despite a strong selection. The most common evolutionary adaptation to strong selection in these populations grown in sulfate limitation is determined by clonal interference, with adaptive variants both persisting and replacing one another.

Radiosensitivity and TP 53, EGFR amplification and LOH10 analysis of primary glioma cell cultures

International Nuclear Information System (INIS)

Gerlach, B.; Harder, A.H.; Slotman, B.J.; Sminia, P.; Hulsebos, T.J.M.; Leenstra, S.; Peter Vandertop, W.; Hartmann, K.A.

2002-01-01

Aim: Determination of in-vitro radiosensitivity and genetic alterations of cell cultures derived from human glioma biopsy tissue and established glioma cell lines. Material and Methods: Fresh brain tumor specimens of six patients were processed to early passage cell cultures. In addition the cell lines D 384 and Gli 6 were used. Cell cultures were irradiated with doses from 2 to 10 Gy. Following irradiation, cell survival was determined by clonogenic assay and survival curves were generated. The surviving fractions after 2 Gy (SF2) and 4 Gy (SF4) were used as radiosensitivity parameters. Genetic analysis included determination of the mutational and loss of heterozygosity (LOH) status of TP 53 (exons 5-8), the LOH 10- and epidermal growth factor receptor gene (EGFR) amplification status. Results: The SF2 and SF4 values ranged from 0.54 to 0.88 (mean: 0.70) and from 0.13 to 0.52 (mean: 0.32), respectively. Genetic alterations were found in the Gli 6 cell line and in two primary cell cultures. The genetic profile of Gli 6 showed LOH but no TP 53 mutation, complete LOH 10 and no EGFR amplification. The VU 15 cell culture showed TP 53 mutation but no LOH 10 or EGFR amplification, while VU 24 showed incomplete LOH 10, EGFR amplification and no TP 53 mutation. In the other four cell cultures and D 384 cell line no genetic alterations were diagnosed. Histopathological classification of glioblastoma multiforme and/or genetic alterations resulted in lower radiosensitivity. Conclusion: In this small series of early passage glioma cell cultures low radiosensitivity and alterations in cell regulatory genes were seen. Further testing of biological behavior in larger series of patient-derived material is ongoing. (orig.)

Development of an allele-specific, loop-mediated, isothermal amplification method (AS-LAMP to detect the L1014F kdr-w mutation in Anopheles gambiae s. l.

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Badolo Athanase

2012-07-01

Full Text Available Abstract Background Malaria control relies heavily on treated bed nets and indoor residual spraying with pyrethroid insecticides. Unfortunately, the resistance to pyrethroid insecticides, mainly due to the kdr mutation, is spreading in the main malaria vector Anopheles gambiae s.l., decreasing the insecticides’ efficacy. To manage the insecticide resistance rapidly and flexibly, simple and effective tools for the early detection of resistant mosquitoes are needed. This study aimed to develop an allele-specific, loop-mediated, isothermal amplification (AS-LAMP method to detect the West African-type kdr mutation (kdr-w; L1014F in field-collected mosquitoes. Methods DNA fragments of the wild-type and the mutated kdr gene were used to select the primers and develop the method. The primers were designed with the mutation at the 5’ end of the backward inner primer (BIP. The AS-LAMP method was compared to the AS-PCR method using the genomic DNA of 120 field-collected mosquitoes. Results The AS-LAMP method could discriminate between the wild-type homozygote, the heterozygote, and the kdr-w homozygote within 75 min. The AS-LAMP method has the advantage of being faster and at least as sensitive and specific as the AS-PCR method. Conclusions The AS-LAMP method can be used to detect the kdr mutation for quick decision-making, even in less well-equipped laboratories.

Slagging gasifier refractories. A new pathway to longer refractory life

Energy Technology Data Exchange (ETDEWEB)

Schnake, Mark [Harbinson-Walker Refractories Company, Mexico, MO (United States)

2013-07-01

Solid fuel slagging gasification to convert coal or petroleum coke feedstocks into syngas has rapidly evolved over the last 25 years. The gasifier is a high temperature, high pressure reaction chamber. Operating temperatures are between 1250 and 1575 C. Pressures will be between 20.4 and 68 atm. Syngas has been typically used for chemical feedstocks, fuel for power plants, or for steam and hydrogen generation in other industrial applications. Ash which comes from the solid fuel during gasification has many impurities. It melts during the gasifier reactor operation forming a liquid that penetrates the refractory lining. Given time, the refractory will wear away from thermal spalling, structural spalling, or overheating of the refractory. In some cases, all three wear mechanisms are seen in the same gasifier lining. Industry users have identified refractory life as one major limiting factor in worldwide use of this technology. Users have stated if the refractory liner can increase on-line availability of the gasifier operation, more industry acceptance of this technology is possible. Harbison-Walker Refractories Company will review destructive factors affecting lining life and discuss new refractory materials that have dramatically increased gasifier lining life and reliability. New refractory materials will be presented and supported by field trial results and post mortem analysis.

Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

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Huang, Lei; Ma, Fei; Chapman, Alec; Lu, Sijia; Xie, Xiaoliang Sunney

2015-01-01

We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

Blocking DNA Repair in Advanced BRCA-Mutated Cancer

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In this trial, patients with relapsed or refractory advanced cancer and confirmed BRCA mutations who have not previously been treated with a PARP inhibitor will be given BMN 673 by mouth once a day in 28-day cycles.

Molecular Epidemiology of EGFR Mutations in Asian Patients with Advanced Non-Small-Cell Lung Cancer of Adenocarcinoma Histology – Mainland China Subset Analysis of the PIONEER study

Science.gov (United States)

Shi, Yuankai; Li, Junling; Zhang, Shucai; Wang, Mengzhao; Yang, Shujun; Li, Ning; Wu, Gang; Liu, Wei; Liao, Guoqing; Cai, Kaican; Chen, Liang’an; Zheng, Meizhen; Yu, Ping; Wang, Xiuwen; Liu, Yunpeng; Guo, Qisen; Nie, Ligong; Liu, Jiwei; Han, Xiaohong

2015-01-01

Epidermal growth factor receptor (EGFR) mutations are the strongest response predictors to EGFR tyrosine kinase inhibitors (TKI) therapy, but knowledge of the EGFR mutation frequency on lung adenocarcinoma is still limited to retrospective studies. The PIONEER study (NCT01185314) is a prospective molecular epidemiology study in Asian patients with newly diagnosed advanced lung adenocarcinoma, aiming to prospectively analyze EGFR mutation status in IIIB/IV treatment-naïve lung adenocarcinomas in Asia. We report the mainland China subset results. Eligible patients (≥20 yrs old, IIIB/IV adenocarcinoma and treatment-naïve) were registered in 17 hospitals in mainland China. EGFR was tested for mutations by amplification refractory mutation system using biopsy samples. Demographic and clinical characteristics were collected for subgroup analyses. A total of 747 patients were registered. Successful EGFR mutation analysis was performed in 741, with an overall mutation rate of 50.2%. The EGFR active mutation rate is 48.0% (with 1.3% of combined active and resistance mutations). Tobacco use (>30 pack-year vs. 0–10 pack-year, OR 0.27, 95%CI: 0.17–0.42) and regional lymph nodes involvement (N3 vs. N0, OR 0.47, 95%CI: 0.29–0.76) were independent predictors of EGFR mutation in multivariate analysis. However, even in regular smokers, the EGFR mutation frequency was 35.3%. The EGFR mutation frequency was similar between diverse biopsy sites and techniques. The overall EGFR mutation frequency of the mainland China subset was 50.2%, independently associated with the intensity of tobacco use and regional lymph nodes involvement. The relatively high frequency of EGFR mutations in the mainland China subset suggest that any effort to obtain tissue sample for EGFR mutation testing should be encouraged. PMID:26599344

An ultrasensitive colorimeter assay strategy for p53 mutation assisted by nicking endonuclease signal amplification.

Science.gov (United States)

Lin, Zhenyu; Yang, Weiqiang; Zhang, Guiyun; Liu, Qida; Qiu, Bin; Cai, Zongwei; Chen, Guonan

2011-08-28

A novel catalytic colorimetric assay assisted by nicking endonuclease signal amplification (NESA) was developed. With the signal amplification, the detection limit of the p53 target gene can be as low as 1 pM, which is nearly 5 orders of magnitude lower than that of other previously reported colorimetric DNA detection strategies based on catalytic DNAzyme.

High alumina refractories

International Nuclear Information System (INIS)

Simao, L.C.; Lopes, A.B.; Galvao Filho, N.B.; Souza, R.B. de

1989-01-01

High alumina refractories with 92 to 96.5% Al 2 O 3 were produced using brown and white fused as aggregate. Those refractories present only alumina-α and mullite as crystalline mineralogical phase. Other physical and chemical characteristics are similar to the ones found in refractories produced in Brazil, Japan and U.S.A. The most important physical and chemical tests used for the characterization of the raw materials and refractories, complemented by those realized at high temperatures, plus X-ray Difractometry and optical microscopy are presented, besides the refractory formulation and main parameters of production [pt

Recurrent mutations in the CDKL5 gene: genotype-phenotype relationships.

Science.gov (United States)

Bahi-Buisson, Nadia; Villeneuve, Nathalie; Caietta, Emilie; Jacquette, Aurélia; Maurey, Helene; Matthijs, Gert; Van Esch, Hilde; Delahaye, Andrée; Moncla, Anne; Milh, Mathieu; Zufferey, Flore; Diebold, Bertrand; Bienvenu, Thierry

2012-07-01

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been described in epileptic encephalopathies in females with infantile spasms with features that overlap with Rett syndrome. With more than 80 reported patients, the phenotype of CDKL5-related encephalopathy is well-defined. The main features consist of seizures starting before 6 months of age, severe intellectual disability with absent speech and hand stereotypies and deceleration of head growth, which resembles Rett syndrome. However, some clinical discrepancies suggested the influence of genetics and/or environmental factors. No genotype-phenotype correlation has been defined and thus there is a need to examine individual mutations. In this study, we analyzed eight recurrent CDKL5 mutations to test whether the clinical phenotype of patients with the same mutation is similar and whether patients with specific CDKL5 mutations have a milder phenotype than those with other CDKL5 mutations. Patients bearing missense mutations in the ATP binding site such as the p.Ala40Val mutation typically walked unaided, had normocephaly, better hand use ability, and less frequent refractory epilepsy when compared to girls with other CDKL5 mutations. In contrast, patients with mutations in the kinase domain (such as p.Arg59X, p.Arg134X, p.Arg178Trp/Pro/Gln, or c.145 + 2T > C) and frameshift mutations in the C-terminal region (such as c.2635_2636delCT) had a more severe phenotype with infantile spasms, refractory epileptic encephalopathy, absolute microcephaly, and inability to walk. It is important for clinicians to have this information when such patients are diagnosed. Copyright © 2012 Wiley Periodicals, Inc.

Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing.

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Karak, Somdatta; Jacobs, Julie S; Kittelmann, Maike; Spalthoff, Christian; Katana, Radoslaw; Sivan-Loukianova, Elena; Schon, Michael A; Kernan, Maurice J; Eberl, Daniel F; Göpfert, Martin C

2015-11-26

Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

Mutation induction by heavy ions

Science.gov (United States)

Kiefer, J.; Stoll, U.; Schneider, E.

1994-10-01

Mutation induction by heavy ions is compared in yeast and mammalian cells. Since mutants can only be recovered in survivors the influence of inactivation cross sections has to be taken into account. It is shown that both the size of the sensitive cellular site as well as track structure play an important role. Another parameter which influences the probability of mutation induction is repair: Contrary to naive assumptions primary radiation damage does not directly lead to mutations but requires modification to reconstitute the genetic machinery so that mutants can survive. The molecular structure of mutations was analyzed after exposure to deuterons by amplification with the aid of polymerase chain reaction. The results-although preliminary-demonstrate that even with densely ionizing particles a large fraction does not carry big deletions which suggests that point mutations may also be induced by heavy ions.

Merits of using andalusite-based refractories compared to bauxite-based refractories

OpenAIRE

Nyoka, M.; Brazier, D.; Courtney, T.; Parry, R.A.

2013-01-01

Historically bauxite-based refractories have been used in applications where andalusite-based refractories could work. Bauxite-based refractories were chosen over andalusite-based refractories mainly because of the availability of low-cost Chinese bauxite and also because many furnaces were designed by international companies that cannot easily access high-quality products. Currently, the availability of low-cost bauxite is under threat as a result of high export duties and tariffs as well as...

Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

Directory of Open Access Journals (Sweden)

Ellis David

2009-08-01

Full Text Available Abstract Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. Results The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96% isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78% fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates, G448E (n = 3, G307S (n = 3, K143R (n = 3 and Y123H, S405F and R467K (each n = 1. DNA sequencing revealed a novel substitution, G450V, in one isolate. Conclusion The sensitive RCA

Clinical features and course of refractory anemia with ring sideroblasts associated with marked thrombocytosis

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Broseus, Julien; Florensa, Lourdes; Zipperer, Esther; Schnittger, Susanne; Malcovati, Luca; Richebourg, Steven; Lippert, Eric; Cermak, Jaroslav; Evans, Jyoti; Mounier, Morgane; Raya, José Maria; Bailly, François; Gattermann, Norbert; Haferlach, Torsten; Garand, Richard; Allou, Kaoutar; Besses, Carlos; Germing, Ulrich; Haferlach, Claudia; Travaglino, Erica; Luno, Elisa; Pinan, Maria Angeles; Arenillas, Leonor; Rozman, Maria; Perez Sirvent, Maria Luz; Favre, Bernardine; Guy, Julien; Alonso, Esther; Ahwij, Nuhri; Jerez, Andrés; Hermouet, Sylvie; Maynadié, Marc; Cazzola, Mario; Girodon, François

2012-01-01

Background Refractory anemia with ring sideroblasts associated with marked thrombocytosis was proposed as a provisional entity in the 2001 World Health Organization classification of myeloid neoplasms and also in the 2008 version, but its existence as a single entity is contested. We wish to define the clinical features of this rare myelodysplastic/myeloproliferative neoplasm and to compare its clinical outcome with that of refractory anemia with ring sideroblasts and essential thrombocythemia. Design and Methods We conducted a collaborative retrospective study across Europe. Our database included 200 patients diagnosed with refractory anemia with ring sideroblasts and marked thrombocytosis. For each of these patients, each patient diagnosed with refractory anemia with ring sideroblasts was matched for age and sex. At the same time, a cohort of 454 patients with essential thrombocythemia was used to compare outcomes of the two diseases. Results In patients with refractory anemia with ring sideroblasts and marked thrombocytosis, depending on the Janus Kinase 2 V617F mutational status (positive or negative) or platelet threshold (over or below 600×109/L), no difference in survival was noted. However, these patients had shorter overall survival and leukemia-free survival with a lower risk of thrombotic complications than did patients with essential thrombocythemia (P<0.001) but better survival (P<0.001) and a higher risk of thrombosis (P=0.039) than patients with refractory anemia with ring sideroblasts. Conclusions The clinical course of refractory anemia with ring sideroblasts and marked thrombocytosis is better than that of refractory anemia with ring sideroblasts and worse than that of essential thrombocythemia. The higher risk of thrombotic events in this disorder suggests that anti-platelet therapy might be considered in this subset of patients. From a clinical point of view, it appears to be important to consider refractory anemia with ring sideroblasts and

Improved Method for Direct Detection of Environmental Microorganisms Using an Amplification of 16S rDNA Region

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Tsujimura, M.; Akutsu, J.; Zhang, Z.; Sasaki, M.; Tajima, H.; Kawarabayasi, Y.

2004-12-01

The thermostable proteins or enzymes were expected to be capable to be utilized in many areas of industries. Many thermophilic microorganisms, which possess the thermostable proteins or enzymes, were identified from the extreme environment. However, many unidentified and uncultivable microorganisms are still remaining in the environment on the earth. It is generally said that the cultivable microorganisms are less than 1% of entire microorganisms living in the earth, remaining over 99% are still uncultivable. As an approach to the uncultivable microorganisms, the PCR amplification of 16S rDNA region using primer sets designed from the conserved region has been generally utilized for detection and community analysis of microorganism in the environment. However, the facts, that PCR amplification introduces the mutation in the amplified DNA fragment and efficiency of PCR amplification is depend on the sequences of primer sets, indicated that the improving of PCR analysis was necessary for more correct detection of microorganisms. As the result of evaluation for the quality of DNA polymerases, sequences of primers used for amplification and conditions of PCR amplification, the DNA polymerase, the primer set and the conditions for amplification, which did not amplify the DNA fragment from the DNA contaminated within the DNA polymerase itself, were successfully selected. Also the rate of mutation in the DNA fragment amplified was evaluated using this conditions and the genomic DNA from cultivable microbes as a template. The result indicated the rate of mutation introduced by PCR was approximately 0.1% to 0.125%. The improved method using these conditions and error rate calculated was applied for the analysis of microorganisms in the geothermal environment. The result indicated that four kinds of dominant microorganisms, including both of bacteria and archaea, were alive within soil in the hot spring in Tohoku Area. We would like to apply this improved method to detection

Evidence of high-elevation amplification versus Arctic amplification.

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Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

2016-01-12

Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world's high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction.

Comparative study of different methodologies to detect the JAK2 V617F mutation in chronic BCR-ABL1 negative myeloproliferative neoplasms

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Alline Didone

2016-04-01

Full Text Available Objectives: A mutation in the JAK2 gene, V617F, has been identified in several BCR-ABL1 negative myeloproliferative neoplasms (MPN: polycythemia vera (PV, essential thrombocythemia (ET, and primary myelofibrosis (PMF. Defining the presence or absence of this mutation is an essential part of clinical diagnostic algorithms and patient management. Here, we aimed to evaluate the performance of three PCR-based assays: Amplification Refractory Mutation System (ARMS, High-Resolution Melting analysis (HRM, and Sanger direct sequencing, and compare their results with those obtained by a PCR restriction fragment polymorphism assay (PCR-RFLP. Design and methods: We used blood samples from 136 patients (PV=20; PMF=20; ET=28, and other MPN suspected cases=68. Results: Comparable results were observed among the four assays in patients with PV, PMF, and MPN suspected cases. In patients with a diagnosis of ET, the JAK2 V617F mutation was detected in 67.8% of them by the PCR-ARMS and PCR-HRM assay and in 64% of them by the conventional Sanger sequence approach. The PCR-ARMS and PCR-HRM assays were 100% concordant. With these tests, only one of the 20 patients with ET and one of the three patients with clinically suspected MPN gave different results compared with those obtained by the PCR-RFLP. Conclusions: Our results have demonstrated that the PCR-ARMS and PCR-HRM assays could detect the JAK2 V617F mutation effectively in MPN patients, but PCR-HRM assays are rapid and the most cost-effective procedures. Keywords: Myeloproliferative, JAK2 V617F, Mutation, Wild type, Screening

Unstable mutations: cause of some neurological hereditary diseases

International Nuclear Information System (INIS)

Cuenca Berger, P.; Morales Montero, F.

1999-01-01

Unstable mutations or amplification of triplets constitute a kind of genetic alteration discovered during the last decade. They had been found inside or near genes important for the normal neurological function of the human being. In some cases, the presence of the amplification causes the inactivation of the gene or the synthesis of a new product which functions different from the original protein. Some common characteristics of diseases caused by the amplification of triplets are that it affects the nervous system and are degenerative in nature. The expression of the manifestations varies according to age. Most of them show genetic anticipation in which the severity of the manifestations increases with each generation and appear at an earlier age. In most cases, the severity of the symptoms is correlated positively to the size of the amplification. The diagnosis of an affected individual in a family may indicate the presence of an altered gene in other relatives. These relatives may not present evident signs of the illness either because it is of late onset or because they carry premutations. The molecular diagnosis of these mutations is important to estimate the risk of developing the disease and/or of transmitting the illness to the descendants and to eliminate the fears of healthy relatives who have inherited normal copies of the gene. (Author) [es

Refractory Materials for Flame Deflector Protection System Corrosion Control: Refractory Ceramics Literature Survey

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Calle, Luz Marina; Hintze, Paul E.; Parlier, Christopher R.; Curran, Jerome P.; Kolody, Mark; Perusich, Stephen; Whitten, Mary C.; Trejo, David; Zidek, Jason; Sampson, Jeffrey W.;

Disabilities caused by unstable mutations in Costa Rica

OpenAIRE

Morales Montero, Fernando; Cuenca Berger, Patricia; Castro Volio, Isabel

2004-01-01

Myotonic dystrophy and fragile X syndrome are two genetically determined relatively common disabilities. Both are examples of a new type of mutation mechanism called unstable or dynamic mutations, triple repeats expansions or DNA amplification. Fragile X syndrome is recognized as the main cause of hereditary mental retardation and myotonic dystrophy is considered the most common muscular dystrophy of adults. This is a prospective non randomized study of clinically affected people,...

Refractory vasculitis

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Rutgers, Bram; Kallenberg, Cees G. M.

Refractory vasculitis occurs in 4-5% of patients with anti-neutrophil cytoplasmic antibody associated vasculitis (AAV). Differences between therapies used for refractory disease are mostly reflected in the percentages of complete and partial remissions, but also in the number of serious side

Pharmacotherapy for Refractory and Super-Refractory Status Epilepticus in Adults.

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Holtkamp, Martin

2018-03-01

Patients with prolonged seizures that do not respond to intravenous benzodiazepines and a second-line anticonvulsant suffer from refractory status epilepticus and those with seizures that do not respond to continuous intravenous anesthetic anticonvulsants suffer from super-refractory status epilepticus. Both conditions are associated with significant morbidity and mortality. A strict pharmacological treatment regimen is urgently required, but the level of evidence for the available drugs is very low. Refractory complex focal status epilepticus generally does not require anesthetics, but all intravenous non-anesthetizing anticonvulsants may be used. Most descriptive data are available for levetiracetam, phenytoin and valproate. Refractory generalized convulsive status epilepticus is a life-threatening emergency, and long-term clinical consequences are eminent. Administration of intravenous anesthetics is mandatory, and drugs acting at the inhibitory gamma-aminobutyric acid (GABA) A receptor such as midazolam, propofol and thiopental/pentobarbital are recommended without preference for one of those. One in five patients with anesthetic treatment does not respond and has super-refractory status epilepticus. With sustained seizure activity, excitatory N-methyl-d-aspartate (NMDA) receptors are increasingly expressed post-synaptically. Ketamine is an antagonist at this receptor and may prove efficient in some patients at later stages. Neurosteroids such as allopregnanolone increase sensitivity at GABA A receptors; a Phase 1/2 trial demonstrated safety and tolerability, but randomized controlled data failed to demonstrate efficacy. Adjunct ketogenic diet may contribute to termination of difficult-to-treat status epilepticus. Randomized controlled trials are needed to increase evidence for treatment of refractory and super-refractory status epilepticus, but there are multiple obstacles for realization. Hitherto, prospective multicenter registries for pharmacological

The three stages of epilepsy in patients with CDKL5 mutations.

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Bahi-Buisson, Nadia; Kaminska, Anna; Boddaert, Nathalie; Rio, Marlène; Afenjar, Alexandra; Gérard, Marion; Giuliano, Fabienne; Motte, Jacques; Héron, Delphine; Morel, Marie Ange N'guyen; Plouin, Perrine; Richelme, Christian; des Portes, Vincent; Dulac, Olivier; Philippe, Christophe; Chiron, Catherine; Nabbout, Rima; Bienvenu, Thierry

2008-06-01

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene are responsible for a severe encephalopathy with early epilepsy. So far, the electroclinical phenotype remains largely unknown and no clear genotype-phenotype correlations have been established. To characterize the epilepsy associated with CDKL5 mutations and to look for a relationship between the genotype and the course of epilepsy. We retrospectively analyzed the electroclinical phenotypes of 12 patients aged from 2.5 to 19 years diagnosed with pathogenic CDKL5 mutations and one patient with a novel intronic sequence variation of uncertain pathogenicity and examined whether the severity of the epilepsy was linked to the type and location of mutations. The epilepsy course reveals three successive stages: (Stage I) early epilepsy (onset 1-10 weeks) with normal interictal electroencephalogram (EEG) (10/13) despite frequent convulsive seizures; (Stage II) epileptic encephalopathy with infantile spasms (8/8) and hypsarrhythmia (8/8). At the age of evaluation, seven patients were seizure free and six had developed refractory epilepsy (stage III) with tonic seizures and myoclonia (5/6). Interestingly, the patients carrying a CDKL5 mutations causing a truncation of the catalytic domain tended to develop a more frequent refractory epilepsy than patients with mutations located downstream (4/6, 66.6% versus 1/6, 16%) although, these trends are not yet significant. Our data contribute to a better definition of the epileptic phenotype in CDKL5 mutations, and might give some clues to a potential relationship between the phenotype and the genotype in these patients.

Mutation analysis of β-thalassemia in East-Western Indian population: a recent molecular approach

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Shah, Parth S; Shah, Nidhi D; Ray, Hari Shankar P; Khatri, Nikunj B; Vaghasia, Ketan K; Raval, Rutvik J; Shah, Sandip C; Rao, Mandava V

2017-01-01

Background β-Thalassemia is the most prevalent genetic disorder in India. Its traits and coinheritance vary from mild to severe conditions, resulting in thalassemia minor, intermediate, and major, depending upon many factors. Purpose The objective of this study was to identify the incidence of β-thalassemia traits, their coinheritance, and mutations, as well as to support the patients already diagnosed with β-thalassemia in East-Western Indian population for better management. Patients and methods Seventy-five referral cases for β-thalassemia were analyzed for various β-thalassemia traits, heterozygosity, and homozygosity conditions. Blood phenotypic parameters using cell counter and capillary electrophoresis were investigated. Analyses of eight common mutations of thalassemia in India were carried out using polymerase chain reaction-amplification refractory mutation system, end point polymerase chain reaction, and DNA sequencing methods. Results Of these (75) referral cases from East-Western Indian region, 68 were positive for β-thalassemia (90.67%). The majority of case types were of β-thalassemia minor (49, 65.33%), followed by HbE traits (6, 8.0%) and β-thalassemia major, including heterozygous and homozygous (5, 6.66%; 4, 5.33%) types and then HbE homozygous (2, 2.66%), as well as one each of the HbE/β-thalassemia and HbD/β-thalassemia (1, 1.34%) combination. Mutation analysis also revealed that the highest frequency of mutation was c.92+5G>C (41, 60.29%) followed by deletion 619bp (9, 13.23%) and c.79G>A (8, 11.76%) in our study group. Five cases (nos. 24, 27, 33, 58, and 71) exhibited coinheritance between β0/β+ (2), β0/β D (1), and c.124_127delTTCT/β+ or β0(2) affecting the Rajasthani and Gujarati populations in our study of the Western region of India. Conclusion We strongly recommend these Western populations for genetic screening before adopting reproductive technologies and interracial marital relations. PMID:28546763

Spectrum of mutations in homozygous familial hypercholesterolemia in India, with four novel mutations.

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Setia, Nitika; Saxena, Renu; Arora, Anjali; Verma, Ishwar C

2016-12-01

Homozygous familial hypercholesterolemia (FH) is a rare but serious, inherited disorder of lipid metabolism characterized by very high total and LDL cholesterol levels from birth. It presents as cutaneous and tendon xanthomas since childhood, with or without cardiac involvement. FH is commonly caused by mutations in three genes, i.e. LDL receptor (LDLR), apolipoprotein B (ApoB) and PCSK9. We aimed to determine the spectrum of mutations in cases of homozygous FH in Asian Indians and evaluate if there was any similarity to the mutations observed in Caucasians. Sixteen homozygous FH subjects from eleven families were analyzed for mutations by Sanger sequencing. Large rearrangements in LDLR gene were evaluated by multiplex ligation probe dependent amplification (MLPA) technique. Ten mutations were observed in LDLR gene, of which four mutations were novel. No mutation was detected in ApoB gene and common PCSK9 mutation (p.D374Y). Fourteen cases had homozygous mutations; one had compound heterozygous mutation, while no mutation was detected in one clinically homozygous case. We report an interesting "Triple hit" case with features of homozygous FH. The spectrum of mutations in the Asian Indian population is quite heterogeneous. Of the mutations identified, 40% were novel. No mutation was observed in exons 3, 9 and 14 of LDLR gene, which are considered to be hot spots in studies done on Asian Indians in South Africa. Early detection followed by aggressive therapy, and cascade screening of extended families has been initiated to reduce the morbidity and mortality in these patients. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Clinical features and gene mutational spectrum of CDKL5-related diseases in a cohort of Chinese patients.

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Zhao, Ying; Zhang, Xiaoying; Bao, Xinhua; Zhang, Qingping; Zhang, Jingjing; Cao, Guangna; Zhang, Jie; Li, Jiarui; Wei, Liping; Pan, Hong; Wu, Xiru

2014-02-25

Mutations in the cyclin-dependent kinase-like 5 (CDKL5) (NM_003159.2) gene have been associated with early-onset epileptic encephalopathies or Hanefeld variants of RTT(Rett syndrome). In order to clarify the CDKL5 genotype-phenotype correlations in Chinese patients, CDKL5 mutational screening in cases with early-onset epileptic encephalopathies and RTT without MECP2 mutation were performed. The detailed clinical information including clinical manifestation, electroencephalogram (EEG), magnetic resonance imaging (MRI), blood, urine amino acid and organic acid screening of 102 Chinese patients with early-onset epileptic encephalopathies and RTT were collected. CDKL5 gene mutations were analyzed by PCR, direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The patterns of X-chromosome inactivation (XCI) were studied in the female patients with CDKL5 gene mutation. De novo CDKL5 gene mutations were found in ten patients including one missense mutation (c.533G > A, p.R178Q) which had been reported, two splicing mutations (ISV6 + 1A > G, ISV13 + 1A > G), three micro-deletions (c.1111delC, c.2360delA, c.234delA), two insertions (c.1791 ins G, c.891_892 ins TT in a pair of twins) and one nonsense mutation (c.1375C > T, p.Q459X). Out of ten patients, 7 of 9 females with Hanefeld variants of RTT and the remaining 2 females with early onset epileptic encephalopathy, were detected while only one male with infantile spasms was detected. The common features of all female patients with CDKL5 gene mutations included refractory seizures starting before 4 months of age, severe psychomotor retardation, Rett-like features such as hand stereotypies, deceleration of head growth after birth and poor prognosis. In contrast, the only one male patient with CDKL5 mutation showed no obvious Rett-like features as females in our cohort. The X-chromosome inactivation patterns of all the female patients were random. Mutations in CDKL5 gene are responsible for 7 with

Animal products and K-ras codon 12 and 13 mutations in colon carcinomas

NARCIS (Netherlands)

Kampman, E.; Voskuil, D.W.; Kraats, A.A. van; Balder, H.F.; Muijen, G.N.P. van; Goldbohm, R.A.; Veer, P. van 't

2000-01-01

K-ras gene mutations (codons 12 and 13) were determined by PCR-based mutant allele-specific amplification (MASA) in tumour tissue of 185 colon cancer patients: 36% harboured mutations, of which 82% were located in codon 12. High intakes of animal protein, calcium and poultry were differently

Application of Single Strand Conformational Polymorphism (PCR-SSCP) in Identification of Some Beta-Globin Gene Mutations in A Group of Egyptian Beta-Thalassemia Patients and Carriers

International Nuclear Information System (INIS)

Somaya, E.T.; Soliman, M.D

2010-01-01

The present study investigated whether the single-strand conformational polymorphism (SSCP) method could be employed to identify (rather than simply detect) four of the most common beta-globin gene mutations in the Egyptian population: IVS-I-110, IVS-I-6, the IVS-I-1, and Codon 39. Using DNA from 90 beta-thalassemia patients and carriers, by PCR the appropriate 238-bp region of the human beta-globin gene was amplified, the reaction products (Single-stranded DNA) were analyzed by none denaturing polyacrylamide gel electrophoresis, and the bands visualized by silver staining. Single-stranded DNA (ssDNA) fragments showed reproducible pattern of bands that were characteristic of the mutations present. With the use of control samples containing six of the 10 possible combinations of the four beta-globin gene mutations under study, we were able to predict the mutations present in 23 out of 90 (26.4%) of the patients studied. These predictions were confirmed independently by the amplification refractory mutation system (ARMS) method. It is concluded that this non-radioactive PCR-SSCP method can be used to reliably identify mutations in beta-thalassemia patients, provided that suitable controls are available. However, usefulness of this method for determining the genotype of beta-thalassaemic individuals is obviously limited by the great number of controls required. Moreover, the ability to detect mutations by SSCP is in general lower compared to other methods, ARMS, DGGE or DHPLC, which are reported to detect 49.5% to 73% of the mutations present. The SSCP method is nevertheless much easier to employ than other methods and is especially successful for beta-thalassemia carriers. This method would thus be particularly useful for an initial screening of target groups (prenatal diagnosis)

Autoradiographic study of corrosion of refractories

International Nuclear Information System (INIS)

Lisenenkova, S.B.; Kucheryavyi, M.N.; Bursteva, T.A.

1988-01-01

A comparative study was made of the character of the interaction between a container-glass melt consisting of sodium calcium silicate and refractories in various furnace sections using an autoradiographic method. Static tests were conducted on specimens of the following refractories: chrome-aluminum-zircon, Bakor 41, corundum, a high alumina refractory, and a refractory based on tin dioxide. The specimens were activated by calcium 45. Autoradiography and photomicrography indicated that an intrinsic feature of all refractories was that calcium from the melt penetrated the refractories along the weak link; for fused-cast refractories, the glass phase; and for sintered refractories, through the binder and cracks

Comparison of HER2 gene amplification and KRAS alteration in eyelid sebaceous carcinomas with that in other eyelid tumors.

Science.gov (United States)

Kwon, Mi Jung; Shin, Hyung Sik; Nam, Eun Sook; Cho, Seong Jin; Lee, Min Joung; Lee, Samuel; Park, Hye-Rim

2015-05-01

Eyelid sebaceous carcinoma (SC) represents a highly aggressive malignancy. Despite the poor prognosis, genetic alterations as potential molecular targets are not available. KRAS mutation and HER2 gene amplification may be candidates related to their genetic alterations. We examined the HER2 and KRAS alteration status in eyelid SCs and compared it with that in other eyelid tumors. The controversial topics of the human papillomavirus (HPV) and p16 expression were also investigated. HER2 amplification was determined by silver in situ hybridization, while immunohistochemistry was performed to study protein expressions in 14 SCs and controls, including 23 other eyelid malignancies and 14 benign tumors. Peptide nucleic acid-mediated PCR clamping and direct sequencing were used to detect KRAS mutations. HER2 protein overexpression was observed in 85.7% (12/14) of the SCs, of which two-thirds showed HER2 gene amplification. HER2 protein overexpression and HER2 amplification were found more frequently in eyelid SCs than in other eyelid tumors. All SCs harbored wild type KRAS genes. No HPV infections were identified in the SCs. Nevertheless, p16 overexpression was found in 71.4% (10/14) of SCs, irrespective of the status of HPV infection. Furthermore, p16 overexpression in eyelid SCs was also significantly higher than that in other eyelid tumors. HER2 protein overexpression, HER2 gene amplifications, and wild type KRAS genes are common in eyelid SCs. HER2 gene amplification may represent potential therapeutic targets for the treatment of eyelid SCs. Copyright © 2014 Elsevier GmbH. All rights reserved.

Response to parenteral iron therapy distinguish unexplained refractory iron deficiency anemia from iron-refractory iron deficiency anemia.

Science.gov (United States)

Akin, M; Sarbay, H; Guler, S; Balci, Y I; Polat, A

2016-04-01

We evaluated that response to parenteral iron therapy could be helpful in distinguishing the types of iron deficiency anemia. This study analyzed responses to IV iron sucrose therapy of 15 children with unexplained refractory iron deficiency anemia (URIDA). We compared the results at diagnosis, 6 weeks and 6 months after the therapy. Results were compared with responses of 11 patients' results with iron-refractory iron deficiency anemia (IRIDA) from our previous study. Six weeks after the start of treatment, ferritin, MCV, MCH and Hb values were in normal range in 10 patients. The increase in Hb, MCH, MCV, and ferritin values ranged 2.6-3.5 g/dL, 1.7-4.2 pg, 2-9 fL, and 13-25 ng/mL, respectively. In five patients, Hb, MCH, and MCV mean (range) values [11.2 g/dL (11-12.2), 24.5 pg (24-25.6), and 67 fL (65-70)] were nearly normal but ferritin mean (range) values [9.8 ng/mL (8-11)] were below normal. Six weeks after the start of treatment, Hb, MCH, MCV and ferritin values of patients with IRIDA were increased. The increase in Hb, MCH, MCV, and ferritin values ranged 0.8-2.7 g/dL, 1.7-4.2 pg, 2-9 fL, and 13-25 ng/mL, respectively. IRIDA is only partially responsive to parenteral iron supplementation. In conclusion, this study demonstrated that the response to intravenous iron therapy for the URIDA cases improved blood parameters more effectively than hereditary IRIDA. Response to parenteral iron therapy would be helpful to distinguish unexplained refractory IDA from hereditary IRIDA for clinicians who do not have access to hepcidin or TMPRS6 mutation analysis. © 2016 John Wiley & Sons Ltd.

Targeted deep sequencing of mucinous ovarian tumors reveals multiple overlapping RAS-pathway activating mutations in borderline and cancerous neoplasms

International Nuclear Information System (INIS)

Mackenzie, Robertson; Kommoss, Stefan; Winterhoff, Boris J.; Kipp, Benjamin R.; Garcia, Joaquin J.; Voss, Jesse; Halling, Kevin; Karnezis, Anthony; Senz, Janine; Yang, Winnie; Prigge, Elena-Sophie; Reuschenbach, Miriam; Doeberitz, Magnus Von Knebel; Gilks, Blake C.; Huntsman, David G.; Bakkum-Gamez, Jamie; McAlpine, Jessica N.; Anglesio, Michael S.

2015-01-01

Mucinous ovarian tumors represent a distinct histotype of epithelial ovarian cancer. The rarest (2-4 % of ovarian carcinomas) of the five major histotypes, their genomic landscape remains poorly described. We undertook hotspot sequencing of 50 genes commonly mutated in human cancer across 69 mucinous ovarian tumors. Our goals were to establish the overall frequency of cancer-hotspot mutations across a large cohort, especially those tumors previously thought to be “RAS-pathway alteration negative”, using highly-sensitive next-generation sequencing as well as further explore a small number of cases with apparent heterogeneity in RAS-pathway activating alterations. Using the Ion Torrent PGM platform, we performed next generation sequencing analysis using the v2 Cancer Hotspot Panel. Regions of disparate ERBB2-amplification status were sequenced independently for two mucinous carcinoma (MC) cases, previously established as showing ERBB2 amplification/overexpression heterogeneity, to assess the hypothesis of subclonal populations containing either KRAS mutation or ERBB2 amplification independently or simultaneously. We detected mutations in KRAS, TP53, CDKN2A, PIK3CA, PTEN, BRAF, FGFR2, STK11, CTNNB1, SRC, SMAD4, GNA11 and ERBB2. KRAS mutations remain the most frequently observed alteration among MC (64.9 %) and mucinous borderline tumors (MBOT) (92.3 %). TP53 mutation occurred more frequently in carcinomas than borderline tumors (56.8 % and 11.5 %, respectively), and combined IHC and mutation data suggest alterations occur in approximately 68 % of MC and as many as 20 % of MBOT. Proven and potential RAS-pathway activating changes were observed in all but one MC. Concurrent ERBB2 amplification and KRAS mutation were observed in a substantial number of cases (7/63 total), as was co-occurrence of KRAS and BRAF mutations (one case). Microdissection of ERBB2-amplified regions of tumors harboring KRAS mutation suggests these alterations are occurring in the same cell

Renal Cell Carcinoma With Chromosome 6p Amplification Including the TFEB Gene: A Novel Mechanism of Tumor Pathogenesis?

Science.gov (United States)

Williamson, Sean R; Grignon, David J; Cheng, Liang; Favazza, Laura; Gondim, Dibson D; Carskadon, Shannon; Gupta, Nilesh S; Chitale, Dhananjay A; Kalyana-Sundaram, Shanker; Palanisamy, Nallasivam

2017-03-01

Amplification of chromosome 6p has been implicated in aggressive behavior in several cancers, but has not been characterized in renal cell carcinoma (RCC). We identified 9 renal tumors with amplification of chromosome 6p including the TFEB gene, 3 by fluorescence in situ hybridization, and 6 from the Cancer Genome Atlas (TCGA) databases. Patients' ages were 28 to 78 years (median, 61 y). Most tumors were high stage (7/9 pT3a, 2/9 pN1). Using immunohistochemistry, 2/4 were positive for melanocytic markers and cathepsin K. Novel TFEB fusions were reported by TCGA in 2; however, due to a small composition of fusion transcripts compared with full-length transcripts (0.5/174 and 3.3/132 FPKM), we hypothesize that these represent secondary fusions due to amplification. Five specimens (4 TCGA, 1 fluorescence in situ hybridization) had concurrent chromosome 3p copy number loss or VHL deletion. However, these did not resemble clear cell RCC, had negative carbonic anhydrase IX labeling, lacked VHL mutation, and had papillary or unclassified histology (2/4 had gain of chromosome 7 or 17). One tumor each had somatic FH mutation and SMARCB1 mutation. Chromosome 6p amplification including TFEB is a previously unrecognized cytogenetic alteration in RCC, associated with heterogenous tubulopapillary eosinophilic and clear cell histology. The combined constellation of features does not fit cleanly into an existing tumor category (unclassified), most closely resembling papillary or translocation RCC. The tendency for high tumor stage, varied tubulopapillary morphology, and a subset with melanocytic marker positivity suggests the possibility of a unique tumor type, despite some variation in appearance and genetics.

RNA amplification for successful gene profiling analysis

Directory of Open Access Journals (Sweden)

Wang Ena

2005-07-01

Full Text Available Abstract The study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene

Improved acid tolerance of Lactobacillus pentosus by error-prone whole genome amplification.

Science.gov (United States)

Ye, Lidan; Zhao, Hua; Li, Zhi; Wu, Jin Chuan

2013-05-01

Acid tolerance of Lactobacillus pentosus ATCC 8041 was improved by error-prone amplification of its genomic DNA using random primers and Taq DNA polymerase. The resulting amplification products were transferred into wild-type L. pentosus by electroporation and the transformants were screened for growth on low-pH agar plates. After only one round of mutation, one mutant (MT3) was identified that was able to completely consume 20 g/L of glucose to produce lactic acid at a yield of 95% in 1L MRS medium at pH 3.8 within 36 h, whereas no growth or lactic acid production was observed for the wild-type strain under the same conditions. The acid tolerance of mutant MT3 remained genetically stable for at least 25 subcultures. Therefore, the error-prone whole genome amplification technique is a very powerful tool for improving phenotypes of this lactic acid bacterium and may also be applicable for other microorganisms. Copyright © 2012 Elsevier Ltd. All rights reserved.

Sensitive and fast mutation detection by solid phase chemical cleavage

DEFF Research Database (Denmark)

Hansen, Lise Lotte; Justesen, Just; Kruse, Torben A

1996-01-01

We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly...... reduced compared to the conventional chemical cleavage method (CCM), and even by using a uniformly labelled probe, the exact position and nature of the mutation can be revealed. The SpCCM is suitable for automatization using a workstation to carry out the reactions and a fluorescent detection-based DNA...

Spectrum of mutations in the renin-angiotensin system genes in autosomal recessive renal tubular dysgenesis

DEFF Research Database (Denmark)

Gribouval, Olivier; Morinière, Vincent; Pawtowski, Audrey

2012-01-01

, pulmonary hypoplasia, and refractory arterial hypotension. The disease is linked to mutations in the genes encoding several components of the renin-angiotensin system (RAS): AGT (angiotensinogen), REN (renin), ACE (angiotensin-converting enzyme), and AGTR1 (angiotensin II receptor type 1). Here, we review...... the series of 54 distinct mutations identified in 48 unrelated families. Most of them are novel and ACE mutations are the most frequent, observed in two-thirds of families (64.6%). The severity of the clinical course was similar whatever the mutated gene, which underlines the importance of a functional RAS...

Filaggrin compound heterozygous patients carry mutations in trans position

DEFF Research Database (Denmark)

Carlsen, Berit C; Meldgaard, Michael; Johansen, Jeanne D

2013-01-01

by means of allele-specific PCR amplification and analysis of PCR products by agarose gel electrophoresis. All R501X/2282del4 compound heterozygous samples collected over a 4-year period of routine FLG mutation testing were investigated. In total, 37 samples were tested. All thirty-seven R501X/2282del4......More than 40 null mutations in the filaggrin (FLG) gene are described. It is therefore possible to find two different null mutations in one individual (compound heterozygosity). It has been generally perceived that homozygous and compound heterozygous individuals were genotypically comparable......; however, this has not been scientifically investigated. Two different FLG null mutations in the same individual may be in trans position, meaning that each mutation locates to a different allele functionally equivalent to homozygosity, or may be in cis position, meaning that both mutations locate...

PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference.

Science.gov (United States)

Wimmer, Katharina; Wernstedt, Annekatrin

2014-01-01

The presence of highly homologous pseudocopies can compromise the mutation analysis of a gene of interest. In particular, when using PCR-based strategies, pseudogene co-amplification has to be effectively prevented. This is often achieved by using primers designed to be parental gene specific according to the reference sequence and by applying stringent PCR conditions. However, there are cases in which this approach is of limited utility. For example, it has been shown that the PMS2 gene exchanges sequences with one of its pseudogenes, named PMS2CL. This results in functional PMS2 alleles containing pseudogene-derived sequences at their 3'-end and in nonfunctional PMS2CL pseudogene alleles that contain gene-derived sequences. Hence, the paralogues cannot be distinguished according to the reference sequence. This shortcoming can be effectively circumvented by using direct cDNA sequencing. This approach is based on the selective amplification of PMS2 transcripts in two overlapping 1.6-kb RT-PCR products. In addition to avoiding pseudogene co-amplification and allele dropout, this method has also the advantage that it allows to effectively identify deletions, splice mutations, and de novo retrotransposon insertions that escape the detection of most DNA-based mutation analysis protocols.

Dramatic Response with Single-Agent Ibrutinib in Multiply Relapsed Marginal Zone Lymphoma with MYD88L265P Mutation

Directory of Open Access Journals (Sweden)

Ryan C. Lynch

2017-09-01

Full Text Available The B-cell receptor signaling pathway is important in the lymphomagenesis of many lymphomas, including marginal zone lymphoma (MZL. Herein we describe a case of extranodal MZL refractory to multiple lines of therapy. The presence of an IgM paraprotein prompted further evaluation, and the patient was found to have an MYD88L265P mutation. Treatment with ibrutinib led to a dramatic response with prompt resolution of symptoms and significant improvement in measurable sites of disease. The excellent response to ibrutinib in our patient with MYD88L265P-mutated refractory MZL supports a biological rationale for its use.

Amplification factor variable amplifier

NARCIS (Netherlands)

Akitsugu, Oshita; Nauta, Bram

2007-01-01

PROBLEM TO BE SOLVED: To provide an amplification factor variable amplifier capable of achieving temperature compensation of an amplification factor over a wide variable amplification factor range. ; SOLUTION: A Gilbert type amplification factor variable amplifier 11 amplifies an input signal and

Refractoriness in human atria

DEFF Research Database (Denmark)

Skibsbye, Lasse; Jespersen, Thomas; Christ, Torsten

2016-01-01

BACKGROUND: Refractoriness of cardiac cells limits maximum frequency of electrical activity and protects the heart from tonic contractions. Short refractory periods support major arrhythmogenic substrates and augmentation of refractoriness is therefore seen as a main mechanism of antiarrhythmic...... drugs. Cardiomyocyte excitability depends on availability of sodium channels, which involves both time- and voltage-dependent recovery from inactivation. This study therefore aims to characterise how sodium channel inactivation affects refractoriness in human atria. METHODS AND RESULTS: Steady......-state activation and inactivation parameters of sodium channels measured in vitro in isolated human atrial cardiomyocytes were used to parameterise a mathematical human atrial cell model. Action potential data were acquired from human atrial trabeculae of patients in either sinus rhythm or chronic atrial...

Amplification factor variable amplifier

NARCIS (Netherlands)

Akitsugu, Oshita; Nauta, Bram

2010-01-01

PROBLEM TO BE SOLVED: To provide an amplification factor variable amplifier capable of achieving temperature compensation of an amplification factor over a wide variable amplification factor range. ;SOLUTION: A Gilbert type amplification factor variable amplifier 11 amplifies an input signal and can

Distinct Histopathologic and Molecular Alterations in Inflammatory Bowel Disease-Associated Intestinal Adenocarcinoma: c-MYC Amplification is Common and Associated with Mucinous/Signet Ring Cell Differentiation.

Science.gov (United States)

Hartman, Douglas J; Binion, David G; Regueiro, Miguel D; Miller, Caitlyn; Herbst, Cameron; Pai, Reetesh K

2018-05-17

Chronic idiopathic inflammatory bowel disease (IBD) is a significant risk factor for the development of intestinal adenocarcinoma. The underlying molecular alterations in IBD-associated intestinal adenocarcinoma remain largely unknown. We compared the clinicopathologic and molecular features of 35 patients with 47 IBD-associated intestinal adenocarcinomas with a consecutive series of 451 patients with sporadic colorectal carcinoma identified at our institution and published data on sporadic colorectal carcinoma. c-MYC amplification was the most frequent molecular alteration identified in 33% of IBD-associated intestinal adenocarcinoma that is a significantly higher frequency than in sporadic colorectal carcinoma (8%) (P = 0.0001). Compared to sporadic colorectal carcinoma, IBD-associated intestinal adenocarcinomas more frequently demonstrated mucinous differentiation (60% vs 25%, P < 0.001) and signet ring cell differentiation (28% vs 4%, P < 0.001). Mucinous and signet ring cell differentiation were significantly associated with the presence of c-MYC amplification (both with P < 0.05). HER2 positivity (11%), KRAS exon 2 or 3 mutation (10%), and IDH1 mutation (7%) were less commonly observed in IBD-associated intestinal adenocarcinoma. There was an association between poor survival and HER2 status with 3 of 4 patients having HER2-positive adenocarcinoma dead of disease at last clinical follow-up; however, no statistically significant survival effect was identified for any of the molecular alterations identified. We demonstrate that IBD-associated intestinal adenocarcinomas have a high frequency of c-MYC amplification that is associated with mucinous and signet ring cell differentiation. Many of the identified molecular alterations have potential therapeutic relevance, including HER2 amplification, IDH1 mutation, and low frequency KRAS mutation.

Plasma spraying of refractory metals and refractory hard materials. State of the art

International Nuclear Information System (INIS)

Eschnauer, H.; Lugscheider, E.; Jaeger, D.

1989-01-01

Suitable spraying processes for manufacturing refractory metals, refractory hard materials as well as spray materials with refractory components are the VPS- and IPS-spraying techniques. The advantages of these special spraying process variations are described. The reactive spraying materials are systematically organized. The characteristical properties used in purpose of improving the substrate surfaces are explained. Finally some examples of the latest results of research concerning plasma spraying of reactive materials are shown. 16 refs., 10 figs. (Author)

[Investigation of RNA viral genome amplification by multiple displacement amplification technique].

Science.gov (United States)

Pang, Zheng; Li, Jian-Dong; Li, Chuan; Liang, Mi-Fang; Li, De-Xin

2013-06-01

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

Mutation spectrum of Chinese patients with Bartter syndrome.

Science.gov (United States)

Han, Yue; Lin, Yi; Sun, Qing; Wang, Shujuan; Gao, Yanxia; Shao, Leping

2017-11-24

Bartter syndrome (BS) has been rarely reported in Chinese population except for a few case reports. This investigation was aimed to analyze the mutations of the causal genes in sixteen Chinese patients with BS, and review their followup and treatment. Identify mutations by the next generation sequencing and the multiplex ligation-dependent probe amplification (MLPA). Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. 15 different CLCNKB gene mutations were identified in fourteen patients with BS, including 11 novel ones. A novel missense mutation and a novel small deletion were found from SLC12A1 gene. A novel gross deletion was found in CLCNKA gene. A recurrent missense mutation was identified from BSND gene. We found that the whole gene deletion mutation of CLCNKB gene was the most frequent mutation (32%), and the rate of gross deletion was up to 50 percent in this group of Chinese patients. The present study has found 19 mutations, including 14 novel ones, which would enrich the human gene mutation database (HGMD) and provide valuable references to the genetic counseling and diagnosis of the Chinese population.

Of Mice and Men: Empirical Support for the Population-Based Social Epistasis Amplification Model (a Comment on ).

Science.gov (United States)

Sarraf, Matthew Alexandar; Woodley Of Menie, Michael Anthony

2017-01-01

This commentary article offers new perspective on recent research investigating the behavioral and social ecological effects of a mutation related to autism spectrum disorders in mice. The authors explain the consistency of this research on mice with predictions advanced by a theory of the role of mutations in altering interorganismal gene-gene interactions (social epistasis) in social species including humans, known as the social epistasis amplification model. The potential significance of the mouse research for understanding contemporary human behavioral trends is explored.

Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

Science.gov (United States)

Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

2015-02-01

In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

Characterization of cancer-associated missense mutations in MDM2

OpenAIRE

Chauhan, Krishna M.; Ramakrishnan, Gopalakrishnan; Kollareddy, Madhusudhan; Martinez, Luis A.

2015-01-01

MDM2 is an E3 ubiquitin ligase that binds the N-terminus of p53 and promotes its ubiquitin-dependent degradation. Elevated levels of MDM2 due to overexpression or gene amplification can contribute to tumor development by suppressing p53 activity. Since MDM2 is an oncogene, we explored the possibility that other genetic lesions, namely missense mutations, might alter its activities. We selected mutations in MDM2 that reside in one of the 4 key regions of the protein: p53 binding domain, acidic...

Theoretical and practical aspects about corrosion of refractories used in steel metallurgy: part 3: characterization of commercial refractories

International Nuclear Information System (INIS)

Braganca, S.R.

2012-01-01

In this study, it was reviewed the main aspects found in the literature about refractories corrosion, evaluating the feasibility of certain tests and relating them with experimental results. The physical properties and microstructure of commercial refractories were analyzed, considering the differences between them and the quality implications and probable life of the refractory. Thus, it was studied the various types of refractories used as lining on steel ladle. Magnesia-carbon and doloma-carbon refractories were analyzed, highlighting the differences between them. The examined refractory showed characteristics favoring high resistance to corrosion process, presenting a series of properties to be selected in accordance with industry practice. (author)

Presence of two alternative kdr-like mutations, L1014F and L1014S, and a novel mutation, V1010L, in the voltage gated Na+ channel of Anopheles culicifacies from Orissa, India

Directory of Open Access Journals (Sweden)

Bhatt Rajendra M

2010-05-01

Full Text Available Abstract Background Knockdown resistance in insects resulting from mutation(s in the voltage gated Na+ channel (VGSC is one of the mechanisms of resistance against DDT and pyrethroids. Recently a point mutation leading to Leu-to-Phe substitution in the VGSC at residue 1014, a most common kdr mutation in insects, was reported in Anopheles culicifacies-a major malaria vector in the Indian subcontinent. This study reports the presence of two additional amino acid substitutions in the VGSC of an An. culicifacies population from Malkangiri district of Orissa, India. Methods Anopheles culicifacies sensu lato (s.l. samples, collected from a population of Malkangiri district of Orissa (India, were sequenced for part of the second transmembrane segment of VGSC and analyzed for the presence of non-synonymous mutations. A new primer introduced restriction analysis-PCR (PIRA-PCR was developed for the detection of the new mutation L1014S. The An. culicifacies population was genotyped for the presence of L1014F substitution by an amplification refractory mutation system (ARMS and for L1014S substitutions by using a new PIRA-PCR developed in this study. The results were validated through DNA sequencing. Results DNA sequencing of An. culicifacies individuals collected from district Malkangiri revealed the presence of three amino acid substitutions in the IIS6 transmembrane segments of VGSC, each one resulting from a single point mutation. Two alternative point mutations, 3042A>T transversion or 3041T>C transition, were found at residue L1014 leading to Leu (TTA-to-Phe (TTT or -Ser (TCA changes, respectively. A third and novel substitution, Val (GTG-to-Leu (TTG or CTG, was identified at residue V1010 resulting from either of the two transversions–3028G>T or 3028G>C. The L1014S substitution co-existed with V1010L in all the samples analyzed irrespective of the type of point mutation associated with the latter. The PIRA-PCR strategy developed for the

Dynamics of 103K/N and 184M/V HIV-1 drug resistant populations: relative comparison in plasma virus RNA versus CD45RO+T cell proviral DNA

DEFF Research Database (Denmark)

Jakobsen, Martin Roelsgaard; Tolstrup, M; Bertelsen, L

2007-01-01

on real-time PCR and amplification refractory mutation system (ARMS). RESULTS: The 103N and 184V mutations were not detected in patients with stable low viremia. Patients previously exposed to mono or dual therapy often carried minor viral populations of either one or both mutations in plasma. The viral......BACKGROUND: Viral populations defined by 103K/N and 184M/V as linked or single mutations in the HIV-1 reverse transcriptase gene were investigated in plasma samples and compared with previous findings in the CD45RO(+)T cell compartment. OBJECTIVE: To develop an ARMS assay for plasma virions...... an unequal distribution of linked-mutation populations in plasma and CD45RO(+)T cells. Furthermore, the linked 103N-184V mutation may be more fit than the single 184V mutation and this linked population emerges rapidly under inadequate drug pressure. Udgivelsesdato: 2007-Jul...

Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification.

Science.gov (United States)

De Brouwer, Sara; De Preter, Katleen; Kumps, Candy; Zabrocki, Piotr; Porcu, Michaël; Westerhout, Ellen M; Lakeman, Arjan; Vandesompele, Jo; Hoebeeck, Jasmien; Van Maerken, Tom; De Paepe, Anne; Laureys, Geneviève; Schulte, Johannes H; Schramm, Alexander; Van Den Broecke, Caroline; Vermeulen, Joëlle; Van Roy, Nadine; Beiske, Klaus; Renard, Marleen; Noguera, Rosa; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Kogner, Per; Martinsson, Tommy; Nakagawara, Akira; Ohira, Miki; Caron, Huib; Eggert, Angelika; Cools, Jan; Versteeg, Rogier; Speleman, Frank

2010-09-01

Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression. The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis. ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival. ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants.

DNAzyme Feedback Amplification: Relaying Molecular Recognition to Exponential DNA Amplification.

Science.gov (United States)

Liu, Meng; Yin, Qingxin; McConnell, Erin M; Chang, Yangyang; Brennan, John D; Li, Yingfu

2018-03-26

Technologies capable of linking DNA amplification to molecular recognition are very desirable for ultrasensitive biosensing applications. We have developed a simple but powerful isothermal DNA amplification method, termed DNAzyme feedback amplification (DFA), that is capable of relaying molecular recognition to exponential DNA amplification. The method incorporates both an RNA-cleaving DNAzyme (RCD) and rolling circle amplification (RCA) carried out by a special DNA polymerase using a circular DNA template. DFA begins with a stimulus-dependent RCA reaction, producing tandemly linked RCDs in long-chain DNA products. These RCDs cleave an RNA-containing DNA sequence to form additional primers that hybridize to the circular DNA molecule, giving rise to DNA assemblies that act as the new inputs for RCA. The RCA reaction and the cleavage event keep on feeding each other autonomously, resulting in exponential growth of repetitive DNA sequences that can be easily detected. This method can be used for the detection of both nucleic acid based targets and non-nucleic acid analytes. In this article, we discuss the conceptual framework of the feedback amplification approach, the essential features of this method as well as remaining challenges and possible solutions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

c-Ha-ras BamHI RFLP in human urothelial tumors and point mutations in hot codons

International Nuclear Information System (INIS)

Weismanova, E; Skovraga, M.; Kaluz, S.

1993-01-01

High-molecular weights DNAs from 30 bladder and renal cell carcinomas (RCC) were isolated and the c-Ha-ras the c-Ha-ras gene BamHI RFLP was examined. Amplification of c-Ha-ras with normal localization with regard to the size of alleles was found only in the case. One of the normally localized c-Ha-ras allele termed RCC c-H-ras of a length of about 6.6 kbp was cloned and an oncogene-activating point mutation was identified using two restriction enzymes. After comparison of CfrI and Cfr10I cleavage maps of RCC c-Ha-ras to complete nucleotide sequences of EJ/T24 c-Ha-ras oncogene and its normal counterpart, a point mutation was identified within codon 11 or 12. The use of CfrI and Cfr10I is of value for clinical practice in identification of point mutations in c-Ha-ras PCR product in neoplasia accompanied by somatic mutation of c-Ha-ras. The correlation among c-Ha-ras allele, amplification/loss, presence of point mutation and progression of neoplasia is discussed. (author)

Heteroduplex analysis of the dystrophin gene: Application to point mutation and carrier detection

Energy Technology Data Exchange (ETDEWEB)

Prior, T.W.; Papp, A.C.; Snyder, P.J.; Sedra, M.S.; Western, L.M.; Bartolo, C.; Mendell, J.R. [Ohio State Univ., Columbus, OH (United States); Moxley, R.T. [Univ. of Rochester Medical Center, NY (United States)

1994-03-01

Approximately one-third of Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, the authors identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. The authors conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing. 29 refs., 4 figs.

Mutations in SYNGAP1 Cause Intellectual Disability, Autism, and a Specific Form of Epilepsy by Inducing Haploinsufficiency

DEFF Research Database (Denmark)

Berryer, Martin H; Hamdan, Fadi F; Klitten, Laura L

2013-01-01

De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP-activating protein, cause nonsyndromic intellectual disability (NSID). All disease-causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID...... also showed ataxia, autism, and a specific form of generalized epilepsy that can be refractory to treatment. All of these mutations occurred de novo, except c.283dupC, which was inherited from a father who is a mosaic. Biolistic transfection of wild-type SYNGAP1 in pyramidal cells from cortical...

Evaluation of the reusing of MGO-C refractory brick in refractory mixes

International Nuclear Information System (INIS)

Silva, R.D.S. da; Braganca, S.R.

2012-01-01

The residue from the use of MgO-C refractories in electric arc furnace presents, mostly, magnesium oxide in its composition and some slag contamination and impurities from the process of electrofusion scrap iron/steel. In this study, it was studied the characteristics of this residue and reused through its introduction into a commercial refractory mix, employed as material of coating and repair. This refractory mix was tested by thermogravimetric analysis, compressive strength, evaluation of plasticity and porosity as well as aspects of its installation, such as adhesion in situ. Results showed that there potential for reuse with the introduction of the waste in commercial mix was 30%, with little loss of compressive strength and plasticity. (author)

Novel mutations underlying argininosuccinic aciduria in Saudi Arabia

Directory of Open Access Journals (Sweden)

Rashed Mohamed S

2010-03-01

Full Text Available Abstract Background Argininosuccinic aciduria (ASAuria is an autosomal recessive disorder of the urea cycle relatively common in Saudi Arabia as a consequence of extensive consanguinity. It is the most common urea cycle disorder identified in the Saudi population, which therefore prioritizes the need to delineate the underlying molecular defects leading to disease. Findings We utilized Whole Genome Amplification (WGA, PCR and direct sequencing to identify mutations underlying ASAuria cases diagnosed by our institution. A missense mutation that accounts for 50% of Saudi ASAuria patients was recently reported by our laboratory. In this study we report a further six novel mutations (and one previously reported found in Saudi patients with ASAuria. The novel four missense, one nonsense and one splice-site mutation were confirmed by their absence in >300 chromosomes from the normal population. Pathogenicity of the novel splice-site mutation was also confirmed using reverse transcriptase-PCR analysis. Cross species amino acid conservation at the substituted residues described were observed in some but not all instances. Conclusions Together, the eight mutations described by our laboratory, encompass >90% of ASAuria patients in Saudi Arabia and add to about 45 other ASAuria mutations reported worldwide.

Mutations of the Norrie gene in Korean ROP infants.

Science.gov (United States)

Kim, Jeong Hun; Yu, Young Suk; Kim, Jiyeon; Park, Seong Sup

2002-12-01

The present study was conducted to evaluate if there is a Norrie disease gene (ND gene) mutation involved in the retinopathy of prematurity (ROP), and to identify the possibility of a genetic abnormality that may be linked to the presence of ROP. Nineteen premature Korean infants, with a low birth weight (1500 g or less) or low gestational age (32 weeks or less), were included in the study. Eighteen infants had ROP, and the other did not. Genomic DNA was isolated from the peripheral blood leukocytes of these patients, and all three exons and their flanking areas, all known ND gene mutations regions, were evaluated following amplification by a polymerase chain reaction, but no ND gene mutations were detected. Any disagreement between the relationship of ROP to the ND gene mutation will need to be clarified by further investigation.

Genomic Amplification of an Endogenous Retrovirus in Zebrafish T-Cell Malignancies

Directory of Open Access Journals (Sweden)

J. Kimble Frazer

2012-01-01

Full Text Available Genomic instability plays a crucial role in oncogenesis. Somatically acquired mutations can disable some genes and inappropriately activate others. In addition, chromosomal rearrangements can amplify, delete, or even fuse genes, altering their functions and contributing to malignant phenotypes. Using array comparative genomic hybridization (aCGH, a technique to detect numeric variations between different DNA samples, we examined genomes from zebrafish (Danio rerio T-cell leukemias of three cancer-prone lines. In all malignancies tested, we identified recurring amplifications of a zebrafish endogenous retrovirus. This retrovirus, ZFERV, was first identified due to high expression of proviral transcripts in thymic tissue from larval and adult fish. We confirmed ZFERV amplifications by quantitative PCR analyses of DNA from wild-type fish tissue and normal and malignant D. rerio T cells. We also quantified ZFERV RNA expression and found that normal and neoplastic T cells both produce retrovirally encoded transcripts, but most cancers show dramatically increased transcription. In aggregate, these data imply that ZFERV amplification and transcription may be related to T-cell leukemogenesis. Based on these data and ZFERV’s phylogenetic relation to viruses of the murine-leukemia-related virus class of gammaretroviridae, we posit that ZFERV may be oncogenic via an insertional mutagenesis mechanism.

Compatibility of refractory materials with boiling sodium

International Nuclear Information System (INIS)

Meacham, S.A.

1976-01-01

The program employed to determine the compatibility of commercially available refractories with boiling sodium is described. The effects of impurities contained within the refractory material, and their relations with the refractory's physical stability are discussed. Also, since consideration of refractories for use as an insulating material within Liquid Metal Fast Breeder Reactor Plants (LMFBR's) is currently under investigation; recommendations, based upon this program, are presented

REFRACTORY THROMBOCYTOPENIA AND NEUTROPENIA: A DIAGNOSTIC CHALLENGE

OpenAIRE

Emmanuel Gyan; François Dreyfus; Pierre Fenaux

2015-01-01

Background. The 2008 WHO classification identified refractory cytopenia with unilineage dysplasia (RCUD) as a composite entity encompassing refractory anemia, refractory thrombocytopenia (RT), and refractory neutropenia (RN), characterized by 10% or more dysplastic cells in the bone marrow respective lineage. The diagnosis of RT and RN is complicated by several factors.  Diagnosing RT first requires exclusion of familial thrombocytopenia, chronic auto-immune thrombocytopenia, concomitant medi...

National mutation study among Danish patients with hereditary haemorrhagic telangiectasia

DEFF Research Database (Denmark)

Tørring, P M; Brusgaard, K; Ousager, L B

2014-01-01

carry mutations in the ENG, ACVRL1 or SMAD4 genes. Here, we report on the genetic heterogeneity in the Danish national HHT population and address the prevalence of pulmonary arteriovenous malformations (PAVM). Probands of 107 apparently unrelated families received genetic testing, including sequencing...... and multiplex ligation-dependent probe amplification (MLPA) analyses of ENG, ACVRL1 and SMAD4. These 107 families included 320 patients confirmed to have HHT either clinically or genetically. In 89% of the probands (n=95), a mutation was identified. We detected 64 unique mutations of which 27 (41%) were novel....... Large deletions were identified in ENG and ACVRL1. The prevalence of PAVM was 52.3% in patients with an ENG mutation and 12.9% in the ACVRL1 mutation carriers. We diagnosed 80% of the patients clinically, fulfilling the Curaçao criteria, and those remaining were diagnosed by genetic testing...

Centrosome Amplification Increases Single-Cell Branching in Post-mitotic Cells.

Science.gov (United States)

Ricolo, Delia; Deligiannaki, Myrto; Casanova, Jordi; Araújo, Sofia J

2016-10-24

Centrosome amplification is a hallmark of cancer, although we are still far from understanding how this process affects tumorigenesis [1, 2]. Besides the contribution of supernumerary centrosomes to mitotic defects, their biological effects in the post-mitotic cell are not well known. Here, we exploit the effects of centrosome amplification in post-mitotic cells during single-cell branching. We show that Drosophila tracheal cells with extra centrosomes branch more than wild-type cells. We found that mutations in Rca1 and CycA affect subcellular branching, causing tracheal tip cells to form more than one subcellular lumen. We show that Rca1 and CycA post-mitotic cells have supernumerary centrosomes and that other mutant conditions that increase centrosome number also show excess of subcellular lumen branching. Furthermore, we show that de novo lumen formation is impaired in mutant embryos with fewer centrioles. The data presented here define a requirement for the centrosome as a microtubule-organizing center (MTOC) for the initiation of subcellular lumen formation. We propose that centrosomes are necessary to drive subcellular lumen formation. In addition, centrosome amplification increases single-cell branching, a process parallel to capillary sprouting in blood vessels [3]. These results shed new light on how centrosomes can contribute to pathology independently of mitotic defects. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Novel Homozygous MYO7A Mutation: Case Report

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Mahsa Ahmadi

2018-05-01

Full Text Available MYO7A is an unconventional myosin that is essential for ordinary hearing and vision; mutations in the MYO7A gene result in Usher syndrome type 1B and other disorders. In this manuscript, we reported a mutation (c.4705delA in exon 35, causing the alteration of a Ser amino acid to Ala at codon 1569 (p.H2027del located within the first FERMdomain of the human protein myosin VIIA. This mutation involved in the pathogenesis of hearing loss, congenital night blindness, muscular weakness, skin problem, and difficulty in keeping balance in the 13-year-old female. After checkup the patient’s DNA was extracted from peripheral blood and amplification was performed by PCR. Sequencing method was performed for identification of the mutation. The c.4705delA mutation in exon 35 was found in the patient in heterozygosis form; this means that her mother and father were carriers. This mutation is located on the tail of the myosinVIIA protein and is associated with several disorders.

Refractory bin for burning

Energy Technology Data Exchange (ETDEWEB)

McPherson, D.L.; McPherson, T.L.

1989-12-26

This patent describes a refractory bin. It has a generally rectangular horizontal cross sectional configuration. It has wall structures each comprising an upper and a lower pair of elongated horizontal vertically spaced generally parallel support beams each having a vertical flange defining a support edge along its upper surface, a first generally rectangular refractory panel arranged with its lower edge at the bottom of the bin and with its outer surface in flat face contacting relation with the vertical flanges of the lower pair of support beams, a plurality of brackets each having a horizontal part and a vertical part and being secured to the outer surface of the first refractory panel.

Talimogene Laherparepvec and Nivolumab in Treating Patients With Refractory Lymphomas or Advanced or Refractory Non-melanoma Skin Cancers

Science.gov (United States)

2018-05-21

Adenoid Cystic Carcinoma; Adnexal Carcinoma; Apocrine Carcinoma; Eccrine Porocarcinoma; Extraocular Cutaneous Sebaceous Carcinoma; Hidradenocarcinoma; Keratoacanthoma; Malignant Sweat Gland Neoplasm; Merkel Cell Carcinoma; Microcystic Adnexal Carcinoma; NK-Cell Lymphoma, Unclassifiable; Non-Melanomatous Lesion; Paget Disease; Papillary Adenocarcinoma; Primary Cutaneous Mucinous Carcinoma; Refractory Anaplastic Large Cell Lymphoma; Refractory Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma; Refractory Mycosis Fungoides; Refractory Primary Cutaneous T-Cell Non-Hodgkin Lymphoma; Refractory T-Cell Non-Hodgkin Lymphoma; Sezary Syndrome; Signet Ring Cell Carcinoma; Skin Basal Cell Carcinoma; Skin Basosquamous Cell Carcinoma; Skin Squamous Cell Carcinoma; Spiradenocarcinoma; Squamous Cell Carcinoma of Unknown Primary Origin; Stage III Skin Cancer; Stage IV Skin Cancer; Sweat Gland Carcinoma; Trichilemmocarcinoma; Vulvar Squamous Cell Carcinoma

Amplification of HER2 is a marker for global genomic instability

Directory of Open Access Journals (Sweden)

Love Brad

2008-10-01

Full Text Available Abstract Background Genomic alterations of the proto-oncogene c-erbB-2 (HER-2/neu are associated with aggressive behavior and poor prognosis in patients with breast cancer. The variable clinical outcomes seen in patients with similar HER2 status, given similar treatments, suggests that the effects of amplification of HER2 can be influenced by other genetic changes. To assess the broader genomic implications of structural changes at the HER2 locus, we investigated relationships between genomic instability and HER2 status in patients with invasive breast cancer. Methods HER2 status was determined using the PathVysion® assay. DNA was extracted after laser microdissection from the 181 paraffin-embedded HER2 amplified (n = 39 or HER2 negative (n = 142 tumor specimens with sufficient tumor available to perform molecular analysis. Allelic imbalance (AI was assessed using a panel of microsatellite markers representing 26 chromosomal regions commonly altered in breast cancer. Student t-tests and partial correlations were used to investigate relationships between genomic instability and HER2 status. Results The frequency of AI was significantly higher (P P Conclusion The poor prognosis associated with HER2 amplification may be attributed to global genomic instability as cells with high frequencies of chromosomal alterations have been associated with increased cellular proliferation and aggressive behavior. In addition, high levels of DNA damage may render tumor cells refractory to treatment. In addition, specific alterations at chromosomes 11q13, 16q22-q24, and 18q21, all of which have been associated with aggressive tumor behavior, may serve as genetic modifiers to HER2 amplification. These data not only improve our understanding of HER in breast pathogenesis but may allow more accurate risk profiles and better treatment options to be developed.

Emergency Surgery for Refractory Status Epilepticus.

Science.gov (United States)

Botre, Abhijeet; Udani, Vrajesh; Desai, Neelu; Jagadish, Spoorthy; Sankhe, Milind

2017-08-15

Management of refractory status epilepticus in children is extremely challenging. Two children with medically refractory status epilepticus, both of whom had lesional pathology on MRI and concordant data on EEG and PET scan. Emergency hemispherotomy performed in both patients. A complete, sustained seizure freedom obtained postoperatively. Emergency surgery is a treatment option in selected cases of drug refractory status epilepticus with lesional pathology and concordant data.

Highly frequent promoter methylation and PIK3CA amplification in non-small cell lung cancer (NSCLC)

International Nuclear Information System (INIS)

Ji, Meiju; Guan, Haixia; Gao, Cuixia; Shi, Bingyin; Hou, Peng

2011-01-01

Lung cancer is the leading cause of cancer-related death worldwide. Genetic and epigenetic alterations have been identified frequently in lung cancer, such as promoter methylation, gene mutations and genomic amplification. However, the interaction between genetic and epigenetic events and their significance in lung tumorigenesis remains poorly understood. We determined the promoter methylation of 6 genes and PIK3CA amplification using quantitative methylation-specific PCR (Q-MSP) and real-time quantitative PCR, respectively, and explore the association of promoter methylation with PIK3CA amplification in a large cohort of clinically well-characterized non-small cell lung cancer (NSCLC). Highly frequent promoter methylation was observed in NSCLC. With 100% diagnostic specificity, excellent sensitivity, ranging from 45.8 to 84.1%, was found for each of the 6 genes. The promoter methylation was associated with histologic type. Methylation of CALCA, CDH1, DAPK1, and EVX2 was more common in squamous cell carcinomas (SCC) compared to adenocarcinomas (ADC). Conversely, there was a trend toward a higher frequency of RASSF1A methylation in ADC than SCC. In addition, PIK3CA amplification was frequently found in NSCLC, and was associated with certain clinicopathologic features, such as smoking history, histologic type and pleural indentation. Importantly, aberrant promoter methylation of certain genes was significantly associated with PIK3CA amplification. Our data showed highly frequent promoter methylation and PIK3CA amplification in Chinese NSCLC population, and first demonstrated the associations of gene methylation with PIK3CA amplification, suggesting that these epigenetic events may be a consequence of overactivation of PI3K/Akt pathway

Updates in Refractory Status Epilepticus

Science.gov (United States)

Mahulikar, Advait; Suchdev, Kushak; Shah, Aashit

2018-01-01

Refractory status epilepticus is defined as persistent seizures despite appropriate use of two intravenous medications, one of which is a benzodiazepine. It can be seen in up to 40% of cases of status epilepticus with an acute symptomatic etiology as the most likely cause. New-onset refractory status epilepticus (NORSE) is a recently coined term for refractory status epilepticus where no apparent cause is found after initial testing. A large proportion of NORSE cases are eventually found to have an autoimmune etiology needing immunomodulatory treatment. Management of refractory status epilepticus involves treatment of an underlying etiology in addition to intravenous anesthetics and antiepileptic drugs. Alternative treatment options including diet therapies, electroconvulsive therapy, and surgical resection in case of a focal lesion should be considered. Short-term and long-term outcomes tend to be poor with significant morbidity and mortality with only one-third of patients reaching baseline neurological status. PMID:29854452

Characterization of Eleusine indica with gene mutation or amplification in EPSPS to glyphosate.

Science.gov (United States)

Chen, Jingchao; Jiang, Cuilan; Huang, Hongjuan; Wei, Shouhui; Huang, Zhaofeng; Wang, Huimin; Zhao, Dandan; Zhang, Chaoxian

2017-11-01

The evolution of weed-resistant species threatens the sustainable use of glyphosate, which is the most important herbicide widely used in agriculture worldwide. Moreover, the high glyphosate resistance (>180-fold based on LD 50 ) of Eleusine indica found in Malaysia, which carries a double mutation in its 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), made the control of this species more difficult. By contrast, the same species carrying the same double mutation in EPSPS (T102I+P106S) but found in China only shows a resistance level of not more than 14-fold based on GR 50 . The resistance level of this population is four times higher than that of the population carrying a single mutation (P106L). Although the members of this population survive under a high glyphosate dosage of 10,080gaeha -1 , their growth was significantly inhibited by glyphosate under the recommend dose (840gaeha -1 ), where in the fresh weight was 85.4% of the control. EPSPS expression, relative copy number, and EPSPS activity in this population were similar to those of the susceptible population. In addition, the expression of two glutathione transferase (GST) genes (GST-U8 and GST-23) and the enzyme activity of the GST in this population did not significantly differ from those of the susceptible population. This finding is important in elucidating the resistance of the naturally evolved glyphosate-resistant (GR) weed species carrying a double mutation in EPSPS to glyphosate. Copyright © 2017. Published by Elsevier Inc.

Meta-analysis of Neuroblastomas Reveals a Skewed ALK Mutation Spectrum in Tumors with MYCN Amplification

NARCIS (Netherlands)

de Brouwer, Sara; de Preter, Katleen; Kumps, Candy; Zabrocki, Piotr; Porcu, Michaël; Westerhout, Ellen M.; Lakeman, Arjan; Vandesompele, Jo; Hoebeeck, Jasmien; van Maerken, Tom; de Paepe, Anne; Laureys, Geneviève; Schulte, Johannes H.; Schramm, Alexander; van den Broecke, Caroline; Vermeulen, Joëlle; van Roy, Nadine; Beiske, Klaus; Renard, Marleen; Noguera, Rosa; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Kogner, Per; Martinsson, Tommy; Nakagawara, Akira; Ohira, Miki; Caron, Huib N.; Eggert, Angelika; Cools, Jan; Versteeg, Rogier; Speleman, Frank

2010-01-01

Purpose: Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic

Mutational profiling of non-small-cell lung cancer patients resistant to first-generation EGFR tyrosine kinase inhibitors using next generation sequencing

Science.gov (United States)

Jin, Ying; Shao, Yang; Shi, Xun; Lou, Guangyuan; Zhang, Yiping; Wu, Xue; Tong, Xiaoling; Yu, Xinmin

2016-01-01

Patients with advanced non-small-cell lung cancer (NSCLC) harboring sensitive epithelial growth factor receptor (EGFR) mutations invariably develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Identification of actionable genetic alterations conferring drug-resistance can be helpful for guiding the subsequent treatment decision. One of the major resistant mechanisms is secondary EGFR-T790M mutation. Other mechanisms, such as HER2 and MET amplifications, and PIK3CA mutations, were also reported. However, the mechanisms in the remaining patients are still unknown. In this study, we performed mutational profiling in a cohort of 83 NSCLC patients with TKI-sensitizing EGFR mutations at diagnosis and acquired resistance to three different first-generation EGFR TKIs using targeted next generation sequencing (NGS) of 416 cancer-related genes. In total, we identified 322 genetic alterations with a median of 3 mutations per patient. 61% of patients still exhibit TKI-sensitizing EGFR mutations, and 36% of patients acquired EGFR-T790M. Besides other known resistance mechanisms, we identified TET2 mutations in 12% of patients. Interestingly, we also observed SOX2 amplification in EGFR-T790M negative patients, which are restricted to Icotinib treatment resistance, a drug widely used in Chinese NSCLC patients. Our study uncovered mutational profiles of NSCLC patients with first-generation EGFR TKIs resistance with potential therapeutic implications. PMID:27528220

Mutations in the Norrie disease gene.

Science.gov (United States)

Schuback, D E; Chen, Z Y; Craig, I W; Breakefield, X O; Sims, K B

1995-01-01

We report our experience to date in mutation identification in the Norrie disease (ND) gene. We carried out mutational analysis in 26 kindreds in an attempt to identify regions presumed critical to protein function and potentially correlated with generation of the disease phenotype. All coding exons, as well as noncoding regions of exons 1 and 2, 636 nucleotides in the noncoding region of exon 3, and 197 nucleotides of 5' flanking sequence, were analyzed for single-strand conformation polymorphisms (SSCP) by polymerase chain reaction (PCR) amplification of genomic DNA. DNA fragments that showed altered SSCP band mobilities were sequenced to locate the specific mutations. In addition to three previously described submicroscopic deletions encompassing the entire ND gene, we have now identified 6 intragenic deletions, 8 missense (seven point mutations, one 9-bp deletion), 6 nonsense (three point mutations, three single bp deletions/frameshift) and one 10-bp insertion, creating an expanded repeat in the 5' noncoding region of exon 1. Thus, mutations have been identified in a total of 24 of 26 (92%) of the kindreds we have studied to date. With the exception of two different mutations, each found in two apparently unrelated kindreds, these mutations are unique and expand the genotype database. Localization of the majority of point mutations at or near cysteine residues, potentially critical in protein tertiary structure, supports a previous protein model for norrin as member of a cystine knot growth factor family (Meitinger et al., 1993). Genotype-phenotype correlations were not evident with the limited clinical data available, except in the cases of larger submicroscopic deletions associated with a more severe neurologic syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in PIK3CA are infrequent in neuroblastoma

International Nuclear Information System (INIS)

Dam, Vincent; Morgan, Brian T; Mazanek, Pavel; Hogarty, Michael D

2006-01-01

Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessed PIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as these have not been previously reported. Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutations within the C2, helical and kinase domain 'hot spots' where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations to assess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studied to date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. These data suggest that activating

Comparison of the Soil Dynamic Amplification Factor and Soil Amplification by Using Microtremor and MASW Methods Respectively

Science.gov (United States)

Tuncel, Aykut; Cevdet Özdag, Özkan; Pamuk, Eren; Akgün, Mustafa

2017-12-01

Single Station Microtremor method, which is widely used nowadays, is an effective and easy applicable method. In this study, dynamic amplification factor distributions of the study area were obtained using scenario earthquake parameters with single station microtremor data gathered at 112 points. In addition, a surface wave active method, which is known as MASW (Multichannel Analysis of Surface Waves), was applied at 43 profiles to calculate the soil amplification values. Dynamic amplification factor (DAF), soil amplification, the predominant soil period (PSP), geology and topography data of the study area were analysed together. Dynamic amplification factor and soil amplification values were obtained 2 or higher at about sea level parts of the study area which are generally composed of alluvial units. Additionally, in high altitude regions that are composed of volcanic rocks, relatively lower dynamic amplification factor and soil amplification values were obtained. The minimum amplification value in the study area was 1.15, while the maximum amplification value was 3.05 according to the dynamic amplification results and the soil amplification values were between 1.16 and 3.85 in harmony. It is seen that the obtained DAF values and the soil amplification values calculated from the seismic velocities are very similar to each other numerically and regionally. Because of this, it is concluded that the values of the soil amplification obtained by the MASW method and the calculated DAF values in this study are in harmony with each other. Although the depths of research in these two calculation methods are different from each other, the similarity of the results allows us to arrive at the result of how effective the ground layer is on the amplification. It has a great importance to calculate the amplification values and other dynamic parameters by in situ measurements for a planned plot because geological units can vary even at very short distances in heterogeneously

Amplification of HER2 is a marker for global genomic instability

International Nuclear Information System (INIS)

Ellsworth, Rachel E; Ellsworth, Darrell L; Patney, Heather L; Deyarmin, Brenda; Love, Brad; Hooke, Jeffrey A; Shriver, Craig D

2008-01-01

Genomic alterations of the proto-oncogene c-erbB-2 (HER-2/neu) are associated with aggressive behavior and poor prognosis in patients with breast cancer. The variable clinical outcomes seen in patients with similar HER2 status, given similar treatments, suggests that the effects of amplification of HER2 can be influenced by other genetic changes. To assess the broader genomic implications of structural changes at the HER2 locus, we investigated relationships between genomic instability and HER2 status in patients with invasive breast cancer. HER2 status was determined using the PathVysion ® assay. DNA was extracted after laser microdissection from the 181 paraffin-embedded HER2 amplified (n = 39) or HER2 negative (n = 142) tumor specimens with sufficient tumor available to perform molecular analysis. Allelic imbalance (AI) was assessed using a panel of microsatellite markers representing 26 chromosomal regions commonly altered in breast cancer. Student t-tests and partial correlations were used to investigate relationships between genomic instability and HER2 status. The frequency of AI was significantly higher (P < 0.005) in HER2 amplified (27%) compared to HER2 negative tumors (19%). Samples with HER2 amplification showed significantly higher levels of AI (P < 0.05) at chromosomes 11q23, 16q22-q24 and 18q21. Partial correlations including ER status and tumor grade supported associations between HER2 status and alterations at 11q13.1, 16q22-q24 and 18q21. The poor prognosis associated with HER2 amplification may be attributed to global genomic instability as cells with high frequencies of chromosomal alterations have been associated with increased cellular proliferation and aggressive behavior. In addition, high levels of DNA damage may render tumor cells refractory to treatment. In addition, specific alterations at chromosomes 11q13, 16q22-q24, and 18q21, all of which have been associated with aggressive tumor behavior, may serve as genetic modifiers to HER2

[Identification of a novel GPR143 mutation in a Chinese family affected with X-linked ocular albinism].

Science.gov (United States)

Zhao, Qi; Guan, Menglong; Wang, Ling; Liao, Yong; Li-Ling, Jesse; Wan, Huajing

2017-04-10

To detect mutation of GPR143 gene in a Chinese patient affected with ocular albinism. Peripheral blood samples were collected from the proband and his parents. The coding regions of the GPR143 gene were subjected to PCR amplification and Sanger sequencing. A previously unreported mutation (c.758T>A) was found in exon 6 of the GPR143 gene in the proband and his mother. The same mutation was not found in his father. As predicted, the mutation has resulted in a stop codon, causing premature termination of protein translation. A novel mutation of the GPR143 gene related to X-linked ocular albinism has been identified.

Refractory status epilepticus

Directory of Open Access Journals (Sweden)

Sanjay P Singh

2014-01-01

Full Text Available Refractory status epilepticus is a potentially life-threatening medical emergency. It requires early diagnosis and treatment. There is a lack of consensus upon its semantic definition of whether it is status epilepticus that continues despite treatment with benzodiazepine and one antiepileptic medication (AED, i.e., Lorazepam + phenytoin. Others regard refractory status epilepticus as failure of benzodiazepine and 2 antiepileptic medications, i.e., Lorazepam + phenytoin + phenobarb. Up to 30% patients in SE fail to respond to two antiepileptic drugs (AEDs and 15% continue to have seizure activity despite use of three drugs. Mechanisms that have made the treatment even more challenging are GABA-R that is internalized during status epilepticus and upregulation of multidrug transporter proteins. All patients of refractory status epilepticus require continuous EEG monitoring. There are three main agents used in the treatment of RSE. These include pentobarbital or thiopental, midazolam and propofol. RSE was shown to result in mortality in 35% cases, 39.13% of patients were left with severe neurological deficits, while another 13% had mild neurological deficits.

Refractory status epilepticus

Science.gov (United States)

Singh, Sanjay P; Agarwal, Shubhi; Faulkner, M

2014-01-01

Refractory status epilepticus is a potentially life-threatening medical emergency. It requires early diagnosis and treatment. There is a lack of consensus upon its semantic definition of whether it is status epilepticus that continues despite treatment with benzodiazepine and one antiepileptic medication (AED), i.e., Lorazepam + phenytoin. Others regard refractory status epilepticus as failure of benzodiazepine and 2 antiepileptic medications, i.e., Lorazepam + phenytoin + phenobarb. Up to 30% patients in SE fail to respond to two antiepileptic drugs (AEDs) and 15% continue to have seizure activity despite use of three drugs. Mechanisms that have made the treatment even more challenging are GABA-R that is internalized during status epilepticus and upregulation of multidrug transporter proteins. All patients of refractory status epilepticus require continuous EEG monitoring. There are three main agents used in the treatment of RSE. These include pentobarbital or thiopental, midazolam and propofol. RSE was shown to result in mortality in 35% cases, 39.13% of patients were left with severe neurological deficits, while another 13% had mild neurological deficits. PMID:24791086

Development of refractory concrete for extreme conditions

International Nuclear Information System (INIS)

Pundiene, I; Antonovich, V; Stonys, R; Demidova-Buiziniene, I

2011-01-01

Comparative analysis is provided for the properties of medium-cement refractory concrete with microsilica based on mullite filler in relation to different type of deflocculant. The effect of different deflocculants on refractory concrete structure formation, hydration, rheology, strength and heat resistance is discussed. Corrosion resistance test, determined that samples with hybrid deflocculant showed better resistance for slag penetration than samples with only the sodium tripolyphosphate or polycarboxylate ether deflocculant. Moreover, a composition of hybrid deflocculant let to control the rate of the hydration process and to get features of refractory refractory concrete.

High-Density Infrared Surface Treatments of Refractories

Energy Technology Data Exchange (ETDEWEB)

Tiegs, T.N.

2005-03-31

Refractory materials play a crucial role in all energy-intensive industries and are truly a crosscutting technology for the Industries of the Future (IOF). One of the major mechanisms for the degradation of refractories and a general decrease in their performance has been the penetration and corrosion by molten metals or glass. Methods and materials that would reduce the penetration, wetting, and corrosive chemistry would significantly improve refractory performance and also maintain the quality of the processed liquid, be it metal or glass. This report presents the results of an R&D project aimed at investigating the use of high-density infrared (HDI) heating to surface treat refractories to improve their performance. The project was a joint effort between Oak Ridge National Laboratory (ORNL) and the University of Missouri-Rolla (UMR). HDI is capable of heating the near-surface region of materials to very high temperatures where sintering, diffusion, and melting can occur. The intended benefits of HDI processing of refractories were to (1) reduce surface porosity (by essentially sealing the surface to prevent liquid penetration), (2) allow surface chemistry changes to be performed by bonding an adherent coating onto the underlying refractory (in order to inhibit wetting and/or improve corrosion resistance), and (3) produce noncontact refractories with high-emissivity surface coatings.

Genetics Home Reference: iron-refractory iron deficiency anemia

Science.gov (United States)

... refractory iron deficiency anemia Iron-refractory iron deficiency anemia Printable PDF Open All Close All Enable Javascript ... expand/collapse boxes. Description Iron-refractory iron deficiency anemia is one of many types of anemia , which ...

Mutation analysis of β-thalassemia in East-Western Indian population: a recent molecular approach

Directory of Open Access Journals (Sweden)

Shah PS

2017-05-01

Full Text Available Parth S Shah,1 Nidhi D Shah,2 Hari Shankar P Ray,3 Nikunj B Khatri,3 Ketan K Vaghasia,3 Rutvik J Raval,4 Sandip C Shah,3 Mandava V Rao5 1Department of Medicine, Lahey Hospital and Medical Center, Boston, MA, 2Department of Pediatrics, Nassau University Medical Centre, New York, NY, USA; 3Supratech Micropath Laboratory and Research Institute, 4Department of Zoology, 5School of Sciences, Gujarat University, Ahmedabad, Gujarat, India Background: β-Thalassemia is the most prevalent genetic disorder in India. Its traits and coinheritance vary from mild to severe conditions, resulting in thalassemia minor, intermediate, and major, depending upon many factors.Purpose: The objective of this study was to identify the incidence of β-thalassemia traits, their coinheritance, and mutations, as well as to support the patients already diagnosed with β-thalassemia in East-Western Indian population for better management.Patients and methods: Seventy-five referral cases for β-thalassemia were analyzed for various β-thalassemia traits, heterozygosity, and homozygosity conditions. Blood phenotypic parameters using cell counter and capillary electrophoresis were investigated. Analyses of eight common mutations of thalassemia in India were carried out using polymerase chain reaction-amplification refractory mutation system, end point polymerase chain reaction, and DNA sequencing methods.Results: Of these (75 referral cases from East-Western Indian region, 68 were positive for β-thalassemia (90.67%. The majority of case types were of β-thalassemia minor (49, 65.33%, followed by HbE traits (6, 8.0% and β-thalassemia major, including heterozygous and homozygous (5, 6.66%; 4, 5.33% types and then HbE homozygous (2, 2.66%, as well as one each of the HbE/β-thalassemia and HbD/β-thalassemia (1, 1.34% combination. Mutation analysis also revealed that the highest frequency of mutation was c.92+5G>C (41, 60.29% followed by deletion 619bp (9, 13.23% and c.79G>A (8, 11

Spectrum of K ras mutations in Pakistani colorectal cancer patients

International Nuclear Information System (INIS)

Murtaza, B.N.; Bibi, A.; Rashid, M.U.; Khan, Y.I.; Chaudri, M.S.; Shakoori, A.R.

2013-01-01

The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients

Spectrum of K ras mutations in Pakistani colorectal cancer patients

Energy Technology Data Exchange (ETDEWEB)

Murtaza, B.N.; Bibi, A. [School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore (Pakistan); Rashid, M.U.; Khan, Y.I. [Shaukat Khanum Memorial Cancer Hospital and Research Centre, Johar Town, Lahore (Pakistan); Chaudri, M.S. [Services Institute of Medical Sciences, Lahore (Pakistan); Shakoori, A.R. [School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore (Pakistan)

2013-11-29

The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients.

Refractory hypertension: definition, prevalence, and patient characteristics.

Science.gov (United States)

Acelajado, Maria Czarina; Pisoni, Roberto; Dudenbostel, Tanja; Dell'Italia, Louis J; Cartmill, Falynn; Zhang, Bin; Cofield, Stacey S; Oparil, Suzanne; Calhoun, David A

2012-01-01

Among patients with resistant hypertension (RHTN), there are those whose blood pressure (BP) remains uncontrolled in spite of maximal medical therapy. This retrospective analysis aims to characterize these patients with refractory hypertension. Refractory hypertension was defined as BP that remained uncontrolled after ≥3 visits to a hypertension clinic within a minimum 6-month follow-up period. Of the 304 patients referred for RHTN, 29 (9.5%) remained refractory to treatment. Patients with refractory hypertension and those with controlled RHTN had similar aldosterone levels and plasma renin activity (PRA). Patients with refractory hypertension had higher baseline BP (175±23/97±15 mm Hg vs 158±25/89±15 mm Hg; P=.001/.005) and heart rate, and higher rates of prior stroke and congestive heart failure. During follow-up, the BP of patients with refractory hypertension remained uncontrolled (168.4±14.8/93.8±17.7 mm Hg) in spite of use of an average of 6 antihypertensive medications, while those of patients with controlled RHTN decreased to 129.3±11.2/77.6±10.8 mm Hg. Spironolactone reduced the BP by 12.9±17.8/6.6±13.7 mm Hg in patients with refractory hypertension and by 24.1±16.7/9.2±12.0 mm Hg in patients with controlled RHTN. In patients with RHTN, approximately 10% remain refractory to treatment. Similar aldosterone and PRA levels and a diminished response to spironolactone suggest that aldosterone excess does not explain the treatment failure. © 2011 Wiley Periodicals, Inc.

Punctate white matter lesions in full-term infants with neonatal seizures associated with SLC13A5 mutations

NARCIS (Netherlands)

Weeke, Lauren C; Brilstra, Eva; Braun, Kees P; Zonneveld-Huijssoon, Evelien; Salomons, Gajja S; Koeleman, BPC; van Gassen, Koen L; van Straaten, Henrica L; Craiu, Dana; de Vries, Linda S

INTRODUCTION: Early-onset epileptic encephalopathy caused by biallelic SLC13A5 mutations is characterized by seizure onset in the first days of life, refractory epilepsy and developmental delay. Little detailed information about the brain MRI features is available in these patients. METHODS:

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

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Yanagihara Kazuyoshi

2009-06-01

Full Text Available Abstract Background Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS, which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. Methods DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. Results DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20 of gastric cancer cell lines (8–18-fold amplification and 4.7% (4/86 of primary gastric tumors (8–50-fold amplification. KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

International Nuclear Information System (INIS)

Mita, Hiroaki; Yanagihara, Kazuyoshi; Fujita, Masahiro; Hosokawa, Masao; Kusano, Masanobu; Sabau, Sorin Vasile; Tatsumi, Haruyuki; Imai, Kohzoh; Shinomura, Yasuhisa; Tokino, Takashi; Toyota, Minoru; Aoki, Fumio; Akashi, Hirofumi; Maruyama, Reo; Sasaki, Yasushi; Suzuki, Hiromu; Idogawa, Masashi; Kashima, Lisa

2009-01-01

Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and

A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2.

Science.gov (United States)

Li, Jianli; Dai, Hongzheng; Feng, Yanming; Tang, Jia; Chen, Stella; Tian, Xia; Gorman, Elizabeth; Schmitt, Eric S; Hansen, Terah A A; Wang, Jing; Plon, Sharon E; Zhang, Victor Wei; Wong, Lee-Jun C

2015-09-01

Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 3' region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant.

Science.gov (United States)

Dormeyer, Miriam; Lübke, Anastasia L; Müller, Peter; Lentes, Sabine; Reuß, Daniel R; Thürmer, Andrea; Stülke, Jörg; Daniel, Rolf; Brantl, Sabine; Commichau, Fabian M

2017-06-01

Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Use of Human Tissue to Assess the Oncogenic Activity of Melanoma-Associated Mutations

OpenAIRE

Chudnovsky, Yakov; Adams, Amy E.; Robbins, Paul B.; Lin, Qun; Khavari, Paul A.

2005-01-01

Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence1,2. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Ras and Raf induction can occur via active N-Ras and B-Raf mutants as well as by gene amplification3–5. Activation of PI3K pathway components occurs by PTEN loss and by AKT amplification6–8. Melanomas also commonly display impairment of p16INK4A-CDK4-Rb and ARF-HDM2-p53 tumor s...

Refractories for Industrial Processing. Opportunities for Improved Energy Efficiency

Energy Technology Data Exchange (ETDEWEB)

Hemrick, James G. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Hayden, H. Wayne [Metals Manufacture Process and Controls Technology, Inc., Oak Ridge, TN (United States); Angelini, Peter [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Moore, Robert E. [R.E. Moore Associates, Maricopa, AZ (United States); Headrick, William L. [R.E. Moore Associates, Maricopa, AZ (United States)

2005-01-01

Refractories are a class of materials of critical importance to manufacturing industries with high-temperature unit processes. This study describes industrial refractory applications and identifies refractory performance barriers to energy efficiency for processing. The report provides recommendations for R&D pathways leading to improved refractories for energy-efficient manufacturing and processing.

Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling.

Directory of Open Access Journals (Sweden)

Swati Tomar

Full Text Available Retinoblastoma (RB is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59 while only 42.4% (25/59 of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9% of tumors screened. There were 3 cases (5.1% in which no mutations could be detected and germline mutations were detected in 19.5% (8/41 of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59 of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and

Punctate white matter lesions in full-term infants with neonatal seizures associated with SLC13A5 mutations

NARCIS (Netherlands)

Weeke, Lauren C.; Brilstra, Eva; Braun, Kees P.; Zonneveld-Huijssoon, Evelien; Salomons, Gajja S.; Koeleman, Bobby P; van Gassen, Koen L. I.; van Straaten, Henrica L.; Craiu, Dana; de Vries, Linda S.

Introduction: Early-onset epileptic encephalopathy caused by biallelic SLC13A5 mutations is characterized by seizure onset in the first days of life, refractory epilepsy and develop mental delay. Little detailed information about the brain MRI features is available in these patients. Methods:

Dramatic effect of levetiracetam in early-onset epileptic encephalopathy due to STXBP1 mutation.

Science.gov (United States)

Dilena, Robertino; Striano, Pasquale; Traverso, Monica; Viri, Maurizio; Cristofori, Gloria; Tadini, Laura; Barbieri, Sergio; Romeo, Antonino; Zara, Federico

2016-01-01

Syntaxin Binding Protein 1 (STXBP1) mutations determine a central neurotransmission dysfunction through impairment of the synaptic vesicle release, thus causing a spectrum of phenotypes varying from syndromic and non-syndromic epilepsy to intellectual disability of variable degree. Among the antiepileptic drugs, levetiracetam has a unique mechanism of action binding SV2A, a glycoprotein of the synaptic vesicle release machinery. We report a 1-month-old boy manifesting an epileptic encephalopathy with clonic seizures refractory to phenobarbital, pyridoxine and phenytoin that presented a dramatic response to levetiracetam with full epilepsy control and EEG normalization. Genetic analysis identified a novel de novo heterozygous mutation (c.[922A>T]p.[Lys308(∗)]) in the STXBP1 gene that severely affects the protein. The observation of a dramatic efficacy of levetiracetam in a case of STXBP1 epileptic encephalopathy refractory to other antiepileptic drugs and considerations regarding the specific mechanism of action of levetiracetam modulating the same system affected by STXBP1 mutations support the hypothesis that this drug may be able to reverse specifically the disease epileptogenic abnormalities. Further clinical observations and laboratory studies are needed to confirm this hypothesis and eventually lead to consider levetiracetam as the first choice treatment of patients with suspected or confirmed STXBP1-related epilepsies. Copyright © 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Improved multiplex ligation-dependent probe amplification analysis identifies a deleterious PMS2 allele generated by recombination with crossover between PMS2 and PMS2CL

OpenAIRE

Wernstedt, Annekatrin; Valtorta, Emanuele; Armelao, Franco; Togni, Roberto; Girlando, Salvatore; Baudis, Michael; Heinimann, Karl; Messiaen, Ludwine; Staehli, Noemie; Zschocke, Johannes; Marra, Giancarlo; Wimmer, Katharina

2012-01-01

Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which – owing to extensive interparalog sequence exchange – closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal number...

GBM-associated mutations and altered protein expression are more common in young patients.

Science.gov (United States)

Ferguson, Sherise D; Xiu, Joanne; Weathers, Shiao-Pei; Zhou, Shouhao; Kesari, Santosh; Weiss, Stephanie E; Verhaak, Roeland G; Hohl, Raymond J; Barger, Geoffrey R; Reddy, Sandeep K; Heimberger, Amy B

2016-10-25

Geriatric glioblastoma (GBM) patients have a poorer prognosis than younger patients, but IDH1/2 mutations (more common in younger patients) confer a favorable prognosis. We compared key GBM molecular alterations between an elderly (age ≥ 70) and younger (18 GBM cohort compared to the older cohort (P GBM cohort, younger patients had significantly more mutations in PDGFRA, PTPN11, SMARCA4, BRAF and TP53. GBMs from 178 elderly patients and 197 young patients were analyzed using DNA sequencing, immunohistochemistry, in situ hybridization, and MGMT-methylation assay to ascertain mutational and amplification/expressional status. Significant molecular differences occurred in GBMs from elderly and young patients. Except for the older cohort's more frequent PTEN mutation and MGMT methylation, younger patients had a higher frequency of potential therapeutic targets.

Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

Directory of Open Access Journals (Sweden)

Barbara Ziegler

2011-11-01

Full Text Available This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip. Biochip hybridization identified 17 (21% samples to carry a KRAS mutation of which 16 (33% were adenocarcinomas and 1 (3% was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples.

Evolution of BCR/ABL gene mutation in CML is time dependent and dependent on the pressure exerted by tyrosine kinase inhibitor.

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Shantashri Vaidya

Full Text Available BACKGROUND: Mutations in the ABL kinase domain and SH3-SH2 domain of the BCR/ABL gene and amplification of the Philadelphia chromosome are the two important BCR/ABL dependent mechanisms of imatinib resistance. Here, we intended to study the role played by TKI, imatinib, in selection of gene mutations and development of chromosomal abnormalities in Indian CML patients. METHODS: Direct sequencing methodology was employed to detect mutations and conventional cytogenetics was done to identify Philadelphia duplication. RESULTS: Among the different mechanisms of imatinib resistance, kinase domain mutations (39% of the BCR/ABL gene were seen to be more prevalent, followed by mutations in the SH3-SH2 domain (4% and then BCR/ABL amplification with the least frequency (1%. The median duration of occurrence of mutation was significantly shorter for patients with front line imatinib than those pre-treated with hydroxyurea. Patients with high Sokal score (p = 0.003 showed significantly higher incidence of mutations, as compared to patients with low/intermediate score. Impact of mutations on the clinical outcome in AP and BC was observed to be insignificant. Of the 94 imatinib resistant patients, only 1 patient exhibited duplication of Philadelphia chromosome, suggesting a less frequent occurrence of this abnormality in Indian CML patients. CONCLUSION: Close monitoring at regular intervals and proper analysis of the disease resistance would facilitate early detection of resistance and thus aid in the selection of the most appropriate therapy.

CNS germinomas are characterized by global demethylation, chromosomal instability and mutational activation of the Kit-, Ras/Raf/Erk- and Akt-pathways

Science.gov (United States)

Schulte, Simone Laura; Waha, Andreas; Steiger, Barbara; Denkhaus, Dorota; Dörner, Evelyn; Calaminus, Gabriele; Leuschner, Ivo; Pietsch, Torsten

2016-01-01

CNS germinomas represent a unique germ cell tumor entity characterized by undifferentiated tumor cells and a high response rate to current treatment protocols. Limited information is available on their underlying genomic, epigenetic and biological alterations. We performed a genome-wide analysis of genomic copy number alterations in 49 CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry for methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members. Ras/Erk and Akt pathway activation was analyzed by immunostaining with antibodies against phospho-Erk, phosho-Akt, phospho-mTOR and phospho-S6. All germinomas coexpressed Oct4 and Kit but showed an extensive global DNA demethylation compared to other tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in all tumors with a high frequency of regional gains and losses including high level gene amplifications. Activating mutations of KIT exons 11, 13, and 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS and RRAS2 indicated mutational activation of crucial signaling pathways. Co-activation of Ras/Erk and Akt pathways was present in 83% of germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding has a striking coincidence with extensive genomic instability. In addition, mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy. PMID:27391150

Miniaturized isothermal nucleic acid amplification, a review.

Science.gov (United States)

Asiello, Peter J; Baeumner, Antje J

2011-04-21

Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

Tungsten and refractory metals 3, proceedings

International Nuclear Information System (INIS)

Bose, A.; Dowding, R.J.

1996-01-01

The Third International Conference on Tungsten and Refractory Metals was held in Greater Washington DC at the McLean Hilton, McLean Virginia, on November 15--16, 1995. This meeting was the third in a series of conferences held in the Washington DC area. The first meeting was in 1992 and was entitled ''International Conference on Tungsten and Tungsten Alloys.'' In 1994, the scope of the meeting was expanded to include other refractory metals such as molybdenum, iridium, rhenium, tantalum and niobium. The tremendous success of that meeting was the primary motivation for this Conference. The broader scope (the inclusion of other refractory metals and alloys) of the Conference was kept intact for this meeting. In fact, it was felt that the developments in the technology of these materials required a common forum for the interchange of current research information. The papers presented in this meeting examined the rapid advancements in the technology of refractory metals, with special emphasis on the processing, structure, and properties. Among the properties there was emphasis on both quasi-static and dynamic rates. Another topic that received considerable interest was the area of refractory carbides and tungsten-copper composites. One day of concurrent session was necessary to accommodate all of the presentations

Potential refractory alloy requirements for space nuclear power applications

International Nuclear Information System (INIS)

Cooper, R.H. Jr.

1984-01-01

In reviewing design requirements for refractory alloys for space nuclear applications, several key points are identified. First, the successful utilization of refractory alloys is considered an enabling requirement for the successful deployment of high efficiency, lightweight, and small space nuclear systems. Second, the recapture of refractory alloy nuclear technology developed in the 1960s and early 1970s appears to be a pacing activity in the successful utilization of refractory alloys. Third, the successful application of refractory alloys for space nuclear applications will present a significant challenge to both the materials and the systems design communities

Mutation analysis with random DNA identifiers (MARDI) catalogs Pig-a mutations in heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats.

Science.gov (United States)

Revollo, Javier R; Crabtree, Nathaniel M; Pearce, Mason G; Pacheco-Martinez, M Monserrat; Dobrovolsky, Vasily N

2016-03-01

Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a "clone-by-clone" approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high-fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS- and PCR-induced sequencing artifacts. When applied to the Pig-a cDNA analysis of heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats, MARDI detected nearly all Pig-a mutations that were previously identified by conventional clone-by-clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non-transcribed DNA strand. © 2015 Wiley Periodicals, Inc.

Refractories for steel-works

International Nuclear Information System (INIS)

Villanova, R.A.; Galant, C.L.; Haas, C.; Rosenbaum, V.

The routine procedures utilized for quality control of refractory materials used by PIRATINI's steel-works, are presented ' under an objetive and practical maner. The attention of the paper is concentrated upon the following' refractory types with higher consume: silicon-aluminous; aluminous; basic magnesia; basic chrom-magnesia. All steps of utilization are described, including specification, supplies programation, storage; sampling; physical tests, and also aplication procedures. Results from routine analysis during a six month period, by ' means of X-Ray Quantometry, using the fusion pearls procedure, are presented compared with Atomic Absorption [pt

Identification of nine genomic regions of amplification in urothelial carcinoma, correlation with stage, and potential prognostic and therapeutic value.

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Yvonne Chekaluk

Full Text Available We performed a genome wide analysis of 164 urothelial carcinoma samples and 27 bladder cancer cell lines to identify copy number changes associated with disease characteristics, and examined the association of amplification events with stage and grade of disease. Multiplex inversion probe (MIP analysis, a recently developed genomic technique, was used to study 80 urothelial carcinomas to identify mutations and copy number changes. Selected amplification events were then analyzed in a validation cohort of 84 bladder cancers by multiplex ligation-dependent probe assay (MLPA. In the MIP analysis, 44 regions of significant copy number change were identified using GISTIC. Nine gene-containing regions of amplification were selected for validation in the second cohort by MLPA. Amplification events at these 9 genomic regions were found to correlate strongly with stage, being seen in only 2 of 23 (9% Ta grade 1 or 1-2 cancers, in contrast to 31 of 61 (51% Ta grade 3 and T2 grade 2 cancers, p<0.001. These observations suggest that analysis of genomic amplification of these 9 regions might help distinguish non-invasive from invasive urothelial carcinoma, although further study is required. Both MIP and MLPA methods perform well on formalin-fixed paraffin-embedded DNA, enhancing their potential clinical use. Furthermore several of the amplified genes identified here (ERBB2, MDM2, CCND1 are potential therapeutic targets.

Development of AZS refractories for the glass industry

International Nuclear Information System (INIS)

Guzman, A.M.; Rodriguez, P.

2004-01-01

Refractory materials can support high temperatures, thermal strength and the contact with aggressive environments, for this reason they are widely used in the cement, glass and steel industry. Commercial AZS (alumina-zirconia-silica) refractories are a good alternative in refractory materials for the glass industry' because they can support the aggressive conditions during liquid processing of glass. However, another problem encountered in glass industry is contamination by refractory' material that fall into the molten glass, which can produce a series of defects in the final product. This research was conducted to develop new formulations of AZS refractories with different amounts of ZrO 2 with the purpose of improving the characteristics, properties and the work conditions in the glass melting furnaces and, at the same time, lower the costs this type of refractories. The results obtained indicate that the composition with low content of ZrO 2 can provide better properties than the commercial product, with some modifications in the particle size distribution. Copyright (2004) AD-TECH - International Foundation for the Advancement of Technology Ltd

Gradual Loss of ACTH Due to a Novel Mutation in LHX4: Comprehensive Mutation Screening in Japanese Patients with Congenital Hypopituitarism

Science.gov (United States)

Takagi, Masaki; Ishii, Tomohiro; Inokuchi, Mikako; Amano, Naoko; Narumi, Satoshi; Asakura, Yumi; Muroya, Koji; Hasegawa, Yukihiro; Adachi, Masanori; Hasegawa, Tomonobu

2012-01-01

Mutations in transcription factors genes, which are well regulated spatially and temporally in the pituitary gland, result in congenital hypopituitarism (CH) in humans. The prevalence of CH attributable to transcription factor mutations appears to be rare and varies among populations. This study aimed to define the prevalence of CH in terms of nine CH-associated genes among Japanese patients. We enrolled 91 Japanese CH patients for DNA sequencing of POU1F1, PROP1, HESX1, LHX3, LHX4, SOX2, SOX3, OTX2, and GLI2. Additionally, gene copy numbers for POU1F1, PROP1, HESX1, LHX3, and LHX4 were examined by multiplex ligation-dependent probe amplification. The gene regulatory properties of mutant LHX4 proteins were characterized in vitro. We identified two novel heterozygous LHX4 mutations, namely c.249-1G>A, p.V75I, and one common POU1F1 mutation, p.R271W. The patient harboring the c.249-1G>A mutation exhibited isolated growth hormone deficiency at diagnosis and a gradual loss of ACTH, whereas the patient with the p.V75I mutation exhibited multiple pituitary hormone deficiency. In vitro experiments showed that both LHX4 mutations were associated with an impairment of the transactivation capacities of POU1F1 andαGSU, without any dominant-negative effects. The total mutation prevalence in Japanese CH patients was 3.3%. This study is the first to describe, a gradual loss of ACTH in a patient carrying an LHX4 mutation. Careful monitoring of hypothalamic–pituitary -adrenal function is recommended for CH patients with LHX4 mutations. PMID:23029363

Gene amplification in carcinogenesis

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Lucimari Bizari

2006-01-01

Full Text Available Gene amplification increases the number of genes in a genome and can give rise to karyotype abnormalities called double minutes (DM and homogeneously staining regions (HSR, both of which have been widely observed in human tumors but are also known to play a major role during embryonic development due to the fact that they are responsible for the programmed increase of gene expression. The etiology of gene amplification during carcinogenesis is not yet completely understood but can be considered a result of genetic instability. Gene amplification leads to an increase in protein expression and provides a selective advantage during cell growth. Oncogenes such as CCND1, c-MET, c-MYC, ERBB2, EGFR and MDM2 are amplified in human tumors and can be associated with increased expression of their respective proteins or not. In general, gene amplification is associated with more aggressive tumors, metastases, resistance to chemotherapy and a decrease in the period during which the patient stays free of the disease. This review discusses the major role of gene amplification in the progression of carcinomas, formation of genetic markers and as possible therapeutic targets for the development of drugs for the treatment of some types of tumors.

Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

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Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

2016-12-01

Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally. Copyright © 2016 Elsevier B.V. All rights reserved.

Refractory migraine in a headache clinic population

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Fernandez-Torron Roberto

2011-08-01

Full Text Available Abstract Background Many migraineurs who seek care in headache clinics are refractory to treatment, despite advances in headache therapies. Epidemiology is poorly characterized, because diagnostic criteria for refractory migraine were not available until recently. We aimed to determine the frequency of refractory migraine in patients attended in the Headache Unit in a tertiary care center, according to recently proposed criteria. Methods The study population consisted of a consecutive sample of 370 patients (60.8% females with a mean age of 43 years (range 14-86 evaluated for the first time in our headache unit over a one-year period (between October 2008 and October 2009. We recorded information on clinical features, previous treatments, Migraine Disability Assessment Score (MIDAS, and final diagnosis. Results Overall migraine and tension-type headache were found in 46.4% and 20.5% of patients, respectively. Refractory migraine was found in 5.1% of patients. In refractory migraineurs, the mean MIDAS score was 96, and 36.8% were medication-overusers. Conclusions Refractory migraine is a relatively common and very disabling condition between the patients attended in a headache unit. The proposed operational criteria may be useful in identifying those patients who require care in headache units, the selection of candidates for combinations of prophylactic drugs or invasive treatments such as neurostimulation, but also to facilitate clinical studies in this patient group.

NOvel Refractory Materials for High Alkali, High Temperature Environments

Energy Technology Data Exchange (ETDEWEB)

Hemrick, J.G.; Griffin, R. (MINTEQ International, Inc.)

2011-08-30

Refractory materials can be limited in their application by many factors including chemical reactions between the service environment and the refractory material, mechanical degradation of the refractory material by the service environment, temperature limitations on the use of a particular refractory material, and the inability to install or repair the refractory material in a cost effective manner or while the vessel was in service. The objective of this project was to address the need for new innovative refractory compositions by developing a family of novel MgO-Al2O3 spinel or other similar magnesia/alumina containing unshaped refractory composition (castables, gunnables, shotcretes, etc) utilizing new aggregate materials, bond systems, protective coatings, and phase formation techniques (in-situ phase formation, altered conversion temperatures, accelerated reactions, etc). This family of refractory compositions would then be tailored for use in high-temperature, highalkaline industrial environments like those found in the aluminum, chemical, forest products, glass, and steel industries. A research team was formed to carry out the proposed work led by Oak Ridge National Laboratory (ORNL) and was comprised of the academic institution Missouri University of Science and Technology (MS&T), and the industrial company MINTEQ International, Inc. (MINTEQ), along with representatives from the aluminum, chemical, glass, and forest products industries. The two goals of this project were to produce novel refractory compositions which will allow for improved energy efficiency and to develop new refractory application techniques which would improve the speed of installation. Also methods of hot installation were sought which would allow for hot repairs and on-line maintenance leading to reduced process downtimes and eliminating the need to cool and reheat process vessels.

SINTERED REFRACTORY TUNGSTEN ALLOYS. Gesinterte hochschmelzende wolframlegierungen

Energy Technology Data Exchange (ETDEWEB)

Kieffer, R.; Sedlatschek, K.; Braun, H.

1971-12-15

Dependence of the melting point of the refractory metals on their positions in the periodic system - alloys of tungsten with other refractory metals - sintering of the alloys - processing of the alloys - technological properties.

Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

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Chen, Qianqian; Chen, Xiaoxiang; Zhang, Sichao; Lan, Ke; Lu, Jian; Zhang, Chiyu

2015-01-01

The development of simple, accurate, rapid and cost-effective technologies for mutation detection is crucial to the early diagnosis and prevention of numerous genetic diseases, pharmacogenetics, and drug resistance. Proofreading PCR (PR-PCR) was developed for mutation detection in 1998 but is rarely applied due to its low efficiency in allele discrimination. Here we developed a modified PR-PCR method using a ddNTP-blocked primer and a mixture of DNA polymerases with and without the 3'-5' proofreading function. The ddNTP-blocked primer exhibited the best blocking efficiency to avoid nonspecific primer extension while the mixture of a tiny amount of high-fidelity DNA polymerase with a routine amount of Taq DNA polymerase provided the best discrimination and amplification effects. The modified PR-PCR method is quite capable of detecting various mutation types, including point mutations and insertions/deletions (indels), and allows discrimination amplification when the mismatch is located within the last eight nucleotides from the 3'-end of the ddNTP-blocked primer. The modified PR-PCR has a sensitivity of 1-5 × 102 copies and a selectivity of 5 × 10-5 mutant among 107 copies of wild-type DNA. It showed a 100% accuracy rate in the detection of P72R germ-line mutation in the TP53 gene among 60 clinical blood samples, and a high potential to detect rifampin-resistant mutations at low frequency in Mycobacterium tuberculosis using an adaptor and a fusion-blocked primer. These results suggest that the modified PR-PCR technique is effective in detection of various mutations or polymorphisms as a simple, sensitive and promising approach. PMID:25915410

Stochastic histories of refractory interstellar dust

International Nuclear Information System (INIS)

Liffman, K.; Chayton, D.D.

1988-01-01

The authors calculate histories for refractory dust particles in the interstellar medium. The double purposes are to learn something of the properties of interstellar dust as a system and to evaluate with specific assumptions the cosmic chemical memory interpretation of a specific class of isotopic anomalies. They assemble the profile of a particle population from a large number of stochastic, or Monte Carlo, histories of single particles, which are necessarily taken to be independent with this approach. They specify probabilities for each of the events that may befall a given particle and unfold its history by a sequence of random numbers. They assume that refractory particles are created only by thermal condensation within stellar material during its ejection from stars, and that these refractory particles can be destroyed only by being sputtered to a size too small for stability or by being incorporated into the formation of new stars. In order to record chemical detail, the authors take each new refractory particle to consist of a superrefractory core plus a more massive refractory mantle. They demonstrate that these superrefractory cores have effective lifetimes much longer than the turnover time of dust mass against sputtering. As examples of cosmic chemical memory they evaluate the 16 O-richness of interstellar aluminum and mechanisms for the 48 Ca/ 50 Ti correlation. Several related consequences of this approach are discussed

Biomaterials in light amplification

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Mysliwiec, Jaroslaw; Cyprych, Konrad; Sznitko, Lech; Miniewicz, Andrzej

2017-03-01

Biologically produced or inspired materials can serve as optical gain media, i.e. they can exhibit the phenomenon of light amplification. Some of these materials, under suitable dye-doping and optical pumping conditions, show lasing phenomena. The emerging branch of research focused on obtaining lasing action in highly disordered and highly light scattering materials, i.e. research on random lasing, is perfectly suited for biological materials. The use of biomaterials in light amplification has been extensively reported in the literature. In this review we attempt to report on progress in the development of biologically derived systems able to show the phenomena of light amplification and random lasing together with the contribution of our group to this field. The rich world of biopolymers modified with molecular aggregates and nanocrystals, and self-organized at the nanoscale, offers a multitude of possibilities for tailoring luminescent and light scattering properties that are not easily replicated in conventional organic or inorganic materials. Of particular importance and interest are light amplification and lasing, or random lasing studies in biological cells and tissues. In this review we will describe nucleic acids and their complexes employed as gain media due to their favorable optical properties and ease of manipulation. We will report on research conducted on various biomaterials showing structural analogy to nucleic acids such as fluorescent proteins, gelatins in which the first distributed feedback laser was realized, and also amyloids or silks, which, due to their dye-doped fiber-like structure, allow for light amplification. Other materials that were investigated in that respect include polysaccharides, like starch exhibiting favorable photostability in comparison to other biomaterials, and chitosan, which forms photonic crystals or cellulose. Light amplification and random lasing was not only observed in processed biomaterials but also in living

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients.

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Avani Solanki

Full Text Available Fanconi anemia (FA, a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C. Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients.

Science.gov (United States)

Solanki, Avani; Mohanty, Purvi; Shukla, Pallavi; Rao, Anita; Ghosh, Kanjaksha; Vundinti, Babu Rao

2016-01-01

Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.

Response properties of the refractory auditory nerve fiber.

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Miller, C A; Abbas, P J; Robinson, B K

2001-09-01

The refractory characteristics of auditory nerve fibers limit their ability to accurately encode temporal information. Therefore, they are relevant to the design of cochlear prostheses. It is also possible that the refractory property could be exploited by prosthetic devices to improve information transfer, as refractoriness may enhance the nerve's stochastic properties. Furthermore, refractory data are needed for the development of accurate computational models of auditory nerve fibers. We applied a two-pulse forward-masking paradigm to a feline model of the human auditory nerve to assess refractory properties of single fibers. Each fiber was driven to refractoriness by a single (masker) current pulse delivered intracochlearly. Properties of firing efficiency, latency, jitter, spike amplitude, and relative spread (a measure of dynamic range and stochasticity) were examined by exciting fibers with a second (probe) pulse and systematically varying the masker-probe interval (MPI). Responses to monophasic cathodic current pulses were analyzed. We estimated the mean absolute refractory period to be about 330 micros and the mean recovery time constant to be about 410 micros. A significant proportion of fibers (13 of 34) responded to the probe pulse with MPIs as short as 500 micros. Spike amplitude decreased with decreasing MPI, a finding relevant to the development of computational nerve-fiber models, interpretation of gross evoked potentials, and models of more central neural processing. A small mean decrement in spike jitter was noted at small MPI values. Some trends (such as spike latency-vs-MPI) varied across fibers, suggesting that sites of excitation varied across fibers. Relative spread was found to increase with decreasing MPI values, providing direct evidence that stochastic properties of fibers are altered under conditions of refractoriness.

Refractory alloy technology for space nuclear power applications

International Nuclear Information System (INIS)

Cooper, R.H. Jr.; Hoffman, E.E.

1984-01-01

Purpose of this symposium is twofold: (1) to review and document the status of refractory alloy technology for structural and fuel-cladding applications in space nuclear power systems, and (2) to identify and document the refractory alloy research and development needs for the SP-100 Program in both the short and the long term. In this symposium, an effort was made to recapture the space reactor refractory alloy technology that was cut off in midstream around 1973 when the national space nuclear reactor program began in the early 1960s, was terminated. The six technical areas covered in the program are compatibility, processing and production, welding and component fabrication, mechanical and physical properties, effects of irradiation, and machinability. The refractory alloys considered are niobium, molybdenum, tantalum, and tungsten. Thirteen of the 14 pages have been abstracted separately. The remaining paper summarizes key needs for further R and D on refractory alloys

HER2 activating mutations are targets for colorectal cancer treatment.

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Kavuri, Shyam M; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M; Migliardi, Giorgia; Searleman, Adam C; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A; Bertotti, Andrea; Bose, Ron

2015-08-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer. ©2015 American Association for Cancer Research.

Two Siblings With a CDKL5 Mutation: Genotype and Phenotype Evaluation.

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Hagebeuk, Eveline E O; Marcelis, Carlo L; Alders, Mariëlle; Kaspers, Ageeth; de Weerd, Al W

2015-10-01

This is the second report of a family with a recurrence of a CDKL5 mutation (c. 283-3_290del) in 2 sisters. Both parents tested negative for the mutation in all tissues, but germline mosaicism is likely. Clinically CDKL5 patients resemble those with Rett syndrome, caused by a MECP2 mutation, who experience a regression, after an initial normal development. Even though both siblings showed a typical CDKL5 phenotype, their presentation is different. From birth, the oldest daughter had a severe developmental delay, feeding problems, and hypotonia and experienced daily refractory seizures. The youngest daughter appeared to be normal until age 3 months. At that age seizures started, deterioration and regression became evident, and an epileptic encephalopathy developed. This report of familial recurrence, with suspected germline mosaicism in a healthy parent, has important consequences for genetic counseling. Although it is not possible to predict an exact recurrence risk, it is likely to be increased. © The Author(s) 2015.

An investigation into mineral processing of north Semnan refractory earth

International Nuclear Information System (INIS)

Aslani, S.; Samin-Bani-Hashemi, H.R.; Taghi-Zadeh, O.

2002-01-01

This paper is dealing with refractory earth of North Semnan. Having an area of 2000 square kilometers, Semnan province is mainly formed by sedimentary rocks with a verity of refractory earth, red earth and kaolin containing heavy minerals. The refractory earth of this area contains a considerable rate of aluminum oxide in shape of dia spore minerals, behemoth and gybsite along with heavy minerals of iron and titanium. To improve the quality of refractory earth, in order to be used in related industries, these minerals have to be separated. To assess the quality of refractory earth of North Semnan as the raw materials of refractory industries, their genesis and mineralogy properties have been precisely studied. Based on the rate of aluminium oxide of the refractory earth of North Semnan mines, a suitable mineral deposit has been selected for more investigation. Using XRD and X RF methods along with electronic and photo microscopes, the refractory earth and heavy minerals of them have been assessed. The elementary laboratory experiments of fragmentation and magnetic separation have been performed. It has been proved that the iron minerals can be separated and, therefore, the quality of the refractory earth can be improved. The separation of titanium minerals has to be investigated with other methods

C-MET overexpression and amplification in gliomas.

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Kwak, Yoonjin; Kim, Seong-Ik; Park, Chul-Kee; Paek, Sun Ha; Lee, Soon-Tae; Park, Sung-Hye

2015-01-01

We investigated c-Met overexpression and MET gene amplification in gliomas to determine their incidence and prognostic significance. c-Met immunohistochemistry and MET gene fluorescence in situ hybridization were carried out on tissue microarrays from 250 patients with gliomas (137 grade IV GBMs and 113 grade II and III diffuse gliomas). Clinicopathological features of these cases were reviewed. c-Met overexpression and MET gene amplification were detected in 13.1% and 5.1% of the GBMs, respectively. All the MET-amplified cases showed c-Met overexpression, but MET amplification was not always concordant with c-Met overexpression. None of grade II and III gliomas demonstrated c-Met overexpression or MET gene amplification. Mean survival of the GBM patients with MET amplification was not significantly different from patients without MET amplification (P=0.155). However, GBM patients with c-Met overexpression survived longer than patients without c-Met overexpression (P=0.035). Although MET amplification was not related to poor GBM prognosis, it is partially associated with the aggressiveness of gliomas, as MET amplification was found only in grade IV, not in grade II and III gliomas. We suggest that MET inhibitor therapy may be beneficial in about 5% GBMs, which was the incidence of MET gene amplification found in the patients included in this study.

[Genetic mutation and clinical features of osteogenesis imperfecta type V].

Science.gov (United States)

Guan, Shizhen; Bai, Xue; Wang, Yi; Liu, Zhigang; Ren, Xiuzhi; Zhang, Tianke; Ju, Mingyan; Li, Keqiu; Li, Guang

2017-12-10

To explore genetic mutations and clinical features of osteogenesis imperfecta type V. Clinical record of five patients (including one familial case) with osteogenesis imperfecta type V were retrospectively analyzed. Peripheral blood samples of the patients, one family member, as well as healthy controls were collected. Mutation of IFITM5 gene was identified by PCR amplification and Sanger sequencing. A heterozygous mutation (c.-14C>T) in the 5-UTR of the IFITM5 gene was identified in all of the patients and one mother. The clinical findings included frequent fractures and spine and/or extremities deformities, absence of dentinogenesis imperfecta, absence of hearing impairment, and blue sclera in 1 case. Radiographic findings revealed calcification of the interosseous membrane between the radius-ulna in all cases. Hyperplastic callus formation was found in 3 cases. Four had radial-head dislocation. A single heterozygous mutation c.-14C>T was found in the 5-UTR of the IFITM5 gene in 5 patients with osteogensis imperfecta type V. The patients showed specific radiological features including calcification of interosseous membrane, hyperplastic callus formation, and radial-head dislocation.

Refractory alloy technology for space nuclear power applications

Energy Technology Data Exchange (ETDEWEB)

Cooper, R.H. Jr.; Hoffman, E.E. (eds.)

1984-01-01

Purpose of this symposium is twofold: (1) to review and document the status of refractory alloy technology for structural and fuel-cladding applications in space nuclear power systems, and (2) to identify and document the refractory alloy research and development needs for the SP-100 Program in both the short and the long term. In this symposium, an effort was made to recapture the space reactor refractory alloy technology that was cut off in midstream around 1973 when the national space nuclear reactor program began in the early 1960s, was terminated. The six technical areas covered in the program are compatibility, processing and production, welding and component fabrication, mechanical and physical properties, effects of irradiation, and machinability. The refractory alloys considered are niobium, molybdenum, tantalum, and tungsten. Thirteen of the 14 pages have been abstracted separately. The remaining paper summarizes key needs for further R and D on refractory alloys. (DLC)

Temperature and Thermal Stress Analysis of Refractory Products

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Shaoyang Shi

2013-05-01

Full Text Available Firstly current status of temperature and thermal stress research of refractory product at home and aboard are analyzed. Finite element model of two classical refractory products is building by using APDL language. Distribution law of temperature and thermal stress of two typical refractory products-ladles and tundish are analyzed and their structures are optimized. Stress of optimal structure is dropped obviously, and operation life is increased effectively.

Characteristics of gene mutation in Chinese patients with hereditary hemochromatosis

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LYU Tingxia

2016-08-01

Full Text Available ObjectiveTo investigate the characteristics of gene mutation in Chinese patients with hereditary hemochromatosis (HH. MethodsA total of 9 patients with HH who visited Beijing Friendship Hospital, Capital Medical University from January 2013 to December 2015 were enrolled. The genomic DNA was extracted, and PCR amplification and Sanger sequencing were performed for all the exons of four genotypes of HH, i.e., HFE (type Ⅰ, HJV (type ⅡA, HAMP (type ⅡB, TFR2 (type Ⅲ, and SLC40A1 (type Ⅳ to analyze gene mutations. A total of 50 healthy subjects were enrolled as control group to analyze the prevalence of identified gene mutations in a healthy population. ResultsOf all patients, 2 had H63D mutation of HFE gene in type Ⅰ HH, 1 had E3D mutation of HJV gene in type ⅡA HH, 2 had I238M mutation of TFR2 gene in type Ⅲ HH, and 1 had IVS 3+10 del GTT splice mutation of SLC40A1 gene in type Ⅳ HH. No patients had C282Y mutation of HFE gene in type Ⅰ HH which was commonly seen in European and American populations. Five patients had no missense mutation or splice mutation. In addition, it was found in a family that a HH patient had E3D mutation of HJV gene, H63D mutation of HFE gene, and I238M mutation of TFR2 gene, but the healthy brother and sister carrying two of these mutations did not had the phenotype of HH. ConclusionHH gene mutations vary significantly across patients of different races, and non-HFE-HH is dominant in the Chinese population. There may be HH genes which are different from known genes, and further investigation is needed.

Characterization of variegate porphyria mutations using a minigene approach.

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Granata, Barbara Xoana; Baralle, Marco; De Conti, Laura; Parera, Victoria; Rossetti, Maria Victoria

2015-01-01

Porphyrias are a group of metabolic diseases that affect the skin and/or nervous system. In 2008, three unrelated patients were diagnosed with variegate porphyria at the CIPYP (Centro de Investigaciones sobre Porfirinas y Porfirias). Sequencing of the protoporphyrinogen oxidase gene, the gene altered in this type of porphyria, revealed three previously undescribed mutations: c.338+3insT, c.807G>A, and c.808-1G>C. As these mutations do not affect the protein sequence, we hypothesized that they might be splicing mutations. RT-PCRs performed on the patient's mRNAs showed normal mRNA or no amplification at all. This result indicated that the aberrant spliced transcript is possibly being degraded. In order to establish whether they were responsible or not for the patient's disease by causing aberrant splicing, we utilized a minigene approach. We found that the three mutations lead to exon skipping; therefore, the abnormal mRNAs are most likely degraded by a mechanism such as nonsense-mediated decay. In conclusion, these mutations are responsible for the disease because they alter the normal splicing pathway, thus providing a functional explanation for the appearance of disease and highlighting the use of minigene assays to complement transcript analysis.

Social amplification of risk

International Nuclear Information System (INIS)

Kasperson, R.E.; Renn, O.; Slovic, P.; Kasperson, J.X.; Emani, S.

1989-01-01

The risks associated with radioactive and other hazardous waste disposal may be expected to interact with societal processes to enlarge or attenuate the consequences of risks and risk events. This article summarizes a conceptual framework that depicts the social amplification of risk. Using a data base of 128 hazard events that have occurred largely over the past ten years, the authors examine the role of physical consequences, media coverage, and public perceptions of risk in generating social and economic impacts. The analysis concludes that social amplification processes substantially shape the nature and magnitude of those impacts but also that such social amplification appears to be systematically related to characteristics of the risks and risk events

Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients

International Nuclear Information System (INIS)

Mihalatos, Markos; Fountzilas, George; Agnantis, Niki J; Nasioulas, Georgios; Apessos, Angela; Dauwerse, Hans; Velissariou, Voula; Psychias, Aristidis; Koliopanos, Alexander; Petropoulos, Konstantinos; Triantafillidis, John K; Danielidis, Ioannis

2005-01-01

Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions / deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation – dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription – Polymerase Chain Reaction). A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations

Comparative evaluation of the locally manufactured low-alumina fireclay refractories

International Nuclear Information System (INIS)

Mohammad, D.; Ahmed, W.; Khan, M.I.

2007-01-01

Properties of domestically produced low-alumina refractories commonly known as medium duty fireclay refractories were studied and a comparative evaluation was performed. Refractory properties such as density, porosity, cold strength, reheat change and deformation at high-temperature were determined. Significant differences were discovered between these products sold as medium duty fireclay refractories. One brand marketed as medium duty refractory was found to be too inferior to be designated as medium duty firebrick. Absence of any standards and lack of awareness on the part of users of the various test methods appear to be the cause of this situation. (author)

Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases

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Pravas Ranjan Sahoo

2016-05-01

Full Text Available India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

Science.gov (United States)

Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

2016-05-01

India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

Directed light fabrication of refractory metals and alloys

International Nuclear Information System (INIS)

Fonseca, J.C.; Lewis, G.K.; Dickerson, P.G.; Nemec, R.B.

1999-01-01

This report covers deposition of refractory pure metals and alloys using the Directed Light Fabrication (DLF) process and represents progress in depositing these materials through September 1998. In extending the DLF process technology to refractory metals for producing fully dense, structurally sound deposits, several problems have become evident. (1) Control of porosity in DLF-deposited refractory metal is difficult because of gases, apparently present in commercially purchased refractory metal powder starting materials. (2) The radiant heat from the molten pool during deposition melts the DLF powder feed nozzle. (3) The high reflectivity of molten refractory metals, at the Nd-YAG laser wavelength (1.06microm), produces damaging back reflections to the optical train and fiber optic delivery system that can terminate DLF processing. (4) The current limits on the maximum available laser power to prevent back reflection damage limit the parameter range available for densification of refractory metals. The work to date concentrated on niobium, W-25Re, and spherodized tungsten. Niobium samples, made from hydride-dehydride powder, had minimal gas porosity and the deposition parameters were optimized; however, test plates were not made at this time. W-25Re samples, containing sodium and potassium from a precipitation process, were made and porosity was a problem for all samples although minimized with some process parameters. Deposits made from potassium reduced tungsten that was plasma spherodized were made with minimized porosity. Results of this work indicate that further gas analysis of starting powders and de-gassing of starting powders and/or gas removal during deposition of refractory metals is required

Determination of HER2 and p53 Mutations by Sequence Analysis Method and EGFR/Chromosome 7 Gene Status by Fluorescence in Situ Hybridization for the Predilection of Targeted Therapy Modalities in Immunohistochemically Triple Negative Breast Carcinomas in Turkish Population.

Science.gov (United States)

Pala, Emel Ebru; Bayol, Umit; Keskin, Elif Usturali; Ozguzer, Alp; Kucuk, Ulku; Ozer, Ozge; Koc, Altug

2015-09-01

Triple negative breast cancer (TNBC), an agressive subtype accounts nearly 15 % of all breast carcinomas. Conventional chemotherapy is the only treatment modality thus new, effective targeted therapy methods have been investigated. Epidermal growth factor receptor (EGFR) inhibitors give hope according to the recent studies results. Also therapeutic agents have been tried against aberrant p53 signal activity as TNBC show high p53 mutation rates. Our aim was to detect the incidence of mutations/amplifications identified in TNBC in our population. Here we used sequence analysis to detect HER2 (exon 18-23), p53 (exon 5-8) mutations; fluorescence in situ hybridization (FISH) method to analyse EGFR/chromosome 7 centromere gene status in 82 immunohistochemically TNBC. Basaloid phenotype was identified in 49 (59.8 %) patients. EGFR amplification was noted in 5 cases (6.1 %). All EGFR amplified cases showed EGFR overexpression by immunohistochemistry (IHC). p53 mutations were identified in 33 (40.2 %) cases. Almost 60 % of the basal like breast cancer cases showed p53 mutation. Only one case showed HER2 mutation (exon 20:g.36830_3). Our results showed that gene amplification is not the unique mechanism in EGFR overexpression. IHC might be used in the decision of anti-EGFR therapy in routine practice. p53 mutation rate was lower than the rates reported in the literature probably due to ethnic differences and low sensitivity of sanger sequences in general mutation screening. We also established the rarity of HER2 mutation in TNBC. In conclusion EGFR and p53 are the major targets in TNBC also for our population.

Clinical study of DMD gene point mutation causing Becker muscular dystrophy

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Ji-qing CAO

2015-07-01

Full Text Available Background  DMD gene point mutation, mainly nonsense mutation, always cause the most severe Duchenne muscular dystrophy (DMD. However, we also observed some cases of Becker muscular dystrophy (BMD carrying DMD point mutation. This paper aims to explore the mechanism of DMD point mutation causing BMD, in order to enhance the understanding of mutation types of BMD.  Methods  Sequence analysis was performed in 11 cases of BMD confirmed by typical clinical manifestations and muscle biopsy. The exon of DMD gene was detected non-deletion or duplication by multiplex ligation-dependent probe amplification (MLPA.  Results  Eleven patients carried 10 mutation types without mutational hotspot. Six patients carried nonsense mutations [c.5002G>T, p.(Glu1668X; c.1615C > T, p.(Arg539X; c.7105G > T, p.(Glu2369X; c.5287C > T, p.(Arg1763X; c.9284T > G, p.(Leu3095X]. One patient carried missense mutation [c.5234G > A, p.(Arg1745His]. Two patients carried frameshift mutations (c.10231dupT, c.10491delC. Two patients carried splicing site mutations (c.4518 + 3A > T, c.649 + 2T > C.  Conclusions  DMD gene point mutation may result in BMD with mild clinical symptoms. When clinical manifestations suggest the possibility of BMD and MLPA reveals non?deletion or duplication mutation of DMD gene, BMD should be considered. Study on the mechanism of DMD point mutation causing BMD is very important for gene therapy of DMD. DOI: 10.3969/j.issn.1672-6731.2015.06.005

Machining refractory alloys: an overview

International Nuclear Information System (INIS)

Christopher, J.D.

1984-01-01

Nontraditional machining is a generic term for those material removal processes that differ drastically from the historic operations such as turning, milling, drilling, tapping, and grinding. The use of primary energy modes other than mechanical, such as thermal, electrical, and chemical, sets these operations apart and reinforces their nontraditional label. Several of these newer processes have been very successful in machining close tolerance parts from refractory materials. This paper provides a general overview of both traditional and nontraditional aspects of machining refractory materials. 11 figures, 7 tables

Progressive age-dependence and frequency difference in the effect of gap junctions on active cochlear amplification and hearing.

Science.gov (United States)

Zong, Liang; Chen, Jin; Zhu, Yan; Zhao, Hong-Bo

2017-07-22

Mutations of Connexin 26 (Cx26, GJB2), which is a predominant gap junction isoform in the cochlea, can induce high incidence of nonsyndromic hearing loss. We previously found that targeted-deletion of Cx26 in supporting Deiters cells and outer pillar cells in the cochlea can influence outer hair cell (OHC) electromotility and reduce active cochlear amplification leading to hearing loss, even though there are no gap junction connexin expressions in the auditory sensory hair cells. Here, we further report that hearing loss and the reduction of active amplification in the Cx26 targeted-deletion mice are progressive and different at high and low frequency regions, first occurring in the high frequency region and then progressively extending to the middle and low frequency regions with mouse age increased. The speed of hearing loss extending was fast in the basal high frequency region and slow in the apical low frequency region, showing a logarithmic function with mouse age. Before postnatal day 25, there were no significant hearing loss and the reduction of active cochlear amplification in the low frequency region. Hearing loss and the reduction of active cochlear amplification also had frequency difference, severe and large in the high frequency regions. These new data indicate that the effect of gap junction on active cochlear amplification is progressive, but, consistent with our previous report, exists in both high and low frequency regions in adulthood. These new data also suggest that cochlear gap junctions may have an important role in age-related hearing loss. Copyright © 2017 Elsevier Inc. All rights reserved.

Production of lightweight refractory material by hydrothermal process

International Nuclear Information System (INIS)

Sulejmani, Ramiz B.

2002-01-01

Many different processes of production of lightweight refractories are well known over the World. Traditional production of lightweight refractories is by addition of combustibles or by a special frothing process. This work is concerned with hydrothermal of lightweight refractories from rice husk ash. The rice husk ash, used in present investigations were from Kocani region, R. Macedonia. The chemical analysis of the rice husk ash shows that it contains 91,8 - 93,7% SiO 2 and some alkaline and alkaline earth oxides. Microscopic and X - ray diffraction examinations of the rice husk ash have shown that it is composed of cristobalite, tridimite and amorphous silica. The composition of the mixture for lightweight refractory brick production is 93,4% rice husk ash and 6,6% Ca(OH) 2 . The mixtures were well mixed, moistened and pressed at 5 - 10 MPa. The hydrothermal reactions between calcium hydroxide and rice husk ash over the temperature range 80 - 160 o C were investigated. The period of autoclave treatment was from 2 to 72 h. After the hydrothermal treatment of the samples, the mineralogical composition, bulk density, density, cold crushing strength, porosity, refractoriness and thermal expansion were examined. Analysing the properties of the obtained samples it can be concluded that from rice husk ash and calcium hydroxide under hydrothermal condition it is possible to obtain lightweight acid refractory material with high quality.(Author)

Interleukin-6 -174 genotype, periodontal disease and adverse pregnancy outcomes: a pilot study.

Science.gov (United States)

Dashash, M; Nugent, J; Baker, P; Tansinda, D; Blinkhorn, F

2008-05-01

This study was undertaken to investigate whether maternal periodontal disease and variant genotypes of IL-6 gene are associated with adverse pregnancy outcomes. A total of 145 pregnant women were recruited from St Mary's Hospital, Manchester, UK. Bleeding on probing (BOP) and pocket depth indices were recorded on all teeth. Amplification refractory mutation system-polymerase chain reaction was used for -174 IL-6 genotyping. Birth weight was assessed using the individualized birth ratio (IBR) with intrauterine growth restriction (IUGR) defined as an IBR below the fifth percentile. The G/G genotype results in more BOP % sites in Caucasian (P periodontal disease, and IUGR.

Prognostic value of plasma EGFR ctDNA in NSCLC patients treated with EGFR-TKIs.

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Chengjuan Zhang

Full Text Available Epidermal growth factor receptor (EGFR specific mutations have been known to improve survival of patients with non-small-cell lung carcinoma (NSCLC. However, whether there are any changes of EGFR mutations after targeted therapy and its clinical significance is unclear. This study was to identify the status of EGFR mutations after targeted therapy and predict the prognostic significance for NSCLC patients.A total of forty-five (45 NSCLC patients who received EGFR-TKI therapy were enrolled. We identified the changes of EGFR mutations in plasma ctDNA by Amplification Refractory Mutation System (ARMS PCR technology.In the 45 cases of NSCLC with EGFR mutations, the EGFR mutation status changed in 26 cases, in which, 12 cases (26.7% from positive to negative, and 14 cases (31.1% from T790M mutation negative to positive after TKI targeted therapy. The T790M occurance group had a shorter Progression -Free-Survival (PFS than the groups of EGFR mutation undetected and EGFR mutation turned out to have no change after EGFR-TKI therapy (p < 0.05.According to this study, it's necessary to closely monitor EGFR mutations during follow-up to predict the prognosis of NSCLC patients who are to receive the TKI targeted therapy.

Directed light fabrication of refractory metals and alloys

International Nuclear Information System (INIS)

Fonseca, J.C.; Lewis, G.K.; Dickerson, P.G.; Nemec, R.B.

1999-01-01

This report covers work performed under Order No. FA0000020 AN Contract DE-AC12-76SN00052 for deposition of refractory pure metals and alloys using the Directed Light Fabrication (DLF) process and represents the progress in depositing these materials through September 1998. In extending the DLF process technology to refractory metals for producing fully dense, structurally sound deposits, several problems have become evident. 1. Control of porosity in DLF-deposited refractory metal is difficult because of gases, apparently present in commercially purchased refractory metal powder starting materials. 2. The radiant heat from the molten pool during deposition melts the DLF powder feed nozzle. 3. The high reflectivity of molten refractory metals, at the Nd-YAG laser wavelength (1.06microm), produces damaging back reflections to the optical train and fiber optic delivery system that can terminate DLF processing. 4. The current limits on the maximum available laser power to prevent back reflection damage limit the parameter range available for densification of refractory metals. The work to date concentrated on niobium, W-25Re, and spherodized tungsten. Niobium samples, made from hydride-dehydride powder, had minimal gas porosity and the deposition parameters were optimized; however, test plates were not made at this time. W-25Re samples, containing sodium and potassium from a precipitation process, were made and porosity was a problem for all samples although minimized with some process parameters. Deposits made from potassium reduced tungsten that was plasma spherodized were made with minimized porosity. Results of this work indicate that further gas analysis of starting powders and de-gassing of starting powders and/or gas removal during deposition of refractory metals is required

Earthquake acceleration amplification based on single microtremor test

Science.gov (United States)

Jaya Syahbana, Arifan; Kurniawan, Rahmat; Soebowo, Eko

2018-02-01

Understanding soil dynamics is needed to understand soil behaviour, including the parameters of earthquake acceleration amplification. Many researchers now conduct single microtremor tests to obtain amplification of velocity and natural periods of soil at test sites. However, these amplification parameters are rarely used, so a method is needed to convert the velocity amplification to acceleration amplification. This paper will discuss the proposed process of changing the value of amplification. The proposed method is to integrate the time histories of the synthetic earthquake acceleration of the soil surface under the deaggregation at that location so the time histories of the velocity earthquake will be obtained. Next is to conduct a “fitting curve” between amplification by a single microtremor test with amplification of the synthetic earthquake velocity time histories. After obtaining the fitting curve time histories of velocity, differentiation will be conducted to obtain fitting curve acceleration time histories. The final step after obtaining the fitting curve is to compare the acceleration of the “fitting curve” against the histories time of the acceleration of synthetic earthquake at bedrocks to obtain single microtremor acceleration amplification factor.

Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies.

Science.gov (United States)

Mairinger, Fabian D; Walter, Robert Fh; Vollbrecht, Claudia; Hager, Thomas; Worm, Karl; Ting, Saskia; Wohlschläger, Jeremias; Zarogoulidis, Paul; Zarogoulidis, Konstantinos; Schmid, Kurt W

2014-01-01

Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. A total of 250 μg DNA (concentration 5 μg/μL) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA.

Spinning of refractory metals

International Nuclear Information System (INIS)

Chang Wenkua; Zheng Han

1989-01-01

The effects of spinning process parameters including max. pass percentage reduction, spinning temperature, feed rate, lubricant and annealing technology on the quality of shaped components are summarized and discussed in the present paper. The above mentioned parameters are adopted in the process of spinning of barrel-shaped and specially shaped components of refractory metals and their alloys W, Mo, Nb, Zr, TZM molybdenum alloy, C-103, C-752 niobium alloy etc. The cause of leading to usual defects of spun products of refractory metals such as lamellar as 'scaling', crack, swelling, wrinkle, etc. have been analysed and the ways to eliminate the defects have been put forward. 8 figs., 5 tabs. (Author)

Migration of TRU-containing slag into candidate refractories for waste management

International Nuclear Information System (INIS)

Seymour, W.C.

1978-11-01

Results of initial tests on transuranic migration into commercial refractories are reported. Five castable refractories and a plastic-bonded refractory were tested using the cup test method. Initial results indicate that castable materials will not survive conditions expected in the slagging pyrolysis unit; plastic refractories appear to have better performance. The major mechanism of migration into the tested refractories is pore penetration. Al 2 O 3 and Al 2 O 3 -Cr 2 O 3 phases appear resistant to chemical diffusion of transuranics. 2 figures, 16 tables

Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies

Directory of Open Access Journals (Sweden)

Mairinger FD

2014-08-01

Full Text Available Fabian D Mairinger,1 Robert FH Walter,2 Claudia Vollbrecht,3 Thomas Hager,1 Karl Worm,1 Saskia Ting,1 Jeremias Wohlschläger,1 Paul Zarogoulidis,4 Konstantinos Zarogoulidis,4 Kurt W Schmid1 1Institute of Pathology, 2Ruhrlandklinik, West German Lung Center, University Hospital Essen, Essen, 3Institute of Pathology, University Hospital Cologne, Cologne, Germany; 4Pulmonary Department, Oncology Unit, G Papanikolaou General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece Background and methods: Isothermal multiple displacement amplification (IMDA can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. Results: A total of 250 µg DNA (concentration 5 µg/µL was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. Conclusion: We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA. Keywords: isothermal multiple displacement amplification, isothermal, whole

Refractory of Furnaces to Reduce Environmental Impact

International Nuclear Information System (INIS)

Hanzawa, Shigeru

2011-01-01

The energy load of furnaces used in the manufacturing process of ceramics is quite large. Most of the environmental impact of ceramics manufacturing is due to the CO 2 produced from this high energy load. To improve this situation, R and D has focused on furnace systems and techniques of control in order to reduce energy load. Since furnaces are comprised of refractory, consideration of their mechanical and thermal characteristics is important. Herein are described several refractory types which were chosen through comparison of the characteristics which contribute to heat capacity reduction, heat insulating reinforcement and high emissivity, thereby improving thermal radiation heat transfer efficiency to the ceramic articles. One selected refractory material which will reduce the environmental impact of a furnace, chosen considering low heat capacity and high emissivity characteristics, is SiC. In this study, thermal radiation heat transfer efficiency improvement and its effect on ceramic articles in the furnace and oxidation behaviour were investigated at 1700K. A high density SiC refractory, built into the furnace at construction, has relatively high oxidation durability and has the ability to reduce environmental impact-CO 2 by 10 percent by decreasing the furnace's energy load. However, new oxidation prevention techniques for SiC will be necessary for long-term use in industrial furnaces, because passive to active oxidation transition behaviour of commercial SiC refractory is coming to close ideal.

Refractory of Furnaces to Reduce Environmental Impact

Science.gov (United States)

Hanzawa, Shigeru

2011-10-01

The energy load of furnaces used in the manufacturing process of ceramics is quite large. Most of the environmental impact of ceramics manufacturing is due to the CO2 produced from this high energy load. To improve this situation, R&D has focused on furnace systems and techniques of control in order to reduce energy load. Since furnaces are comprised of refractory, consideration of their mechanical and thermal characteristics is important. Herein are described several refractory types which were chosen through comparison of the characteristics which contribute to heat capacity reduction, heat insulating reinforcement and high emissivity, thereby improving thermal radiation heat transfer efficiency to the ceramic articles. One selected refractory material which will reduce the environmental impact of a furnace, chosen considering low heat capacity and high emissivity characteristics, is SiC. In this study, thermal radiation heat transfer efficiency improvement and its effect on ceramic articles in the furnace and oxidation behaviour were investigated at 1700K. A high density SiC refractory, built into the furnace at construction, has relatively high oxidation durability and has the ability to reduce environmental impact-CO2 by 10 percent by decreasing the furnace's energy load. However, new oxidation prevention techniques for SiC will be necessary for long-term use in industrial furnaces, because passive to active oxidation transition behaviour of commercial SiC refractory is coming to close ideal.

Development of alumino-silicate refractories in Ghana

International Nuclear Information System (INIS)

Kisiedu, A. K.; Tetteh, D.M.B.; Obiri, H. A.; Brenya, E. F.; Ayensu, A.

2008-01-01

Alumino-silicate (bauxite), andalusite, kaolin and clay were investigated for suitability in production of alumina, mullite and fireclay brick refractories. The raw materials were characterized by X-ray diffraction, differential thermal and silicate analyses. The x-ray diffraction analysis of alumina and mullite refractories fired at 1450 0 C, and fireclay bricks fired at 1350 0 C, indicated presence of corundum and alpha-alumina crystals. The values of thermal (fired) shrinkage, crushing, strength, porosity, water absorption and bulk density determined were 31.1%, 2.3 x 10 3 kg/m 3 , 4.86 x 10 6 N/m 2 and 13.2 % for mullite; 30.2%, 2.4 x 10 3 kg/m 3 , 3.20 x 10 6 N/m 2 and W = 12.8 % for alumina; and 25.2 %, 2.1 x 10 3 kg/m 3 , 2.61 x 10 6 N/m 2 and W = 11.8% for fireclay, respectively. Bauxite, andalusite and special kaolin were identified as potential raw materials for developing alumina and mullite refractories for construction of high temperature kilns and furnaces operating at 1350 0 C. The clay and kaolin minerals could be used to produce fireclay refractories for construction of incinerators operating at maximum temperatures of about 1000 0 C. The performance of the refractories was demonstrated by producing bricks to construct kilns and incinerators for the ceramic industry and hospitals. (au)

[Clinical features and COMP gene mutation in a family with a pseudoachondroplasia child].

Science.gov (United States)

Lu, Chun-Ting; Guo, Li; Zahng, Zhan-Hui; Lin, Wei-Xia; Song, Yuan-Zong; Feng, Lie

2013-11-01

This study aimed to report the clinical characteristics and COMP gene mutation of a family with pseudoachondroplasia (PSACH), a relatively rare spinal and epiphyseal dysplasia that is inherited as an autosomal dominant trait. Clinical information on a 5-year-2-month-old PSACH child and his parents was collected and analyzed. Diagnosis was confirmed by PCR amplification and direct sequencing of all the 19 exons and their flanking sequences of COMP gene, and the mutation was further ascertained by cloning analysis of exon 10. The child presented with short and stubby fingers, bow leg, short limb dwarfism and metaphysic broadening in long bone as well as lumbar lordosis. A mutation c.1048_1116del (p.Asn350_Asp372del) in exon 10, inherited from his father who did not demonstrate any phenotypic feature of PSACH, was detected in the child. PSACH was diagnosed definitively by means of COMP mutation analysis, on the basis of the child's clinical and imaging features. The non-penetrance phenomenon of COMP mutation was described for the first time in PSACH.

Analysis of KIT expression and KIT exon 11 mutations in canine oral malignant melanomas.

Science.gov (United States)

Murakami, A; Mori, T; Sakai, H; Murakami, M; Yanai, T; Hoshino, Y; Maruo, K

2011-09-01

KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases. © 2011 Blackwell Publishing Ltd.

Rare MDM4 gene amplification in colorectal cancer: The principle of a mutually exclusive relationship between MDM alteration and TP53 inactivation is not applicable.

Science.gov (United States)

Suda, Tetsuji; Yoshihara, Mitsuyo; Nakamura, Yoshiyasu; Sekiguchi, Hironobu; Godai, Ten-I; Sugano, Nobuhiro; Tsuchida, Kazuhito; Shiozawa, Manabu; Sakuma, Yuji; Tsuchiya, Eiju; Kameda, Yoichi; Akaike, Makoto; Matsukuma, Shoichi; Miyagi, Yohei

2011-07-01

MDM4, a homolog of MDM2, is considered a key negative regulator of p53. Gene amplification of MDM4 has been identified in a variety of tumors. MDM2 or MDM4 gene amplification is only associated with the wild-type TP53 gene in retinoblastomas, thus the amplification of the two genes is mutually exclusive. Previously, we demonstrated that MDM2 amplification and TP53 alteration were not mutually exclusive in colorectal cancer, and we identified a subset of colorectal cancer patients without alterations in either the TP53 or the MDM2 gene. In this study, we investigated the gene amplification status of MDM4 in the same set of colorectal cancer cases. Unexpectedly, MDM4 amplification was rare, detected in only 1.4% (3 out of 211) of colorectal cancer cases. All the three gene-amplified tumors also harbored TP53-inactivating mutations. This contradicts the simple mutually exclusive relationship observed in retinoblastomas. Surprisingly, two of the three MDM4-amplified tumors also demonstrated MDM2 amplification. Paradoxically, the MDM4 protein levels were decreased in the tumor tissue of the gene-amplified cases compared with levels in the matched normal mucosa. We speculate that MDM4 might play a role in colorectal carcinogenesis that is not limited to negative regulation of p53 in combination with MDM2. The functional significance of MDM4 is still unclear and further studies are needed.

Refractory disease in antineutrophil cytoplasmic antibodies associated vasculitis

NARCIS (Netherlands)

Rutgers, Abraham; Kallenberg, Cornelis

Purpose of review Induction treatment of antineutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV) is not always successful and nonresponding patients are considered refractory. Recent findings Refractory disease should be subdefined to the treatment that was received.

Detection of mutation in isoniazid-resistant Mycobacterium tuberculosis isolates from tuberculosis patients in Belarus

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Bostanabad S

2008-01-01

Full Text Available The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis strains isolated from patients in Belarus. Forty two isoniazid-resistant isolates were identified from sputum of 163 patients with active pulmonary tuberculosis. Drug susceptibility testing was determined by using CDC standard conventional proportional method and BACTEC system. Standard PCR method for detection of isoniazid resistance associated mutations was performed by katG gene amplification and DNA sequencing. Most mutations were found in katG gene codons 315, 316 and 309. Four types of mutations were identified in codon 315: AGC→ACC ( n = 36 85%, AGC→AGG ( n = 1 2.3%, AGC→AAC ( n = 2 4.7%, AGC→GGC ( n = 1 2.3%. One type of mutation was found in codon 316: GGC→AGC ( n = 1841.4%, four types of mutations were detected in codon 309: GGT→GGT ( n = 716.1%, GGT→GCT ( n = 49.2%, GGT→GTC ( n = 36.9%, GGT→GGG ( n = 12.7%. The highest frequency of mutations sharing between primary and secondary infections was found in codon 315.

Protein misfolding cyclic amplification induces the conversion of recombinant prion protein to PrP oligomers causing neuronal apoptosis.

Science.gov (United States)

Yuan, Zhen; Yang, Lifeng; Chen, Baian; Zhu, Ting; Hassan, Mohammad Farooque; Yin, Xiaomin; Zhou, Xiangmei; Zhao, Deming

2015-06-01

The formation of neurotoxic prion protein (PrP) oligomers is thought to be a key step in the development of prion diseases. Recently, it was determined that the sonication and shaking of recombinant PrP can convert PrP monomers into β-state oligomers. Herein, we demonstrate that β-state oligomeric PrP can be generated through protein misfolding cyclic amplification from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP, and that these oligomers can be used for subsequent research into the mechanisms of PrP-induced neurotoxicity. We have characterized protein misfolding cyclic amplification-induced monomer-to-oligomer conversion of PrP from three species using western blotting, circular dichroism, size-exclusion chromatography, and resistance to proteinase K (PK) digestion. We have further shown that all of the resulting β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, whereas the corresponding monomeric PrP were not toxic. In addition, we found that this toxicity is the result of oligomer-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3 in both wild-type and PrP(-/-) cortical neurons. It is our hope that these results may contribute to our understanding of prion transformation within the brain. We found that β-state oligomeric PrPs can be generated through protein misfolding cyclic amplification (PMCA) from recombinant full-length hamster, human, rabbit, and mutated rabbit PrP. β-oligomers are toxic to primary mouse cortical neurons independent of the presence of PrP(C) in the neurons, while the corresponding monomeric PrPs were not toxic. This toxicity is the result of oligomers-induced apoptosis via regulation of Bcl-2, Bax, and caspase-3. These results may contribute to our understanding of prion transformation within the brain. © 2015 International Society for Neurochemistry.

Diagnosis and management of refractory celiac disease: a systematic review.

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Labidi, Asma; Serghini, Meriem; Karoui, Sami; Boubaker, Jalel; Filali, Azza

2013-01-01

Refractory celiac disease is defined by persisting malabsorptive symptoms in spite of a strict gluten free diet for at least 6 to 12 months. Alternatives to gluten free diet seem to be still controversial. To describe the clinical and epidemiologic aspects of refractory celiac disease, and to identify therapeutic options in this condition. Systematic review and critical analysis of observational studies, clinical trials and case reports that focused on diagnosis and management of refractory celiac disease. Refractory celiac disease can be classified as type 1 or type 2 according to the phenotype of intraepithelial lymphocytes. Great complications such as enteropathy-associated T-cell lymphoma may occur in a subgroup of these patients mainly in refractory celiac disease type 2. Curative therapies are still lacking. Refractory celiac disease remains a diagnosis of exclusion. Its prognosis remains still dismal by the absence yet of curative therapies. However, some new treatments seem to hold promise during few cohort-studies.

Modern materials based on refractory compounds

International Nuclear Information System (INIS)

Kosolapova, T.Ya.

1979-01-01

Discussed are the existing methods for synthesizing powders of binary refractory compounds and high-productivity techniques which hold promise as regards the manufacture of highly disperse and pure powders. Plasmochemical synthesis is shown to be an effective method for obtaining practically all carbides, nitrides and borides. A description is given of three main methods for obtaining single crystals of refractory compounds (TiN, TiC, ZrC, ZrB 2 , NbC) fairly perfect in structure and composition. These processes include deposition from vapour-gas phase, melting in arc plasma and crystallization from solutions in metallic melts. The advantages have been shown of the self-propagating high-temperature synthesis of refractory compounds, ensuring the manufacture of products, close in composition to stoichiometric ones simultaneously with forming of items. Mechanical, thermal, abrasive, and resistive characteristics of the above materials are presented

The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans.

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Yang, Huan; Vallandingham, Jim; Shiu, Philip; Li, Hua; Hunter, Craig P; Mak, Ho Yi

2014-04-14

RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments. Copyright © 2014 Elsevier Ltd. All rights reserved.

Coexistence of K-ras mutations and HPV infection in colon cancer

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Tezol Ayda

2006-05-01

Full Text Available Abstract Background Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer. Methods K-ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Stattistical analyses were performed by the chi-square and Fisher's exact tests Results HPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV-positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis. Conclusion Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype.

Structure, preparation and properties of refractory compounds and systems

International Nuclear Information System (INIS)

Holleck, H.; Thuemmler, F.

1977-01-01

At the beginning of this report the possibilities of hardness optimization of refractory carbides are generally discussed. Three papers deal with TaC-basis refractories and hard metals. In particular, carbides with very low nonmetal/metal ratios and composites with hard phases formed by decomposition of tantalum carbonitrides are discussed. Another contribution reports investigations concerning the influence of the microstructure on the hardness of polycristaline mixed carbides. In a series of four papers, results are presented on the work of optimization conventional WC hard metals by introduction of a Fe,Co,Ni-binder: The influence of composition, carbon content and sintering conditions, as well as the wetting behaviour between carbides and binder metals are discussed. Phase relations in the refractory nitride and refractory nitride-binder metal systems as well as phase stabilities of ordered transition metal phases are reported in three papers, fundamental in character. Finally, the work concerning chemical analysis of refractory systems is described. (orig.) [de

Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes.

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Maher, Geoffrey J; McGowan, Simon J; Giannoulatou, Eleni; Verrill, Clare; Goriely, Anne; Wilkie, Andrew O M

2016-03-01

De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

Privacy amplification for quantum key distribution

International Nuclear Information System (INIS)

Watanabe, Yodai

2007-01-01

This paper examines classical privacy amplification using a universal family of hash functions. In quantum key distribution, the adversary's measurement can wait until the choice of hash functions is announced, and so the adversary's information may depend on the choice. Therefore the existing result on classical privacy amplification, which assumes the independence of the choice from the other random variables, is not applicable to this case. This paper provides a security proof of privacy amplification which is valid even when the adversary's information may depend on the choice of hash functions. The compression rate of the proposed privacy amplification can be taken to be the same as that of the existing one with an exponentially small loss in secrecy of a final key. (fast track communication)

Cutaneous hidradenocarcinoma: a clinicopathological, immunohistochemical, and molecular biologic study of 14 cases, including Her2/neu gene expression/amplification, TP53 gene mutation analysis, and t(11;19) translocation.

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Kazakov, Dmitry V; Ivan, Doina; Kutzner, Heinz; Spagnolo, Dominic V; Grossmann, Petr; Vanecek, Tomas; Sima, Radek; Kacerovska, Denisa; Shelekhova, Ksenia V; Denisjuk, Natalja; Hillen, Uwe; Kuroda, Naoto; Mukensnabl, Petr; Danis, Dusan; Michal, Michal

2009-05-01

, in 2 cases, a minority of the neoplastic cells (10%-20%) demonstrated nuclear staining, whereas the remaining 4 cases were negative. Of 9 specimens of hidradenocarcinoma studied for TP53 mutations, 2 harbored mutations, whereas the remaining 7 specimens showed the wild-type sequence. Of 11 specimens studied for translocation t(11;19), 2 cases harbored the translocation. It is concluded that cutaneous hidradenocarcinomas show some microscopic heterogeneity and comprise both low- and high-grade lesions that cytologically are similar to their benign counterpart, the hidradenoma. Within the spectrum of low-grade lesions, there seem to exist tumors almost indistinguishable from hidradenomas but still being capable of regional or distant metastasis. Similar to hidradenomas, hidradenocarcinomas show a t(11;19) translocation, but it is a significantly rarer event. Even rarer is the amplification of the Her2/neu gene. Of note is the relatively low frequency of TP53 mutations despite a high rate of p53 protein expression at the immunohistochemical level.

The spectrum of HNF1A gene mutations in Greek patients with MODY3: relative frequency and identification of seven novel germline mutations.

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Tatsi, Christina; Kanaka-Gantenbein, Christina; Vazeou-Gerassimidi, Adriani; Chrysis, Dionysios; Delis, Dimitrios; Tentolouris, Nikolaos; Dacou-Voutetakis, Catherine; Chrousos, George P; Sertedaki, Amalia

2013-11-01

Maturity-Onset Diabetes of the Young (MODY) is the most common type of monogenic diabetes accounting for 1-2% of the population with diabetes. The relative incidence of HNF1A-MODY (MODY3) is high in European countries; however, data are not available for the Greek population. The aims of this study were to determine the relative frequency of MODY3 in Greece, the type of the mutations observed, and their relation to the phenotype of the patients. Three hundred ninety-five patients were referred to our center because of suspected MODY during a period of 15 yr. The use of Denaturing Gradient Gel Electrophoresis of polymerase chain reaction amplified DNA revealed 72 patients carrying Glucokinase gene mutations (MODY2) and 8 patients carrying HNF1A gene mutations (MODY3). After using strict criteria, 54 patients were selected to be further evaluated by direct sequencing or by multiplex ligation probe amplification (MLPA) for the presence of HNF1A gene mutations. In 16 unrelated patients and 13 of their relatives, 15 mutations were identified in the HNF1A gene. Eight of these mutations were previously reported, whereas seven were novel. Clinical features, such as age of diabetes at diagnosis or severity of hyperglycemia, were not related to the mutation type or location. In our cohort of patients fulfilling strict clinical criteria for MODY, 12% carried an HNF1A gene mutation, suggesting that defects of this gene are responsible for a significant proportion of monogenic diabetes in the Greek population. No clear phenotype-genotype correlations were identified. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

K13 mutations and pfmdr1 copy number variation in Plasmodium falciparum malaria in Myanmar.

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Win, Aye A; Imwong, Mallika; Kyaw, Myat P; Woodrow, Charles J; Chotivanich, Kesinee; Hanboonkunupakarn, Borimas; Pukrittayakamee, Sasithon

2016-02-24

Artemisinin-based combination therapy has been first-line treatment for falciparum malaria in Myanmar since 2005. The wide extent of artemisinin resistance in the Greater Mekong sub-region and the presence of mefloquine resistance at the Myanmar-Thailand border raise concerns over resistance patterns in Myanmar. The availability of molecular markers for resistance to both drugs enables assessment even in remote malaria-endemic areas. A total of 250 dried blood spot samples collected from patients with Plasmodium falciparum malarial infection in five malaria-endemic areas across Myanmar were analysed for kelch 13 sequence (k13) and pfmdr1 copy number variation. K13 mutations in the region corresponding to amino acids 210-726 (including the propeller region of the protein) were detected by nested PCR amplification and sequencing, and pfmdr1 copy number variation by real-time PCR. In two sites, a sub-set of patients were prospectively followed up for assessment of day-3 parasite clearance rates after a standard course of artemether-lumefantrine. K13 mutations and pfmdr1 amplification were successfully analysed in 206 and 218 samples, respectively. Sixty-nine isolates (33.5 %) had mutations within the k13 propeller region with 53 of these (76.8 %) having mutations already known to be associated with artemisinin resistance. F446I (32 isolates) and P574L (15 isolates) were the most common examples. K13 mutation was less common in sites in western border regions (29 of 155 isolates) compared to samples from the east and north (40 of 51 isolates; p Myanmar. There is a low prevalence of parasites with multiple pfmdr1 copies across the country. The efficacy of artemisinin-based combination therapy containing mefloquine and lumefantrine is, therefore, expected to be high, although regular monitoring of efficacy will be important.

Efficient Audio Power Amplification - Challenges

DEFF Research Database (Denmark)

Andersen, Michael Andreas E.

2005-01-01

For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where...

Helicase-dependent amplification of nucleic acids.

Science.gov (United States)

Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

2013-10-11

Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

Refractory versus resistant hypertension: Novel distinctive phenotypes

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Dudenbostel, Tanja; Siddiqui, Mohammed; Gharpure, Nitin; Calhoun, David A.

2017-01-01

Resistant hypertension (RHTN) is relatively common with an estimated prevalence of 10-20% of treated hypertensive patients. It is defined as blood pressure (BP) >140/90 mmHg treated with ≥3 antihypertensive medications, including a diuretic, if tolerated. Refractory hypertension is a novel phenotype of severe antihypertensive treatment failure. The proposed definition for refractory hypertension, i.e. BP >140/90 mmHg with use of ≥5 different antihypertensive medications, including a diuretic and a mineralocorticoid receptor antagonist (MRA) has been applied inconsistently. In comparison to RHTN, refractory hypertension seems to be less prevalent than RHTN. This review focuses on current knowledge about this novel phenotype compared with RHTN including definition, prevalence, mechanisms, characteristics and comorbidities, including cardiovascular risk. In patients with RHTN excess fluid retention is thought to be a common mechanism for the development of RHTN. Recently, evidence has emerged suggesting that refractory hypertension may be more of neurogenic etiology due to increased sympathetic activity as opposed to excess fluid retention. Treatment recommendations for RHTN are generally based on use and intensification of diuretic therapy, especially with the combination of a long-acting thiazide-like diuretic and an MRA. Based on findings from available studies, such an approach does not seem to be a successful strategy to control BP in patients with refractory hypertension and effective sympathetic inhibition in such patients, either with medications and/or device based approaches may be needed. PMID:29034321

Ras mutations are rare in solitary cold and toxic thyroid nodules.

Science.gov (United States)

Krohn, K; Reske, A; Ackermann, F; Müller, A; Paschke, R

2001-08-01

Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

Forming Refractory Insulation On Copper Wire

Science.gov (United States)

Setlock, J.; Roberts, G.

1995-01-01

Alternative insulating process forms flexible coat of uncured refractory insulating material on copper wire. Coated wire formed into coil or other complex shape. Wire-coating apparatus forms "green" coat on copper wire. After wire coiled, heating converts "green" coat to refractory electrical insulator. When cured to final brittle form, insulating material withstands temperatures above melting temperature of wire. Process used to make coils for motors, solenoids, and other electrical devices to be operated at high temperatures.

[Rapid prenatal genetic diagnosis of a fetus with a high risk for Morquio A syndrome].

Science.gov (United States)

Guo, Yi-bin; Ai, Yang; Zhao, Yan; Tang, Jia; Jiang, Wei-ying; Du, Min-lian; Ma, Hua-mei; Zhong, Yan-fang

2012-04-01

To provide rapid and accurate prenatal genetic diagnosis for a fetus with high risk of Morquio A syndrome. Based on ascertained etiology of the proband and genotypes of the parents, particular mutations of the GALNS gene were screened at 10th gestational week with amplification refractory mutation system (ARMS), denaturing high performance liquid chromatography (DHPLC), and direct DNA sequencing. DHPLC screening has identified abnormal double peaks in the PCR products of exons 1 and 10, whilst only a single peak was detected in normal controls. Amplification of ARMS specific primers derived a specific product for the fetus's gene, whilst no similar product was detected in normal controls. Sequencing of PCR products confirmed that exons 1 and 10 of the GALNS gene from the fetus contained a heterozygous paternal c.106-111 del (p.L36-L37 del) deletion and a heterozygous maternal c.1097 T>C (p.L366P) missense mutation, which resulted in a compound heterozygote status. The fetus was diagnosed with Morquio A syndrome and a genotype similar to the proband. Termination of the pregnancy was recommended. Combined ARMS, DHPLC and DNA sequencing are effective for rapid and accurate prenatal diagnosis for fetus with a high risk for Morquio A syndrome. Such methods are particularly suitable for early diagnosis when pathogenesis is clear. Furthermore, combined ARMS and DHPLC are suitable for rapid processing of large numbers of samples for the identification of new mutations.

Osteogenesis imperfecta type 3 in South Africa: Causative mutations in FKBP10

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Alvera Vorster

2017-05-01

Full Text Available Background. A relatively high frequency of autosomal recessively inherited osteogenesis imperfecta (OI type 3 (OI-3 is present in the indigenous black southern African population. Affected persons may be severely handicapped as a result of frequent fractures, progressive deformity of the tubular bones and spinal malalignment. Objective. To delineate the molecular basis for the condition. Methods. Molecular investigations were performed on 91 affected persons from seven diverse ethnolinguistic groups in this population. Results. Following polymerase chain reaction amplification and direct cycle sequencing, FKBP10 mutations were identified in 45.1% (41/91 OI-3-affected persons. The homozygous FKBP10 c.831dupC frameshift mutation was confirmed in 35 affected individuals in the study cohort. Haplotype analysis suggests that this mutation is identical among these OI-3-affected persons by descent, thereby confirming that they had a common ancestor. Compound heterozygosity of this founder mutation was observed, in combination with three different deleterious FKBP10 mutations, in six additional persons in the cohort. Four of these individuals had the c.831delC mutation. Conclusion. The burden of the disorder, both in frequency and severity, warrants the establishment of a dedicated service for molecular diagnostic confirmation and genetic management of persons and families with OI in southern Africa.

High-dose chemotherapy and auto-SCT for relapsed and refractory Hodgkin's lymphoma patients refractory to first-line salvage chemotherapy but responsive to second-line salvage chemotherapy.

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Rauf, Muhammad Shahzad; Maghfoor, Irfan; Elhassan, Tusneem Ahmed M; Akhtar, Saad

2015-01-01

Relapsed or primary refractory Hodgkin's lymphoma (HL) patients refractory to first-line salvage chemotherapy (first salvage) and unable to undergo high-dose chemotherapy (HDC) and autologous stem cell transplant (auto-SCT) have very poor outcome. Some patients are offered second-line salvage chemotherapy (second salvage), if they are responsive and may receive HDC auto-SCT. We identified 31 patients (18 males, 13 females) from 1996-2012 who received second salvage prior to auto-SCT. Median age at auto-SCT is 22 years. Patients were grouped as (1) relapsed-refractory (Rel:Ref): patients with prior complete response (CR) and on relapse found refractory to first salvage and received second salvage and (2) refractory-refractory (Ref:Ref): patients refractory to both primary treatment and first salvage and received second salvage. Median follow-up is 63 months (18-170). Disease status after second salvage prior to HDC was CR 16 %, partial response (PR) 71 % and stable disease 13 %. After HDC auto-SCT, CR:PR: progressive disease was observed in 18 (58 %): four (12 %): nine (29 %) patients, respectively. Five-year overall survival (OS) for whole group is 57 % (Rel:Ref vs. Ref:Ref, 73 % vs. 48 %, p = 0.48). Progression-free survival (PFS) for whole group is 52 % (Rel:Ref vs. Ref:Ref, 73 % vs. 40 % respectively, p = 0.11). Second-line salvage is a valid approach with no long-term side effects for those HL patients who do not respond to first-line salvage chemotherapy and they can be candidate of HDC and stem cell transplant with a high ORR, the PFS and OS in relapse-refractory and refractory-refractory group of patients.

Response to HER2 Inhibition in a Patient With Brain Metastasis With EGFR TKI Acquired Resistance and an HER2 Amplification

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Arenda D. Meedendorp

2018-05-01

Full Text Available A 62-year-old man was referred to our university hospital for treatment of advanced adenocarcinoma of the lung after disease progression on two lines of EGFR TKI and one line of chemotherapy. Fluorescent in situ hybridization analysis upon progression showed an HER2 amplification. At our weekly Molecular Tumor Board (MTB, a decision was made to treat this patient with afatinib, which resulted in a partial response. However, progression was observed with a facial nerve paresis due to a metastasis in the skull. A biopsy of a location in the thorax revealed the presence of an EGFR-T790M mutation associated with acquired resistance, after which treatment with osimertinib was started. After 6 months, disease progression was observed, and a new biopsy was taken from the pelvic bone, which revealed the original amplification of HER2 together with the EGFR-L858R mutation, the EGFR-T790M mutation was not detected. The MTB decided to treat the patient with trastuzumab/paclitaxel. A partial response was observed in different bone lesions, while the skull metastasis with ingrowth in the brain remained stable for 6 months. Because of progression of the bone metastases after 6 months, a biopsy of a lesion in the thorax wall was taken. In this lesion, the EGFR-T790M mutation could be detected again. The MTB advised to start treatment with a combination of osimertinib and afatinib. This resulted in an impressive clinical improvement and a partial response of the bone metastases on the most recent 18-fluorodeoxyglucose positron emission tomography and computer tomography-scan. In conclusion, adjusting treatment to the mutational make-up of the tumor is a great challenge. For optimal treatment response multiple biopsies and re-biopsy upon progression are imperative. As more genes are investigated, treatment decision becomes increasingly difficult, therefore, expert opinions from an MTB is essential.

Refractory myasthenia gravis - clinical profile, comorbidities and response to rituximab.

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Sudulagunta, Sreenivasa Rao; Sepehrar, Mona; Sodalagunta, Mahesh Babu; Settikere Nataraju, Aravinda; Bangalore Raja, Shiva Kumar; Sathyanarayana, Deepak; Gummadi, Siddharth; Burra, Hemanth Kumar

2016-01-01

Introduction: Myasthenia gravis (MG) is an antibody mediated autoimmune neuromuscular disorder characterized by fatigable muscle weakness. A proportion of myasthenia gravis patients are classified as refractory due to non responsiveness to conventional treatment. This retrospective study was done to evaluate clinical profile, epidemiological, laboratory, and features of patients with MG and mode of management using rituximab and complications. Methods: Data of myasthenia gravis patients admitted or presented to outpatient department (previous medical records) with MG between January 2008 and January 2016 were included. A total of 512 patients fulfilled the clinical and diagnostic criteria of myasthenia gravis of which 76 patients met the diagnostic certainty for refractory myasthenia gravis and were evaluated. Results: Out of 76 refractory MG patients, 53 (69.73%) patients fulfilled all the three defined criteria. The median age of onset of the refractory MG group was 36 years with a range of 27-53 years. In our study 25 patients (32.89%) belonged to the age group of 21-30 years. Anti-MuSK antibodies were positive in 8 non-refractory MG patients (2.06%) and 36 refractory MG patients (47.36%). Mean HbA 1C was found to be 8.6±2.33. The dose of administered prednisone decreased by a mean of 59.7% ( p =3.3x10 -8 ) to 94.6% ( p =2.2x10 -14 ) after the third cycle of rituximab treatment. Conclusion: The refractory MG patients are most commonly female with an early age of onset, anti-MuSK antibodies, and thymomas. Refractory MG patients have higher prevalence and poor control (HbA 1C >8%) of diabetes mellitus and dyslipidemia probably due to increased steroid usage. Rituximab is very efficient in treatment of refractory MG with adverse effects being low.

RESULTS OF OUTPATIENT PROGRAM ON EFFECTIVE THERAPY OF REFRACTORY ARTERIAL HYPERTENSION

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M. M. Batyushin

2015-12-01

Full Text Available Aim. To increase in efficacy of antihypertensive therapy in patients with refractory arterial hypertension (HT.Material and methods. Patients with refractory HT were revealed during first month of program. The causes of refractory HT were analyzed. Combined antihypertensive therapy was prescribed to reach target level of blood pressure (BP. This therapy lasted 24 weeks and included angiotensin converting enzyme (ACE inhibitor, thiazid diuretic (indapamide and dihydropyridine calcium antagonist (nifedipine XL.Results. 200 patients with refractory HT were revealed. True refractory HT took place in 59,9% of patients and pseudo refractory HT – in 40,1% of patients. Lack of diuretics or combined antihypertensive therapy were the main reason of insufficient BP control. Proposed 3-drugs therapy resulted in reduction of systolic BP from 190 to 132 Hg mm and diastolic BP from 104 to 81 Hg mm. Target level of BP was reached in 94% patients. There were no side effects which demanded to stop therapy.Conclusion. High incidence of pseudorefractory HT (40,1% is revealed. Significant prevalence of renal disturbances especially chronic interstitial inflammatory could be responsible for refractory HT development. Use of 3-drugs therapy (ACE inhibitor, indapamide and nifedipine XL provides effective control of BP in refractory and pseudorefractory HT.

RESULTS OF OUTPATIENT PROGRAM ON EFFECTIVE THERAPY OF REFRACTORY ARTERIAL HYPERTENSION

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M. M. Batyushin

2007-01-01

Full Text Available Aim. To increase in efficacy of antihypertensive therapy in patients with refractory arterial hypertension (HT.Material and methods. Patients with refractory HT were revealed during first month of program. The causes of refractory HT were analyzed. Combined antihypertensive therapy was prescribed to reach target level of blood pressure (BP. This therapy lasted 24 weeks and included angiotensin converting enzyme (ACE inhibitor, thiazid diuretic (indapamide and dihydropyridine calcium antagonist (nifedipine XL.Results. 200 patients with refractory HT were revealed. True refractory HT took place in 59,9% of patients and pseudo refractory HT – in 40,1% of patients. Lack of diuretics or combined antihypertensive therapy were the main reason of insufficient BP control. Proposed 3-drugs therapy resulted in reduction of systolic BP from 190 to 132 Hg mm and diastolic BP from 104 to 81 Hg mm. Target level of BP was reached in 94% patients. There were no side effects which demanded to stop therapy.Conclusion. High incidence of pseudorefractory HT (40,1% is revealed. Significant prevalence of renal disturbances especially chronic interstitial inflammatory could be responsible for refractory HT development. Use of 3-drugs therapy (ACE inhibitor, indapamide and nifedipine XL provides effective control of BP in refractory and pseudorefractory HT.

Heterozygous M1V variant of ELA-2 gene mutation associated with G-CSF refractory severe congenital neutropenia.

Science.gov (United States)

Setty, Bhuvana A; Yeager, Nicholas D; Bajwa, Rajinder P

2011-09-01

Severe congenital neutropenia is an autosomal recessive disorder characterized by maturation arrest at the promyelocyte/myelocyte phase in the bone marrow, absolute neutrophil count ELA-2 have been described. We report the case of a premature male infant with congenital neutropenia, associated with multiple infections, refractory to treatment with granulocyte colony stimulating factor who subsequently underwent matched sibling donor stem-cell transplant. He was found to be heterozygous for the M1V variant of the ELA-2 gene that we postulate to be causative for his severe neutropenia Copyright © 2011 Wiley-Liss, Inc.

SSR allelic variation of rice variety Hangxiangnuo bred by space mutation

International Nuclear Information System (INIS)

Yang Tifeng; Liu Chuanguang; Pan Dajian; Fan Zhilan; Li Chen; Chen Jianyou; Liu Bin; Jiang Yijun; Gao Yun; Zhou Hanqin

2011-01-01

Hangxiangnuo, an indica fragrant glutinous rice mutant, was induced by space environment. Comparing with its wild type Nanfengnuo, the yield and blast resistance of Hangxiangnuo are improved significantly and the grain shape became slender and with fragrance. To understand the mechanisms of space mutation and identify the changes at molecular level associated with phenotypic variations, SSR allelic variation analysis were performed on Hangxiangnuo and Nanfengnuo in this study. The results showed that 45 loci were polymorphic among the 156 SSR loci tested throughout the genome, the frequency of variation was 28.85%. Among the polymorphic loci, 42 loci only showed variations in the molecular weight of the amplified bands, only on locus increased the number of amplification bands in Hangxiangnuo and two loci were differed by heterozygous loci (with two amplification bands at one locus) detected in Nanfengnuo and homozygous loci in Hangxiangnuo. It suggests that the change of some loci in mutants was due to the normal segregation and recombination of heterozygous loci of the wild type. The variation frequencies among different chromosomes were quite different, with the highest one at 50.00% detected on chromosomes 7, 8 and 12, and the lowest at 6.25% on chromosome 6. The polymorphic loci were clustered on chromosomes throughout the genome indicating that large DNA segments mutation is one of the major variation patterns induced by space environment. Some of reported QTLs involved in grain shape, yield, fragrance and blast resistance were found to be located exactly in the mutated regions. Therefore, further study is needed to confirm that these QTLs are responsible for the trait variations. (authors)

Bone marrow immunophenotyping by flow cytometry in refractory cytopenia of childhood

NARCIS (Netherlands)

A.M. Aalbers (Anna Maartje); M.M. van den Heuvel-Eibrink (Marry); I. Baumann (Irith); M.N. Dworzak (Michael); H. Hasle (Henrik); F. Locatelli (Franco); B. de Moerloose (Barbara); M. Schmugge; E. Mejstříková (Ester); M. Nováková (Michaela); M. Zecca (Marco); C.M. Zwaan (Christian Michel); J.G. te Marvelde (Jeroen); A.W. Langerak (Anton); J.J.M. van Dongen (Jacques); R. Pieters (Rob); C.M. Niemeyer (Charlotte); V.H.J. van der Velden (Vincent)

2015-01-01

textabstractRefractory cytopenia of childhood is the most common type of childhood myelodysplastic syndrome. Because the majority of children with refractory cytopenia have a normal karyotype and a hypocellular bone marrow, differentiating refractory cytopenia from the immune-mediated bone marrow

Screening of Duchenne muscular dystrophy (DMD mutations and investigating its mutational mechanism in Chinese patients.

Directory of Open Access Journals (Sweden)

Chen Chen

Full Text Available Duchenne muscular dystrophy (DMD is a common X-linked recessive disease of muscle degeneration and death. In order to provide accurate and reliable genetic counseling and prenatal diagnosis, we screened DMD mutations in a cohort of 119 Chinese patients using multiplex ligation-dependent probe amplification (MLPA and denaturing high performance liquid chromatography (DHPLC followed by Sanger sequencing. In these unrelated DMD patients, we identified 11 patients with DMD small mutations (9.2% and 81 patients with DMD deletions/duplications (del/dup (68.1%, of which 64 (79.0% were deletions, 16 (19.8% were duplications, and one (1.2% was both deletion and duplication. Furthermore, we analyzed the frequency of DMD breakpoint in the 64 deletion cases by calculating exon-deletion events of certain exon interval that revealed a novel mutation hotspot boundary. To explore why DMD rearrangement breakpoints were predisposed to specific regions (hotspot, we precisely characterized junction sequences of breakpoints at the nucleotide level in 21 patients with exon deleted/duplicated in DMD with a high-resolution SNP microarray assay. There were no exactly recurrent breakpoints and there was also no significant difference between single-exon del/dup and multiple-exon del/dup cases. The data from the current study provided a comprehensive strategy to detect DMD mutations for clinical practice, and identified two deletion hotspots at exon 43-55 and exon 10-23 by calculating exon-deletion events of certain exon interval. Furthermore, this is the first study to characterize DMD breakpoint at the nucleotide level in a Chinese population. Our observations provide better understanding of the mechanism for DMD gene rearrangements.

Efficient audio power amplification - challenges

Energy Technology Data Exchange (ETDEWEB)

Andersen, Michael A.E.

2005-07-01

For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where extensive research and development are needed is covered. (au)

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants.

Science.gov (United States)

Haghshenas, Maryam; Akbari, Mohammad Taghi; Karizi, Shohreh Zare; Deilamani, Faravareh Khordadpoor; Nafissi, Shahriar; Salehi, Zivar

2016-06-01

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50-79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.

Retinal phenotype-genotype correlation of pediatric patients expressing mutations in the Norrie disease gene.

Science.gov (United States)

Wu, Wei-Chi; Drenser, Kimberly; Trese, Michael; Capone, Antonio; Dailey, Wendy

2007-02-01

To correlate the ophthalmic findings of patients with pediatric vitreoretinopathies with mutations occurring in the Norrie disease gene (NDP). One hundred nine subjects with diverse pediatric vitreoretinopathies and 54 control subjects were enrolled in the study. Diagnoses were based on retinal findings at each patient's first examination. Samples of DNA from each patient underwent polymerase chain reaction amplification and direct sequencing of the NDP gene. Eleven male patients expressing mutations in the NDP gene were identified in the test group, whereas the controls demonstrated wild-type NDP. All patients diagnosed as having Norrie disease had mutations in the NDP gene. Four of the patients with Norrie disease had mutations involving a cysteine residue in the cysteine-knot motif. Four patients diagnosed as having familial exudative vitreoretinopathy were found to have noncysteine mutations. One patient with retinopathy of prematurity had a 14-base deletion in the 5' untranslated region (exon 1), and 1 patient with bilateral persistent fetal vasculature syndrome expressed a noncysteine mutation in the second exon. Mutations disrupting the cysteine-knot motif corresponded to severe retinal dysgenesis, whereas patients with noncysteine mutations had varying degrees of avascular peripheral retina, extraretinal vasculature, and subretinal exudate. Patients exhibiting severe retinal dysgenesis should be suspected of carrying a mutation that disrupts the cysteine-knot motif in the NDP gene.

The clinical phenotype of Lynch syndrome due to germ-line PMS2 mutations.

Science.gov (United States)

Senter, Leigha; Clendenning, Mark; Sotamaa, Kaisa; Hampel, Heather; Green, Jane; Potter, John D; Lindblom, Annika; Lagerstedt, Kristina; Thibodeau, Stephen N; Lindor, Noralane M; Young, Joanne; Winship, Ingrid; Dowty, James G; White, Darren M; Hopper, John L; Baglietto, Laura; Jenkins, Mark A; de la Chapelle, Albert

2008-08-01

Although the clinical phenotype of Lynch syndrome (also known as hereditary nonpolyposis colorectal cancer) has been well described, little is known about disease in PMS2 mutation carriers. Now that mutation detection methods can discern mutations in PMS2 from mutations in its pseudogenes, more mutation carriers have been identified. Information about the clinical significance of PMS2 mutations is crucial for appropriate counseling. Here, we report the clinical characteristics of a large series of PMS2 mutation carriers. We performed PMS2 mutation analysis using long-range polymerase chain reaction and multiplex ligation-dependent probe amplification for 99 probands diagnosed with Lynch syndrome-associated tumors showing isolated loss of PMS2 by immunohistochemistry. Penetrance was calculated using a modified segregation analysis adjusting for ascertainment. Germ-line PMS2 mutations were detected in 62% of probands (n = 55 monoallelic; 6 biallelic). Among families with monoallelic PMS2 mutations, 65.5% met revised Bethesda guidelines. Compared with the general population, in mutation carriers, the incidence of colorectal cancer was 5.2-fold higher, and the incidence of endometrial cancer was 7.5-fold higher. In North America, this translates to a cumulative cancer risk to age 70 years of 15%-20% for colorectal cancer, 15% for endometrial cancer, and 25%-32% for any Lynch syndrome-associated cancer. No elevated risk for non-Lynch syndrome-associated cancers was observed. PMS2 mutations contribute significantly to Lynch syndrome, but the penetrance for monoallelic mutation carriers appears to be lower than that for the other mismatch repair genes. Modified counseling and cancer surveillance guidelines for PMS2 mutation carriers are proposed.

Single base pair mutation analysis by PNA directed PCR clamping

DEFF Research Database (Denmark)

Ørum, H.; Nielsen, P.E.; Egholm, M.

1993-01-01

A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity...... allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers....

Colectomy for refractory constipation

DEFF Research Database (Denmark)

Raahave, Dennis; Loud, Franck Bjørn; Christensen, Elsebeth

2010-01-01

OBJECTIVE: This study evaluated the type of colectomy, postoperative complications, functional results, and satisfaction in patients with constipation refractory to conservative therapy. Further, colonic transit time (CTT), faecal load (coprostasis), and colon length (redundancies) were compared ...

Statistical experimental design for refractory coatings

International Nuclear Information System (INIS)

McKinnon, J.A.; Standard, O.C.

2000-01-01

The production of refractory coatings on metal casting moulds is critically dependent on the development of suitable rheological characteristics, such as viscosity and thixotropy, in the initial coating slurry. In this paper, the basic concepts of mixture design and analysis are applied to the formulation of a refractory coating, with illustration by a worked example. Experimental data of coating viscosity versus composition are fitted to a statistical model to obtain a reliable method of predicting the optimal formulation of the coating. Copyright (2000) The Australian Ceramic Society

Typical corrosion of alumina refractory in aluminum reflow oven

International Nuclear Information System (INIS)

Baldo, Jaoa B.

2014-01-01

The refractory linings of furnaces for secondary melting of aluminum, are exposed to intense attack by the molten metal. This occurs, because molten aluminum has a strong reducing power over the refractory oxide components, particularly Fe 2 O 3 , SiO 2 and TiO 2 . In this work, based on X-ray diffraction and scanning electron microscopy, it is presented a post mortem study of the mechanisms that lead to a premature wear of a 80% Al2O3 chemically bonded refractory bricks, used in the metal line of an aluminum re-melting furnace. The SEM analysis demonstrated that the oxides SiO 2 and TiO 2 contained in the refractory were reduced and transformed into their metallic elements causing an intense structural spalling. (author)

Neonatal Marfan Syndrome: Report of a Case with an Inherited Splicing Mutation outside the Neonatal Domain.

Science.gov (United States)

Le Gloan, Laurianne; Hauet, Quentin; David, Albert; Hanna, Nadine; Arfeuille, Chloé; Arnaud, Pauline; Boileau, Catherine; Romefort, Bénédicte; Benbrik, Nadir; Gournay, Véronique; Joram, Nicolas; Baron, Olivier; Isidor, Bertrand

2016-02-01

We report a child and her mother affected by Marfan syndrome. The child presented with a phenotype of neonatal Marfan syndrome, revealed by acute and refractory heart failure, finally leading to death within the first 4 months of life. Her mother had a common clinical presentation. Genetic analysis revealed an inherited FBN1 mutation. This intronic mutation (c.6163+3_6163+6del), undescribed to date, leads to exon 49 skipping, corresponding to in-frame deletion of 42 amino acids (p.Ile2014_Asp2055del). FBN1 next-generation sequencing did not show any argument for mosaicism. Association in the same family of severe neonatal and classical Marfan syndrome illustrates the intrafamilial phenotype variability.

Beta-blockers in cirrhosis and refractory ascites

DEFF Research Database (Denmark)

Kimer, Nina; Feineis, Martin; Møller, Søren

2015-01-01

OBJECTIVE: It is currently discussed if beta-blockers exert harmful effects and increase mortality in patients with cirrhosis and refractory ascites. In this study, we provide an overview of the available literature in this field in combination with a retrospective analysis of 61 patients...... trials (9 trials on propranolol, 1 case-control study and 4 retrospective analyses) were identified. One trial suggested an increased mortality in patients treated with beta-blockers and refractory ascites. The results of the remaining trials were inconclusive. No increase in mortality among beta-blocker......-treated patients was found in the present retrospective analysis. CONCLUSIONS: Treatment with beta-blockers may increase mortality in patients with cirrhosis and refractory ascites. However, the current evidence is sparse and high-quality studies are warranted to clarify the matter....

Identification of a Non-Gatekeeper Hot Spot for Drug-Resistant Mutations in mTOR Kinase.

Science.gov (United States)

Wu, Tzung-Ju; Wang, Xiaowen; Zhang, Yanjie; Meng, Linghua; Kerrigan, John E; Burley, Stephen K; Zheng, X F Steven

2015-04-21

Protein kinases are therapeutic targets for human cancer. However, "gatekeeper" mutations in tyrosine kinases cause acquired clinical resistance, limiting long-term treatment benefits. mTOR is a key cancer driver and drug target. Numerous small-molecule mTOR kinase inhibitors have been developed, with some already in human clinical trials. Given our clinical experience with targeted therapeutics, acquired drug resistance in mTOR is thought likely, but not yet documented. Herein, we describe identification of a hot spot (L2185) for drug-resistant mutations, which is distinct from the gatekeeper site, and a chemical scaffold refractory to drug-resistant mutations. We also provide new insights into mTOR kinase structure and function. The hot spot mutations are potentially useful as surrogate biomarkers for acquired drug resistance in ongoing clinical trials and future treatments and for the design of the next generation of mTOR-targeted drugs. Our study provides a foundation for further research into mTOR kinase function and targeting. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Fire and explosion hazards of refractory compound powders

International Nuclear Information System (INIS)

Krivtsov, V.A.; Kostina, E.S.

1978-01-01

Data on fire and explosion hazards of refractory compound powders (HfC, ZrC,LaB 6 , ZrN, etc.) are presented. It is shown that refractory compounds can be fire- and exposion hazardous in various degrees. Qualitative and quantitative estimations of one of fire-hazard characteristics - smoldering temperature - are presented

Acquired resistance mechanisms to tyrosine kinase inhibitors in lung cancer with activating epidermal growth factor receptor mutation--diversity, ductility, and destiny.

Science.gov (United States)

Suda, Kenichi; Mizuuchi, Hiroshi; Maehara, Yoshihiko; Mitsudomi, Tetsuya

2012-12-01

Lung cancers that harbor somatic activating mutations in the gene for the epidermal growth factor receptor (EGFR) depend on mutant EGFR for their proliferation and survival; therefore, lung cancer patients with EGFR mutations often dramatically respond to orally available EGFR tyrosine kinase inhibitors (TKIs). However, emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. To elucidate and overcome this acquired resistance, multidisciplinary basic and clinical investigational approaches have been applied, using in vitro cell line models or samples obtained from lung cancer patients treated with EGFR-TKIs. These efforts have revealed several acquired resistance mechanisms and candidates, including EGFR secondary mutations (T790M and other rare mutations), MET amplification, PTEN downregulation, CRKL amplification, high-level HGF expression, FAS-NFκB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer. Interestingly, cancer cells harbor potential destiny and ductility together in acquiring resistance to EGFR-TKIs, as shown in in vitro acquired resistance models. Molecular mechanisms of "reversible EGFR-TKI tolerance" that occur in early phase EGFR-TKI exposure have been identified in cell line models. Furthermore, others have reported molecular markers that can predict response to EGFR-TKIs in clinical settings. Deeper understanding of acquired resistance mechanisms to EGFR-TKIs, followed by the development of molecular target drugs that can overcome the resistance, might turn this fatal disease into a chronic disorder.

Missense Mutation in Fam83H Gene in Iranian Patients with Amelogenesis Imperfecta.

Science.gov (United States)

Pourhashemi, S Jalal; Ghandehari Motlagh, Mehdi; Meighani, Ghasem; Ebrahimi Takaloo, Azadeh; Mansouri, Mahsa; Mohandes, Fatemeh; Mirzaii, Maryam; Khoshzaban, Ahad; Moshtaghi, Faranak; Abedkhojasteh, Hoda; Heidari, Mansour

2014-12-01

Amelogenesis Imperfecta (AI) is a disorder of tooth development where there is an abnormal formation of enamel or the external layer of teeth. The aim of this study was to screen mutations in the four most important candidate genes, ENAM, KLK4, MMP20 and FAM83H responsible for amelogenesis imperfect. Geneomic DNA was isolated from five Iranian families with 22 members affected with enamel malformations. The PCR amplifications were typically carried out for amplification the coding regions for AI patients and unaffected family members. The PCR products were subjected to direct sequencing. The pedigree analysis was performed using Cyrillic software. One family had four affected members with autosomal dominant hypocalcified amelogenesis imperfecta (ADHPCAI); pedigree analysis revealed four consanguineous families with 18 patients with autosomal recessive hypoplastic amelogenesis imperfecta (ARHPAI). One non-synonymous single-nucleotide substitution, c.1150T>A, p. Ser 342Thr was identified in the FAM83H, which resulted in ADHCAI. Furthermore, different polymorphisms or unclassified variants were detected in MMP20, ENAM and KLK4. Our results are consistent with other studies and provide further evidence for pathogenic mutations of FAM83H gene. These findings suggest different loci and genes could be implicated in the pathogenesis of AI.

Amplification and sequencing of varicella zoster virus (VZV) gene 4: point mutation in a VZV strain causing chickenpox during pregnancy

International Nuclear Information System (INIS)

Chow, V.T.K.; Lim, K.P.

1997-01-01

The varicella-zoster virus (VZV) causes chickenpox (varicella) as the primary disease and shingles (zoster) as a recurrent manifestation of infection, both being generality benign and self-limiting. While these infections may be severe in adults and even life-threatening in immunosuppressed individuals, they may be amenable to effective antiviral drugs or varicella-zoster immune globulin, provided the treatment is administered early. The prompt diagnosis of VZV infections may be accelerated by rapid, sensitive and specific molecular techniques such as amplification by polymerase chain reaction (PCR) compared with slower and more cumbersome tissue culture and serological procedures. Based on the VZV gene 4 which encodes a transcriptional activator, primers were designed for use in PCR to amplify a target fragment of 381 bp. Distinct diagnostic bands were observed by agarose gel electrophoresis of PCR products of VZV strains isolated from II varicella and 7 zoster patients in Singapore, as well as of the Japanese vaccine Oka strain. The detection sensitivity of this PCR assay was determined to be 1 pg of purified VZV DNA equivalent to about 7,000 viral DNA copies. No target bands were amplified from negative control templates from five related human herpes-viruses and from human DNA. The specificity of the PCR products was ensured by direct cycle DNA sequencing, which revealed complete identity of the 18 VZV isolates with the published European Dumas strain. The strong sequence conservation of the target fragment renders this PCR assay highly reliable for detecting the VZV sequence. Only one VZV strain isolated from a patient with varicella during pregnancy exhibited a Gaga to GAA point mutation at codon 46 of gene 4, culminating in the non-conservative substitution of Ser with Phe. The predicted secondary structure of the mutant polypeptide portrayed a radical alteration, which may influence its function in transcriptional activation. (authors)

Inflammatory peeling skin syndrome caused a novel mutation in CDSN.

Science.gov (United States)

Telem, Dana Fuchs; Israeli, Shirli; Sarig, Ofer; Sprecher, Eli

2012-04-01

Generalized peeling skin syndrome (PSS) is a rare autosomal recessive dermatosis manifesting with continuous exfoliation of the stratum corneum. The inflammatory (type B) subtype of PSS was recently found to be caused by deleterious mutations in the CDSN gene encoding corneodesmosin, a major component of desmosomal junctions in the uppermost layers of the epidermis. In the present study, we assessed a 10-month-old baby, who presented with generalized superficial peeling of the skin. Using PCR amplification and direct sequencing, we identified the third PSS-associated mutation in CDSN, a homozygous 4 bp duplication in the second exon of the gene (c.164_167dup GCCT; p.Thr57ProfsX6). These data further support the notion that corneodesmosin deficiency impairs cell-cell adhesion in the upper epidermis, paving the way for an abnormal inflammatory response due to epidermal barrier disruption.

Gefitinib inhibits invasive phenotype and epithelial-mesenchymal transition in drug-resistant NSCLC cells with MET amplification.

Directory of Open Access Journals (Sweden)

Silvia La Monica

Full Text Available Despite the initial response, all patients with epidermal growth factor receptor (EGFR-mutant non-small cell lung cancer (NSCLC eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs. The EGFR-T790M secondary mutation is responsible for half of acquired resistance cases, while MET amplification has been associated with acquired resistance in about 5-15% of NSCLCs. Clinical findings indicate the retained addiction of resistant tumors on EGFR signaling. Therefore, we evaluated the molecular mechanisms supporting the therapeutic potential of gefitinib maintenance in the HCC827 GR5 NSCLC cell line harbouring MET amplification as acquired resistance mechanism. We demonstrated that resistant cells can proliferate and survive regardless of the presence of gefitinib, whereas the absence of the drug significantly enhanced cell migration and invasion. Moreover, the continuous exposure to gefitinib prevented the epithelial-mesenchymal transition (EMT with increased E-cadherin expression and down-regulation of vimentin and N-cadherin. Importantly, the inhibition of cellular migration was correlated with the suppression of EGFR-dependent Src, STAT5 and p38 signaling as assessed by a specific kinase array, western blot analysis and silencing functional studies. On the contrary, the lack of effect of gefitinib on EGFR phosphorylation in the H1975 cells (EGFR-T790M correlated with the absence of effects on cell migration and invasion. In conclusion, our findings suggest that certain EGFR-mutated patients may still benefit from a second-line therapy including gefitinib based on the specific mechanism underlying tumor cell resistance.

Gypsy Phenylketonuria: A point mutation of the phenylalanine hydroxylase gene in Gypsy families from Slovakia

Energy Technology Data Exchange (ETDEWEB)

Kalanin, J. [Institute for Clinical and Experical Medicine, Praha (Czechoslovakia); Takarada, Y. [Toyobo Research Center, Shiga (Japan); Kagawa, S.; Yamashita, K.; Ohtsuka, N.; Matsuoka, A. [Hyogo College of Medicine, Nishinomiya (Japan)

1994-01-15

A direct mutational analysis of the phenylalanine hydroxylase gene (PAH) in Gypsy families with phenylketonuria (PKU) has not yet been presented. However, they obviously represent a group at high risk for this inherited disease. The authors analyzed the PAH loci of 65 Gypsies originating from Eastern Slovakia by a combination of PCR amplification, direct sequencing and ASO hybridization. These studies uncovered 10 {open_quotes}classical PKU{close_quotes} patients to be homozygous for a R252W (CGG-TGG) transition, and 29 heterozygous carriers of this mutation. Fifteen control Caucasoid PKU patients from the Czech and Slovak Republics were selected. In this group they detected R252W mutation in two subjects (6.67% of all mutant alleles). Both were compound heterozygous for two different mutations. Previous haplotype studies of Welsh Gypsies with PKU were uninformative in the determination of heterozygosity. ASO hybridization served effectively for the consequent analyses in Gypsy PKU-related families and to identify the carriers among the unrelated subjects. 19 refs., 2 figs.

Molecular characterization and diversity of a novel non-autonomous mutator-like transposon family in brassica

International Nuclear Information System (INIS)

Nouroz, F.

2015-01-01

Transposable elements (TEs) are capable of mobilizing from one genomic location to other, with changes in their copy numbers. Mutator-like elements (MULEs) are DNA transposons characterized by 9 bp target site duplications (TSDs), with high variability in sequence and length, and include non-conserved terminal inverted repeats (TIRs). We identified and characterized a family of Mutator-like elements designated as Shahroz. The structural and molecular analyses revealed that family had a small number of mostly defective non-autonomous MULEs and has shown limited activity in the evolutionary history of the Brassica A-genome. The Shahroz elements range in size from 2734 to 3160 bp including 76 bp imperfect TIRs and 9 bp variable TSDs. The individual copies have shown high homology (52-99%) in their entire lengths. The study revealed that the elements are less in numbers but active in Brassica rapa genomes and PCR amplification revealed their specificity and amplification in A-genome containing diploid and polyploids Brassica. The phylogenetic analysis of Brassica MULEs with other plant Mutator elements revealed that no correlation exists between Brassica MULEs and other elements suggesting a separate line of evolution. Analyzing the regions flanking the insertions revealed that the insertions have showed a preference for AT rich regions. The detailed study of these insertions revealed that although less in number and small sizes, they have played a role in Brassica genome evolution by their mobilization. (author)

Immunomodulatory therapy in refractory/recurrent ovarian cancer.

Science.gov (United States)

Chen, Chao-Yu; Lai, Chyong-Huey; Yang, Lan-Yan; Tang, Yun-Hsin; Chou, Hung-Hsueh; Chang, Chee-Jen; Lin, Cheng-Tao

2015-04-01

To investigate the efficacy and toxicity of immunomodulatory therapy (IMT) alone or as an add-on to palliative/salvage chemotherapy in patients with refractory/recurrent epithelial ovarian cancer (EOC). We retrospectively analyzed the efficacy and toxicity of IMT in 15 patients with refractory/recurrent EOC who had previously received multiple chemotherapy regimens. The median age of the patients was 56 years (range, 41-75 years). Three patients were platinum-sensitive, two were platinum-resistant, and the remaining 10 patients were refractory to platinum-based front-line chemotherapy. IMT consisted of picibanil (OK-432) on Day 1, interleukin-2 and/or interferon-α on Day 2 administered by subcutaneous injection (every week or 2-weekly). Five patients never received metronomic oral cyclophosphamide. After IMT, three patients achieved partial remission (PR, lasting for 11 months, ≥ 12 months, and 16 months), and six patients had stable disease (SD). The disease stabilizing rate (PR+SD) was 60% (3/3 in platinum-sensitive and 6/12 in platinum-resistant/refractory patients). The absolute lymphocyte count (ALC) at 1 month after IMT was significantly higher in the PR+SD group (median 1242.0/μL) than in the progression group (median 325.0/μL) (p = 0.012). No ≥ Grade 3 toxicities were observed. The median post-IMT survival time was 12 months (range, 2-39 months). IMT alone or add-on to palliative/salvage chemotherapy for refractory/recurrent EOC achieves a substantial disease stabilizing rate without severe toxicity, which might be a potential option in selected patients. The ALC 1 month after IMT could be an early indicator to disease stabilization. Copyright © 2015. Published by Elsevier B.V.

A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation.

Science.gov (United States)

Ponder, Rebecca G; Fonville, Natalie C; Rosenberg, Susan M

2005-09-16

Special mechanisms of mutation are induced in microbes under growth-limiting stress causing genetic instability, including occasional adaptive mutations that may speed evolution. Both the mutation mechanisms and their control by stress have remained elusive. We provide evidence that the molecular basis for stress-induced mutagenesis in an E. coli model is error-prone DNA double-strand break repair (DSBR). I-SceI-endonuclease-induced DSBs strongly activate stress-induced mutations near the DSB, but not globally. The same proteins are required as for cells without induced DSBs: DSBR proteins, DinB-error-prone polymerase, and the RpoS starvation-stress-response regulator. Mutation is promoted by homology between cut and uncut DNA molecules, supporting a homology-mediated DSBR mechanism. DSBs also promote gene amplification. Finally, DSBs activate mutation only during stationary phase/starvation but will during exponential growth if RpoS is expressed. Our findings reveal an RpoS-controlled switch from high-fidelity to mutagenic DSBR under stress. This limits genetic instability both in time and to localized genome regions, potentially important evolutionary strategies.

Key clinical features to identify girls with CDKL5 mutations.

Science.gov (United States)

Bahi-Buisson, Nadia; Nectoux, Juliette; Rosas-Vargas, Haydeé; Milh, Mathieu; Boddaert, Nathalie; Girard, Benoit; Cances, Claude; Ville, Dorothée; Afenjar, Alexandra; Rio, Marlène; Héron, Delphine; N'guyen Morel, Marie Ange; Arzimanoglou, Alexis; Philippe, Christophe; Jonveaux, Philippe; Chelly, Jamel; Bienvenu, Thierry

2008-10-01

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause infantile spasms as well as Rett syndrome (RTT)-like phenotype. To date, less than 25 different mutations have been reported. So far, there are still little data on the key clinical diagnosis criteria and on the natural history of CDKL5-associated encephalopathy. We screened the entire coding region of CDKL5 for mutations in 183 females with encephalopathy with early seizures by denaturing high liquid performance chromatography and direct sequencing, and we identified in 20 unrelated girls, 18 different mutations including 7 novel mutations. These mutations were identified in eight patients with encephalopathy with RTT-like features, five with infantile spasms and seven with encephalopathy with refractory epilepsy. Early epilepsy with normal interictal EEG and severe hypotonia are the key clinical features in identifying patients likely to have CDKL5 mutations. Our study also indicates that these patients clearly exhibit some RTT features such as deceleration of head growth, stereotypies and hand apraxia and that these RTT features become more evident in older and ambulatory patients. However, some RTT signs are clearly absent such as the so called RTT disease profile (period of nearly normal development followed by regression with loss of acquired fine finger skill in early childhood and characteristic intensive eye communication) and the characteristic evolution of the RTT electroencephalogram. Interestingly, in addition to the overall stereotypical symptomatology (age of onset and evolution of the disease) resulting from CDKL5 mutations, atypical forms of CDKL5-related conditions have also been observed. Our data suggest that phenotypic heterogeneity does not correlate with the nature or the position of the mutations or with the pattern of X-chromosome inactivation, but most probably with the functional transcriptional and/or translational consequences of CDKL5

Prevalence and clinical association of MET gene overexpression and amplification in patients with NSCLC: Results from the European Thoracic Oncology Platform (ETOP) Lungscape project.

Science.gov (United States)

Bubendorf, Lukas; Dafni, Urania; Schöbel, Martin; Finn, Stephen P; Tischler, Verena; Sejda, Aleksandra; Marchetti, Antonio; Thunnissen, Erik; Verbeken, Eric K; Warth, Arne; Sansano, Irene; Cheney, Richard; Speel, Ernst Jan M; Nonaka, Daisuke; Monkhorst, Kim; Hager, Henrik; Martorell, Miguel; Savic, Spasenija; Kerr, Keith M; Tan, Qiang; Tsourti, Zoi; Geiger, Thomas R; Kammler, Roswitha; Schulze, Katja; Das-Gupta, Ashis; Shames, David; Peters, Solange; Stahel, Rolf A

2017-09-01

In a well-defined NSCLC cohort of the ETOP Lungscape program, we explored the epidemiology of IHC MET overexpression and amplification, their inter-correlation, and their association to outcome. Resected NSCLC were assessed for MET gene copy number (GCN) and expression using silver in-situ hybridization (SISH) and immunohistochemistry (IHC) on TMAs in a multicenter setting. MET amplification was defined as MET/centromere ratio≥2 (with average MET GCN≥4), high MET GCN as CGN≥5 and MET IHC+ as ≥2+ intensity in ≥50% of tumor cells. A total of 182 MET IHC+ and EGFR/KRAS WT tumors were analyzed for METex14 skipping mutation. MET IHC+ was found in 23.8% of 2432 patients, significantly associated with female gender, small tumor size, and adenocarcinoma histology. We observed a high inter-laboratory variability in IHC and SISH analysis. MET amplification prevailed in 4.6% and MET GCN≥5 in 4.1% of 1572 patients. MET amplification and MET GCN≥5 were not significantly associated with any tumor characteristics or stage. Both were significantly associated with IHC MET positivity (poverexpression, SISH MET amplification or high MET GCN was found with OS, RFS or TTR. MET overexpression is found in 23.8% of surgically resected NSCLC. MET amplification prevails in 4.6% and is associated with MET overexpression. Both have no influence on prognosis. The large inter-laboratory variability in IHC highlights the challenge of MET IHC analysis in routine practice. Copyright © 2017 Elsevier B.V. All rights reserved.

Clinical implications of somatic mutations in aplastic anemia and myelodysplastic syndrome in genomic age.

Science.gov (United States)

Maciejewski, Jaroslaw P; Balasubramanian, Suresh K

2017-12-08

Recent technological advances in genomics have led to the discovery of new somatic mutations and have brought deeper insights into clonal diversity. This discovery has changed not only the understanding of disease mechanisms but also the diagnostics and clinical management of bone marrow failure. The clinical applications of genomics include enhancement of current prognostic schemas, prediction of sensitivity or refractoriness to treatments, and conceptualization and selective application of targeted therapies. However, beyond these traditional clinical aspects, complex hierarchical clonal architecture has been uncovered and linked to the current concepts of leukemogenesis and stem cell biology. Detection of clonal mutations, otherwise typical of myelodysplastic syndrome, in the course of aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria has led to new pathogenic concepts in these conditions and created a new link between AA and its clonal complications, such as post-AA and paroxysmal nocturnal hemoglobinuria. Distinctions among founder vs subclonal mutations, types of clonal evolution (linear or branching), and biological features of individual mutations (sweeping, persistent, or vanishing) will allow for better predictions of the biologic impact they impart in individual cases. As clonal markers, mutations can be used for monitoring clonal dynamics of the stem cell compartment during physiologic aging, disease processes, and leukemic evolution. © 2016 by The American Society of Hematology. All rights reserved.

[Therapy-resistant and therapy-refractory arterial hypertension].

Science.gov (United States)

Wallbach, M; Koziolek, M J

2018-05-02

Therapy-resistant and therapy-refractory arterial hypertension differ in prevalence, pathogenesis, prognosis and therapy. In both cases, a structured approach is required, with the exclusion of pseudoresistance and, subsequently, secondary hypertension. Resistant hypertension has been reported to be more responsive to intensified diuretic therapy, whereas refractory hypertension is presumed to require sympathoinhibitory therapy. Once the general measures and the drug-based step-up therapy have been exhausted, interventional procedures are available.

HER-2 amplification in tubular carcinoma of the breast.

Science.gov (United States)

Oakley, Gerard J; Tubbs, Raymond R; Crowe, Joseph; Sebek, Bruce; Budd, G Thomas; Patrick, Rebecca J; Procop, Gary W

2006-07-01

The prognostic and therapeutic implications of HER-2 gene amplification and estrogen and progesterone receptor status in breast cancer are well described. To address the relative paucity of information concerning HER-2 amplification for tubular carcinomas, we assessed the frequency of gene amplification in 55 tubular carcinomas of the breast from 54 patients, 5 of which had axillary node metastases. The HER-2 gene copy number was assessed by fluorescence in situ hybridization for the majority of tumors analyzed, whereas estrogen and progesterone receptor status was achieved by immunohistochemical analysis. HER-2 gene amplification was not observed in any of the tumors examined, and most were estrogen receptor-positive. This HER-2 gene amplification frequency was significantly lower than the frequency of gene amplification previously reported for all invasive ductal carcinoma of no special type (P < .01). HER-2 gene amplification likely occurs infrequently, or not at all, in tubular carcinomas of the breast, whereas most express estrogen receptors.

Refractory myasthenia gravis – clinical profile, comorbidities and response to rituximab

Science.gov (United States)

Sudulagunta, Sreenivasa Rao; Sepehrar, Mona; Sodalagunta, Mahesh Babu; Settikere Nataraju, Aravinda; Bangalore Raja, Shiva Kumar; Sathyanarayana, Deepak; Gummadi, Siddharth; Burra, Hemanth Kumar

2016-01-01

Introduction: Myasthenia gravis (MG) is an antibody mediated autoimmune neuromuscular disorder characterized by fatigable muscle weakness. A proportion of myasthenia gravis patients are classified as refractory due to non responsiveness to conventional treatment. This retrospective study was done to evaluate clinical profile, epidemiological, laboratory, and features of patients with MG and mode of management using rituximab and complications. Methods: Data of myasthenia gravis patients admitted or presented to outpatient department (previous medical records) with MG between January 2008 and January 2016 were included. A total of 512 patients fulfilled the clinical and diagnostic criteria of myasthenia gravis of which 76 patients met the diagnostic certainty for refractory myasthenia gravis and were evaluated. Results: Out of 76 refractory MG patients, 53 (69.73%) patients fulfilled all the three defined criteria. The median age of onset of the refractory MG group was 36 years with a range of 27–53 years. In our study 25 patients (32.89%) belonged to the age group of 21–30 years. Anti-MuSK antibodies were positive in 8 non-refractory MG patients (2.06%) and 36 refractory MG patients (47.36%). Mean HbA1C was found to be 8.6±2.33. The dose of administered prednisone decreased by a mean of 59.7% (p=3.3x10–8) to 94.6% (p=2.2x10–14) after the third cycle of rituximab treatment. Conclusion: The refractory MG patients are most commonly female with an early age of onset, anti-MuSK antibodies, and thymomas. Refractory MG patients have higher prevalence and poor control (HbA1C >8%) of diabetes mellitus and dyslipidemia probably due to increased steroid usage. Rituximab is very efficient in treatment of refractory MG with adverse effects being low. PMID:27790079

IMPROVED CORROSION RESISTANCE OF ALUMINA REFRACTORIES

Energy Technology Data Exchange (ETDEWEB)

John P. Hurley; Patty L. Kleven

2001-09-30

The initial objective of this project was to do a literature search to define the problems of refractory selection in the metals and glass industries. The problems fall into three categories: Economic--What do the major problems cost the industries financially? Operational--How do the major problems affect production efficiency and impact the environment? and Scientific--What are the chemical and physical mechanisms that cause the problems to occur? This report presents a summary of these problems. It was used to determine the areas in which the EERC can provide the most assistance through bench-scale and laboratory testing. The final objective of this project was to design and build a bench-scale high-temperature controlled atmosphere dynamic corrosion application furnace (CADCAF). The furnace will be used to evaluate refractory test samples in the presence of flowing corrodents for extended periods, to temperatures of 1600 C under controlled atmospheres. Corrodents will include molten slag, steel, and glass. This test should prove useful for the glass and steel industries when faced with the decision of choosing the best refractory for flowing corrodent conditions.

Spectrum of EGFR gene mutations in Vietnamese patients with non-small cell lung cancer.

Science.gov (United States)

Vu, Hoang Anh; Xinh, Phan Thi; Ha, Hua Thi Ngoc; Hanh, Ngo Thi Tuyet; Bach, Nguyen Duc; Thao, Doan Thi Phuong; Dat, Ngo Quoc; Trung, Nguyen Sao

2016-03-01

Epidermal growth factor receptor (EGFR) mutational status is a crucial biomarker for prediction of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Although these mutations have been well characterized in other countries, little is known about the frequency or spectrum of EGFR mutations in Vietnamese NSCLC patients. Using Sanger DNA sequencing, we investigated mutations in EGFR exons 18-21 from 332 patients diagnosed with NSCLC at University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam. DNA was extracted from formalin-fixed, paraffin-embedded tissues, followed by PCR amplification and sequencing. EGFR mutations were detected in 135 samples (40.7%), of which eight samples carried double mutations. In total, 46 different types of EGFR mutations were found, including six novel mutations (p.K713E, p.K714R, p.P794S, p.R803W, p.P848S, and p.K867E). Among the four exons investigated, exon 19 was most frequently mutated (63 out of 332 patients, 19%), with the p.E746_A750del appearing in 43 samples. Exon 21 was mutated in 56 samples (16.9%), of which 47 were p.L858R. Each of exons 18 and 20 was mutated in 12 samples (3.6%). The frequency of EGFR mutations was higher in females than in males (48.9% vs 35%, P = 0.012), but not statistically different between adenocarcinomas and other histological types of NSCLC (41.3% vs 34.5%, P = 0.478). DNA sequencing detected EGFR mutations with high frequency and revealed a broad spectrum of mutation type in Vietnamese patients with NSCLC. © 2015 Wiley Publishing Asia Pty Ltd.

Clinical and microbiological features of refractory periodontitis subjects.

Science.gov (United States)

Colombo, A P; Haffajee, A D; Dewhirst, F E; Paster, B J; Smith, C M; Cugini, M A; Socransky, S S

1998-02-01

The purpose of this investigation was to compare the clinical parameters and the site prevalence and levels of 40 subgingival species in successfully treated and refractory periodontitis subjects. 94 subjects received scaling and root planing and if needed, periodontal surgery and systemically administered tetracycline. 28 refractory subjects showed mean full mouth attachment loss and/or > 3 sites showing attachment loss > 2.5 mm within 1 year post-therapy. 66 successfully treated subjects showed mean attachment level gain and no sites with attachment loss > 2.5 mm. Baseline subgingival plaque samples were taken from the mesial aspect of each tooth and the presence and levels of 40 subgingival taxa were determined using whole genomic DNA probes and checkerboard DNA-DNA hybridization. The mean levels and % of sites colonized by each species (prevalence) was computed for each subject and differences between groups sought using the Mann-Whitney test. Most of the 40 species tested, including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Bacteroides forsythus, were equally or less prevalent in the refractory group. Prevotella nigrescens was significantly more prevalent in successfully treated subjects, while refractory subjects harbored a larger proportion of Streptococcus species, particularly Streptococcus constellatus. The odds of a subject being refractory was 8.6 (p or = 3.5% of the total DNA probe count. Since few microbiological differences existed between treatment outcome groups using DNA probes to known species, the predominant cultivable microbiota of 33 subgingival samples from 14 refractory subjects was examined. 85% of the 1649 isolates were identified using probes to 69 recognized subgingival species. The remaining unidentified strains were classified by analyzing 16S rRNA gene sequences. Many sequenced isolates were of taxa not considered a common part of the oral microbiota such as Acinetobacter baumanni

Clinical application of somatosensory amplification in psychosomatic medicine

Directory of Open Access Journals (Sweden)

Nakao Mutsuhiro

2007-10-01

Full Text Available Abstract Many patients with somatoform disorders are frequently encountered in psychosomatic clinics as well as in primary care clinics. To assess such patients objectively, the concept of somatosensory amplification may be useful. Somatosensory amplification refers to the tendency to experience a somatic sensation as intense, noxious, and disturbing. It may have a role in a variety of medical conditions characterized by somatic symptoms that are disproportionate to demonstrable organ pathology. It may also explain some of the variability in somatic symptomatology found among different patients with the same serious medical disorder. It has been assessed with a self-report questionnaire, the Somatosensory Amplification Scale. This instrument was developed in a clinical setting in the U.S., and the reliability and validity of the Japanese and Turkish versions have been confirmed as well. Many studies have attempted to clarify the specific role of somatosensory amplification as a pathogenic mechanism in somatization. It has been reported that somatosensory amplification does not correlate with heightened sensitivity to bodily sensations and that emotional reactivity exerts its influence on somatization via a negatively biased reporting style. According to our recent electroencephalographic study, somatosensory amplification appears to reflect some aspects of long-latency cognitive processing rather than short-latency interoceptive sensitivity. The concept of somatosensory amplification can be useful as an indicator of somatization in the therapy of a broad range of disorders, from impaired self-awareness to various psychiatric disorders. It also provides useful information for choosing appropriate pharmacological or psychological therapy. While somatosensory amplification has a role in the presentation of somatic symptoms, it is closely associated with other factors, namely, anxiety, depression, and alexithymia that may also influence the same

ALK gene amplification is associated with poor prognosis in colorectal carcinoma.

Science.gov (United States)

Bavi, P; Jehan, Z; Bu, R; Prabhakaran, S; Al-Sanea, N; Al-Dayel, F; Al-Assiri, M; Al-Halouly, T; Sairafi, R; Uddin, S; Al-Kuraya, K S

2013-11-12

Recently, the anaplastic lymphoma kinase (ALK) has been found to be altered in several solid and haematological tumours. ALK gene copy number changes and mutations in colorectal cancers (CRCs) are not well characterised. We aimed to study the prevalence of ALK copy number changes, translocations, gene mutations and protein expression in 770 CRC patients, and correlate these findings with molecular and clinico-pathological data. ALK gene copy number variations and ALK expression were evaluated by fluorescence in situ hybridisation (FISH) and immunohistochemistry, respectively. Translocations of the ALK gene were not observed; 3.4% (26 out of 756) of the CRC patients tested had an increase in ALK gene copy number either amplification or gain. Interestingly, increased ALK gene copy number alteration was associated with poor prognosis (P=0.0135) and was an independent prognostic marker in multivariate Cox proportional hazards model. The study reveals a significant impact of ALK gene copy number alterations on the outcome of patients with CRC. The findings of our study highlight a potential role of targeting ALK in advanced CRCs by using ALK FISH and ALK IHC as a screening tool to detect ALK alterations. Based on these findings, a potential role of ALK inhibitor as a therapeutic agent in a subset of CRC merits further investigation.

Classification of human cancers based on DNA copy number amplification modeling

Directory of Open Access Journals (Sweden)

Knuutila Sakari

2008-05-01

Full Text Available Abstract Background DNA amplifications alter gene dosage in cancer genomes by multiplying the gene copy number. Amplifications are quintessential in a considerable number of advanced cancers of various anatomical locations. The aims of this study were to classify human cancers based on their amplification patterns, explore the biological and clinical fundamentals behind their amplification-pattern based classification, and understand the characteristics in human genomic architecture that associate with amplification mechanisms. Methods We applied a machine learning approach to model DNA copy number amplifications using a data set of binary amplification records at chromosome sub-band resolution from 4400 cases that represent 82 cancer types. Amplification data was fused with background data: clinical, histological and biological classifications, and cytogenetic annotations. Statistical hypothesis testing was used to mine associations between the data sets. Results Probabilistic clustering of each chromosome identified 111 amplification models and divided the cancer cases into clusters. The distribution of classification terms in the amplification-model based clustering of cancer cases revealed cancer classes that were associated with specific DNA copy number amplification models. Amplification patterns – finite or bounded descriptions of the ranges of the amplifications in the chromosome – were extracted from the clustered data and expressed according to the original cytogenetic nomenclature. This was achieved by maximal frequent itemset mining using the cluster-specific data sets. The boundaries of amplification patterns were shown to be enriched with fragile sites, telomeres, centromeres, and light chromosome bands. Conclusions Our results demonstrate that amplifications are non-random chromosomal changes and specifically selected in tumor tissue microenvironment. Furthermore, statistical evidence showed that specific chromosomal features

EGFR mutations in patients with lung adenocarcinoma in southwest China: are G719S/A and L861Q more likely detected in tumors derived from smokers?

Directory of Open Access Journals (Sweden)

Wang QS

2013-07-01

Full Text Available Qiushi Wang,1 Jianghong Mou,1 Xin Yang,1 Yong He,2 Zengpeng Li,1 Qingya Luo,1 Yanqing Li,1 Li Lin,1 Yu Ma,1 Hualiang Xiao11Department of Pathology, 2Department of Respiration, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of ChinaBackground: The clinical characteristics of epidermal growth factor receptor (EGFR hotspot mutations, such as deletions in exon 19, substitution of L858R in exon 21, and mutations in exon 20, have been widely reported in nonsmall cell lung cancer. However, the clinical features of other low frequency EGFR mutations in these four exons (especially the relationship with smoking history, eg, substitutions of G719S/A/C in exon 18 and L861Q in exon 21, remain unclear. This study investigated the relationship between G719S/A/C and L861Q mutations (in exon 18 and 21 and smoking history.Methods: Specimens from 194 patients with lung adenocarcinoma were analyzed for EGFR mutations in exons 18–21 by high-resolution melting curve analysis and amplification refractory mutation technology to establish the relationship between G719S/A/C and L861Q mutations and smoking history.Results: Ninety-six of 194 tumors (49.5% were confirmed to be EGFR mutation-positive. Among these mutations, 71 of 104 (68.3% were from never smokers, six of 17 (35.3% were from former smokers, and 19 of 73 (26.0% were from current smokers (P < 0.001. The mutation rate in heavy smokers (5/23, 21.7% was significantly lower than in light smokers (20/67, 29.9% and never smokers (71/104, 68.3%, P < 0.001. Seven low frequency EGFR mutations (four substitutions of G719S, and G719 A, respectively, and three of L861Q in exon 21 were identified. Five of these mutations were derived from smokers (one former light smoker, one current heavy smoker, and three current light smokers. Four of these patients had been treated with tyrosine kinase inhibitors and all had a partial response, with median overall

Defining refractory migraine: results of the RHSIS Survey of American Headache Society members.

Science.gov (United States)

Schulman, Elliott A; Peterlin, B Lee; Lake, Alvin E; Lipton, Richard B; Hanlon, Alexandra; Siegel, Sherry; Levin, Morris; Goadsby, Peter J; Markley, Herbert G

2009-04-01

To gauge consensus regarding a proposed definition for refractory migraine proposed by Refractory Headache Special Interest Section, and where its use would be most appropriate. Headache experts have long recognized that a subgroup of headache sufferers remains refractory to treatment. Although different groups have proposed criteria to define refractory migraine, the definition remains controversial. The Refractory Headache Special Interest Section of the American Headache Society developed a definition through a consensus process, assisted by a literature review and initial membership survey. A 12-item questionnaire was distributed at the American Headache Society meeting in 2007 during a platform session and at the Refractory Headache Special Interest Section symposium. The same questionnaire was subsequently sent to all American Headache Society members via e-mail. A total of 151 responses from AHS members form the basis of this report. The survey instrument was designed using Survey Monkey. Frequencies and percentages of the survey were used to describe survey responses. American Headache Society members agreed that a definition for refractory migraine is needed (91%) that it should be added to the International Classification of Headache Disorders-2 (86%), and that refractory forms of non-migraine headache disorders should be defined (87%). Responders believed a refractory migraine definition would be of greatest value in selecting patients for clinical drug trials. The current refractory migraine definition requires a diagnosis of migraine, interference with function or quality of life despite modification of lifestyle factors, and adequate trials of acute and preventive medicines with established efficacy. The proposed criteria for the refractory migraine definition require failing 2 preventive medications to meet the threshold for failure. Although 42% of respondents agreed with the working definition of refractory migraine, 43% favored increasing the

The Role of Refractory Dissolved Organic Matter in Ocean Carbon Sequestration

DEFF Research Database (Denmark)

Jørgensen, Linda

The ocean assimilates a large amount of atmospheric CO2 and is potentially a buffer for climate change. A fraction of the assimilated CO2 is incorporated into algal biomass and further converted into refractory dissolved organic matter (DOM). Carbon bound in refractory DOM has the potential...... studies the prokaryotic production and degradation of oceanic refractory DOM and discusses the reasons for the persistent nature of this large DOM fraction. The first two papers investigate the microbial carbon pump, i.e. prokaryotic transfor-mation of organic carbon into refractory DOM. The results show...... DOM compounds in the ocean are rare—possibly too rare to sustain viable uptake and assimilation. Hence, the dilute concentration of individual compounds is a possible explanation for the apparent refractory nature of most DOM in the ocean. Understanding the mechanisms that control the quality...

Eight previously unidentified mutations found in the OA1 ocular albinism gene

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Dufier Jean-Louis

2006-04-01

Full Text Available Abstract Background Ocular albinism type 1 (OA1 is an X-linked ocular disorder characterized by a severe reduction in visual acuity, nystagmus, hypopigmentation of the retinal pigmented epithelium, foveal hypoplasia, macromelanosomes in pigmented skin and eye cells, and misrouting of the optical tracts. This disease is primarily caused by mutations in the OA1 gene. Methods The ophthalmologic phenotype of the patients and their family members was characterized. We screened for mutations in the OA1 gene by direct sequencing of the nine PCR-amplified exons, and for genomic deletions by PCR-amplification of large DNA fragments. Results We sequenced the nine exons of the OA1 gene in 72 individuals and found ten different mutations in seven unrelated families and three sporadic cases. The ten mutations include an amino acid substitution and a premature stop codon previously reported by our team, and eight previously unidentified mutations: three amino acid substitutions, a duplication, a deletion, an insertion and two splice-site mutations. The use of a novel Taq polymerase enabled us to amplify large genomic fragments covering the OA1 gene. and to detect very likely six distinct large deletions. Furthermore, we were able to confirm that there was no deletion in twenty one patients where no mutation had been found. Conclusion The identified mutations affect highly conserved amino acids, cause frameshifts or alternative splicing, thus affecting folding of the OA1 G protein coupled receptor, interactions of OA1 with its G protein and/or binding with its ligand.

Intraepithelial lymphocytes in refractory celiac disease : lost in transition

NARCIS (Netherlands)

Schmitz, Frederike

2014-01-01

Refractory coeliac disease type II (RCDII) is a severe complication of coeliac disease. Whereas celiac disease can successfully be treated by the strict avoidance of gluten, refractory celiac patients show no remission despite a gluten-free diet. The pathology of RCDII is only partially understood,

Allogeneic Transplant in ELANE and MEFV Mutation Positive Severe Cyclic Neutropenia: Review of Prognostic Factors for Secondary Severe Events

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Onyemaechi N. Okolo

2017-01-01

Full Text Available Objective and Importance. Cyclic neutropenia (CyN is a rare autosomal dominant inherited disorder due to the mutation ELANE primarily affecting bone marrow stem cells and is characterized by recurrent neutropenia every 2 to 4 weeks. Symptoms vary from benign to severe, including death. Postulations on the cause of wide spectrum in symptom presentation include the possibility of other genetic mutations, such as MEFV. Recommended treatment for CyN is G-CSF to keep ANC higher to minimize risk of infection. Case. A 25-year-old male diagnosed with CyN, on G-CSF but worsening quality of life. Pretransplant investigations revealed ELANE mutation positive severe CyN along with familial Mediterranean fever (MEFV mutation. Intervention. Bone marrow transplantation as treatment for dual mutation (ELANE and MEFV mutation positive severe CyN. Conclusion. BMT may be considered as an alternative treatment for severe CyN in patients who are refractory to G-CSF. It is postulated that in our patient the combined mutations (CyN and MEFV may have contributed to the severity of this individual’s symptoms. We suggest CyN patients who present with severe symptoms have evaluation with ELANE mutation testing, Periodic Fever Syndromes Panel, and routine marrow assessment with FISH, conventional cytogenetics, and morphological evaluation for MDS/AML.

Clinical Features of Refractory Ascites in Outpatients

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Wanda Regina Caly

Full Text Available OBJECTIVES: To present the clinical features and outcomes of outpatients who suffer from refractory ascites. METHODS: This prospective observational study consecutively enrolled patients with cirrhotic ascites who submitted to a clinical evaluation, a sodium restriction diet, biochemical blood tests, 24 hour urine tests and an ascitic fluid analysis. All patients received a multidisciplinary evaluation and diuretic treatment. Patients who did not respond to the diuretic treatment were controlled by therapeutic serial paracentesis, and a transjugular intrahepatic portosystemic shunt was indicated for patients who required therapeutic serial paracentesis up to twice a month. RESULTS: The most common etiology of cirrhosis in both groups was alcoholism [49 refractory (R and 11 non-refractory ascites (NR]. The majority of patients in the refractory group had Child-Pugh class B cirrhosis (p=0.034. The nutritional assessment showed protein-energy malnutrition in 81.6% of the patients in the R group and 35.5% of the patients in the NR group, while hepatic encephalopathy, hernia, spontaneous bacterial peritonitis, upper digestive hemorrhage and type 2 hepatorenal syndrome were present in 51%, 44.9%, 38.8%, 38.8% and 26.5% of the patients in the R group and 9.1%, 18.2%, 0%, 0% and 0% of the patients in the NR group, respectively (p=0.016, p=0.173, p=0.012, p=0.012, and p=0.100, respectively. Mortality occurred in 28.6% of the patients in the R group and in 9.1% of the patients in the NR group (p=0.262. CONCLUSION: Patients with refractory ascites were malnourished, suffered from hernias, had a high prevalence of complications and had a high postoperative death frequency, which was mostly due to infectious processes.

Long-range PCR facilitates the identification of PMS2-specific mutations.

Science.gov (United States)

Clendenning, Mark; Hampel, Heather; LaJeunesse, Jennifer; Lindblom, Annika; Lockman, Jan; Nilbert, Mef; Senter, Leigha; Sotamaa, Kaisa; de la Chapelle, Albert

2006-05-01

Mutations within the DNA mismatch repair gene, "postmeiotic segregation increased 2" (PMS2), have been associated with a predisposition to hereditary nonpolyposis colorectal cancer (HNPCC; Lynch syndrome). The presence of a large family of highly homologous PMS2 pseudogenes has made previous attempts to sequence PMS2 very difficult. Here, we describe a novel method that utilizes long-range PCR as a way to preferentially amplify PMS2 and not the pseudogenes. A second, exon-specific, amplification from diluted long-range products enables us to obtain a clean sequence that shows no evidence of pseudogene contamination. This method has been used to screen a cohort of patients whose tumors were negative for the PMS2 protein by immunohistochemistry and had not shown any mutations within the MLH1 gene. Sequencing of the PMS2 gene from 30 colorectal and 11 endometrial cancer patients identified 10 novel sequence changes as well as 17 sequence changes that had previously been identified. In total, putative pathologic mutations were detected in 11 of the 41 families. Among these were five novel mutations, c.705+1G>T, c.736_741del6ins11, c.862_863del, c.1688G>T, and c.2007-1G>A. We conclude that PMS2 mutation detection in selected Lynch syndrome and Lynch syndrome-like patients is both feasible and desirable. Published 2006 Wiley-Liss, Inc.

Gene structure and mutations of glutaryl-coenzyme A dehydrogenase: impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish.

OpenAIRE

Biery, B. J.; Stein, D. E.; Morton, D. H.; Goodman, S. I.

1996-01-01

The structure of the human glutaryl coenzyme A dehydrogenase (GCD) gene was determined to contain 11 exons and to span approximately 7 kb. Fibroblast DNA from 64 unrelated glutaric acidemia type I (GA1) patients was screened for mutations by PCR amplification and analysis of SSCP. Fragments with altered electrophoretic mobility were subcloned and sequenced to detect mutations that caused GA1. This report describes the structure of the GCD gene, as well as point mutations and polymorphisms fou...

Resistant and Refractory Hypertension: Antihypertensive Treatment Resistance vs Treatment Failure

Science.gov (United States)

Siddiqui, Mohammed; Dudenbostel, Tanja; Calhoun, David A.

2017-01-01

Resistant or difficult to treat hypertension is defined as high blood pressure that remains uncontrolled with 3 or more different antihypertensive medications, including a diuretic. Recent definitions also include controlled blood pressure with use of 4 or more medications as also being resistant to treatment. Recently, refractory hypertension, an extreme phenotype of antihypertensive treatment failure has been defined as hypertension uncontrolled with use of 5 or more antihypertensive agents, including a long-acting thiazide diuretic and a mineralocorticoid receptor antagonist. Patients with resistant vs refractory hypertension share similar characteristics and comorbidities, including obesity, African American race, female sex, diabetes, coronary heart disease, chronic kidney disease, and obstructive sleep apnea. Patients with refractory vs resistant hypertension tend to be younger and are more likely to have been diagnosed with congestive heart failure. Refractory hypertension might also differ from resistant hypertension in terms of underlying cause. Preliminary evidence suggests that refractory hypertension is more likely to be neurogenic in etiology (ie, heightened sympathetic tone), vs a volume-dependent hypertension that is more characteristic of resistant hypertension in general. PMID:26514749

Performance assessment of refractory samples in the Los Alamos Controlled Air Incinerator

International Nuclear Information System (INIS)

Hutchins, D.A.; Borduin, L.C.; Koenig, R.A.; Vavruska, J.S.; Warner, C.L.

1986-01-01

A refractory evaluation project was initiated in 1979 to study the performance of six selected refractory materials within the Los Alamos Controlled Air Incinerator (CAI). Determining refractory resistance to thermal shock, chemical attack, and plutonium uptake was of particular interest. The experimental refractories were subjected to a variety of waste materials, including transuranic (TRU) contaminated wastes, highly chlorinated compounds and alkaline metal salts of perchlorate, chlorate, nitrate and oxylate, over the six year period of this study. Results of this study to date indicate that the use of high alumina, and possibly specialty plastic refractories, is advisable for the lining of incinerators used for the thermal destruction of diverse chemical compounds. 12 refs., 4 tabs

HFE gene mutations in coronary atherothrombotic disease

Directory of Open Access Journals (Sweden)

Calado R.T.

2000-01-01

Full Text Available Although iron can catalyze the production of free radicals involved in LDL lipid peroxidation, the contribution of iron overload to atherosclerosis remains controversial. The description of two mutations in the HFE gene (Cys282Tyr and His63Asp related to hereditary hemochromatosis provides an opportunity to address the question of the association between iron overload and atherosclerosis. We investigated the prevalence of HFE mutations in 160 survivors of myocardial infarction with angiographically demonstrated severe coronary atherosclerotic disease, and in 160 age-, gender- and race-matched healthy control subjects. PCR amplification of genomic DNA followed by RsaI and BclI restriction enzyme digestion was used to determine the genotypes. The frequency of the mutant Cys282Tyr allele was identical among patients and controls (0.022; carrier frequency, 4.4%, whereas the mutant His63Asp allele had a frequency of 0.143 (carrier frequency, 27.5% in controls and of 0.134 (carrier frequency, 24.5% in patients. Compound heterozygotes were found in 2 of 160 (1.2% controls and in 1 of 160 (0.6% patients. The finding of a similar prevalence of Cys282Tyr and His63Asp mutations in the HFE gene among controls and patients with coronary atherothrombotic disease, indirectly questions the possibility of an association between hereditary hemochromatosis and atherosclerosis.

White-Coat Effect Is Uncommon in Patients With Refractory Hypertension.

Science.gov (United States)

Siddiqui, Mohammed; Judd, Eric K; Oparil, Suzanne; Calhoun, David A

2017-09-01

Refractory hypertension is a recently described phenotype of antihypertensive treatment failure defined as uncontrolled blood pressure (BP) despite the use of ≥5 different antihypertensive agents, including chlorthalidone and spironolactone. Recent studies indicate that refractory hypertension is uncommon, with a prevalence of ≈5% to 10% of patients referred to a hypertension specialty clinic for uncontrolled hypertension. The prevalence of white-coat effect, that is, uncontrolled automated office BP ≥135/85 mm Hg and controlled out-of-office BP hypertensive patients overall is ≈30% to 40%. The prevalence of white-coat effect among patients with refractory hypertension has not been previously reported. In this prospective evaluation, consecutive patients referred to the University of Alabama at Birmingham Hypertension Clinic for uncontrolled hypertension were enrolled. Refractory hypertension was defined as uncontrolled automated office BP ≥135/85 mm Hg with the use of ≥5 antihypertensive agents, including chlorthalidone and spironolactone. Automated office BP measurements were based on 6 serial readings, done automatically with the use of a BpTRU device unobserved in the clinic. Out-of-office BP measurements were done by 24-hour ambulatory BP monitor. Thirty-four patients were diagnosed with refractory hypertension, of whom 31 had adequate ambulatory BP monitor readings. White-coat effect was present in only 2 patients, or 6.5% of the 31 patients with refractory hypertension, suggesting that white-coat effect is largely absent in patients with refractory hypertension. These findings suggest that white-coat effect is not a common cause of apparent lack of BP control in patients failing maximal antihypertensive treatment. © 2017 American Heart Association, Inc.

Analysis of APC mutation in human ameloblastoma and clinical significance.

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Li, Ning; Liu, Bing; Sui, Chengguang; Jiang, Youhong

2016-01-01

As a highly conserved signaling pathway, Wnt/β-catenin signal transduction pathway plays an important role in many processes. Either in the occurrence or development of tumor, activation of this pathway takes an important place. APC inhibits Wnt/β-catenin pathway to regulate cell proliferation and differentiation. This study aimed to investigate the function of cancer suppressor gene. PCR amplification and sequencing method was used to analyze APC mutations of human clinical specimens. The pathological specimens were collected for PCR and clear electrophoretic bands were obtained after electrophoresis. The gene sequence obtained after purification and sequencing analysis was compared with the known APC gene sequence (NM_000038.5). Base mutations at APC 1543 (T → C), APC-4564 (G → A), APC-5353 (T → G), APC-5550 (T → A) and APC-5969 (G → A) locus existed in 22 (27.5 %), 12 (15 %), 5 (6.25 %), 13 (16.25 %) and 12 patients (15 %), respectively. Gene mutations existed in ameloblastoma, and the mutation loci were 1543 locus (T → C), 4564 locus (G → A), 5353 locus (T → G), 5550 locus (T → A) and 5969 locus (G → A) 15 %, respectively. APC mutation plays a certain role in monitoring the tumor malignant degree as it may indicate the transition process of ameloblastoma malignant phenotype.

Friction measurements of steel on refractory bricks

International Nuclear Information System (INIS)

Eiselstein, L.E.

1981-08-01

During startup or shutdown of a pool-type LMFBR, substantial shear stresses may arise between the base of the steel reactor vessel and the refractory brick support base. The magnitude of these stresses, which result from differences in thermal expansion, can be estimated if the friction coefficient is known. This report describes experiments to determine friction coefficients between 2 1/4 Cr-1Mo steel and several refractory materials and to examine effects to contact pressure, temperature, sliding velocity, lubricants, and surface condition

Germline mutations in PMS2 and MLH1 in individuals with solitary loss of PMS2 expression in colorectal carcinomas from the Colon Cancer Family Registry Cohort.

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Rosty, Christophe; Clendenning, Mark; Walsh, Michael D; Eriksen, Stine V; Southey, Melissa C; Winship, Ingrid M; Macrae, Finlay A; Boussioutas, Alex; Poplawski, Nicola K; Parry, Susan; Arnold, Julie; Young, Joanne P; Casey, Graham; Haile, Robert W; Gallinger, Steven; Le Marchand, Loïc; Newcomb, Polly A; Potter, John D; DeRycke, Melissa; Lindor, Noralane M; Thibodeau, Stephen N; Baron, John A; Win, Aung Ko; Hopper, John L; Jenkins, Mark A; Buchanan, Daniel D

2016-02-19

Immunohistochemistry for DNA mismatch repair proteins is used to screen for Lynch syndrome in individuals with colorectal carcinoma (CRC). Although solitary loss of PMS2 expression is indicative of carrying a germline mutation in PMS2, previous studies reported MLH1 mutation in some cases. We determined the prevalence of MLH1 germline mutations in a large cohort of individuals with a CRC demonstrating solitary loss of PMS2 expression. This cohort study included 88 individuals affected with a PMS2-deficient CRC from the Colon Cancer Family Registry Cohort. Germline PMS2 mutation analysis (long-range PCR and multiplex ligation-dependent probe amplification) was followed by MLH1 mutation testing (Sanger sequencing and multiplex ligation-dependent probe amplification). Of the 66 individuals with complete mutation screening, we identified a pathogenic PMS2 mutation in 49 (74%), a pathogenic MLH1 mutation in 8 (12%) and a MLH1 variant of uncertain clinical significance predicted to be damaging by in silico analysis in 3 (4%); 6 (9%) carried variants likely to have no clinical significance. Missense point mutations accounted for most alterations (83%; 9/11) in MLH1. The MLH1 c.113A> G p.Asn38Ser mutation was found in 2 related individuals. One individual who carried the MLH1 intronic mutation c.677+3A>G p.Gln197Argfs*8 leading to the skipping of exon 8, developed 2 tumours, both of which retained MLH1 expression. A substantial proportion of CRCs with solitary loss of PMS2 expression are associated with a deleterious MLH1 germline mutation supporting the screening for MLH1 in individuals with tumours of this immunophenotype, when no PMS2 mutation has been identified. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Characterization of refractory brick based on local raw material from Lampung Province - Indonesia

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Amin, Muhammad; Suryana, Yayat I.; Isnugroho, Kusno; Aji, Bramantyo B.; Birawidha, David C.; Hendronursito, Yusup

2018-04-01

Refractories are non-metallic inorganic materials that are difficult to melt at high temperatures and used in high-temperature casting industries. Refractories are classified into their constituent mineral feed stocks, refractories having typical plot properties commonly called fire bricks. In the manufacture of refractory bricks that exist in the market during the use of mangrove materials derived from abroad that is from China. In this research the refractory brick materials used are quartz sand, feldspart, kaolin, bentonite, and ball clay. All materials come from local Lampung Province - Indonesia. The experiment, there are 7 kinds of experimental composition, made of plot shape with size 230 mm, 65 mm in thickness, 114 mm height mould using manual press machine with 10 tons power and burning at 1400°C for 5 hours. Refractory brick product is done by physical test in the form of porosity, specific gravity, compressive strength and XRF and SEM characteristics. The result of XRF characteristic of refractory brick composition of 1 to 5 compared to the refractory brick type SK 34 in the market and the result of composition 1 is a composition close to refractory brick composition type SK 34 namely SiO2 is 54.21 %, Al2O3 is 25.38 % and test Physical of Bulk density is 2.25 g/cm3, porosity is 18.98 % and compressive strength is 325 kg/cm2.

Refractory benign esophageal strictures

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Gopal Goyal

2016-01-01

Full Text Available Refractory benign esophageal stricture (RBES is a frequently encountered problem worldwide. These strictures arise from various causes such as corrosive injury, radiation therapy, peptic origin, ablative therapy, and after surgery. Most strictures can be treated successfully with endoscopic dilatation using bougies or balloons, with only a few complications. Those patients who fail after serial dilatation with bougies or balloons will come to the category of refractory strictures. Dilatation combined with intralesional steroid injections can be considered for peptic strictures, whereas incisional therapy has been demonstrated to be effective for short anastomotic strictures. When these therapeutic options do not resolve the stenosis, stent placement should be considered. Self-bougienage can be proposed to a selected group of patients with a proximal stenosis. Most of the patients of RBES respond to above-mentioned treatment and occasional patient may require surgery as the final treatment option. This review aims to provide a comprehensive approach toward endoscopic management of RBESs based on current literature and personal experience.

Contribution of silent mutations to thermal adaptation of RNA bacteriophage Qβ.

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Kashiwagi, Akiko; Sugawara, Ryu; Sano Tsushima, Fumie; Kumagai, Tomofumi; Yomo, Tetsuya

2014-10-01

Changes in protein function and other biological properties, such as RNA structure, are crucial for adaptation of organisms to novel or inhibitory environments. To investigate how mutations that do not alter amino acid sequence may be positively selected, we performed a thermal adaptation experiment using the single-stranded RNA bacteriophage Qβ in which the culture temperature was increased from 37.2°C to 41.2°C and finally to an inhibitory temperature of 43.6°C in a stepwise manner in three independent lines. Whole-genome analysis revealed 31 mutations, including 14 mutations that did not result in amino acid sequence alterations, in this thermal adaptation. Eight of the 31 mutations were observed in all three lines. Reconstruction and fitness analyses of Qβ strains containing only mutations observed in all three lines indicated that five mutations that did not result in amino acid sequence changes but increased the amplification ratio appeared in the course of adaptation to growth at 41.2°C. Moreover, these mutations provided a suitable genetic background for subsequent mutations, altering the fitness contribution from deleterious to beneficial. These results clearly showed that mutations that do not alter the amino acid sequence play important roles in adaptation of this single-stranded RNA virus to elevated temperature. Recent studies using whole-genome analysis technology suggested the importance of mutations that do not alter the amino acid sequence for adaptation of organisms to novel environmental conditions. It is necessary to investigate how these mutations may be positively selected and to determine to what degree such mutations that do not alter amino acid sequences contribute to adaptive evolution. Here, we report the roles of these silent mutations in thermal adaptation of RNA bacteriophage Qβ based on experimental evolution during which Qβ showed adaptation to growth at an inhibitory temperature. Intriguingly, four synonymous mutations and

Illness identity in young adults with refractory epilepsy.

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Luyckx, Koen; Oris, Leen; Raymaekers, Koen; Rassart, Jessica; Moons, Philip; Verdyck, Ludo; Mijnster, Teus; Mark, Ruth E

2018-03-01

Refractory epilepsy is an intrusive condition with important implications for daily functioning in emerging and young adulthood. The present study examined the degree to which refractory epilepsy is integrated in one's identity, and examined how such a sense of illness identity was related to health-related quality of life (HRQOL). A total of 121 18- to 40-year-old patients with refractory epilepsy (56.2% women) completed self-report questionnaires assessing the four illness identity states of acceptance, enrichment, engulfment, and rejection (Illness Identity Questionnaire (IIQ)); HRQOL (Quality of Life in Epilepsy Inventory - 31); and seizure frequency and severity (Liverpool Seizure Severity Scale (LSSS)). Illness identity scores were compared with a sample of 191 patients with a nonneurological chronic disease (congenital heart disease). Hierarchical regression analyses were conducted to assess the predictive value of illness identity for HRQOL when simultaneously controlling for demographic and clinical features. Patients with refractory epilepsy scored higher on rejection and engulfment and lower on acceptance when compared with patients with congenital heart disease. Further, seizure severity and number of medication side-effects were positively related to engulfment and negatively to acceptance. Finally, when simultaneously controlling for various demographic and clinical variables, illness identity significantly predicted HRQOL (with engulfment being the strongest and most consistent predictor). The extent to which patients with refractory epilepsy succeed in integrating their illness into their identity may have important implications for HRQOL. Clinicians should be especially attentive for signs that patients feel engulfed by their epilepsy. Copyright © 2018 Elsevier Inc. All rights reserved.

Ketogenic diet treatment for pediatric super-refractory status epilepticus.

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Appavu, Brian; Vanatta, Lisa; Condie, John; Kerrigan, John F; Jarrar, Randa

2016-10-01

We aimed to study whether ketogenic diet (KD) therapy leads to resolution of super-refractory status epilepticus in pediatric patients without significant harm. A retrospective review was performed at Phoenix Children's Hospital on patients with super-refractory status epilepticus undergoing ketogenic diet therapy from 2011 to 2015. Ten children with super-refractory status epilepticus, ages 2-16 years, were identified. 4/10 patients had immune mediated encephalitis, including Rasmussen encephalitis, anti-N-methyl-d-aspartate receptor encephalitis, and post-infectious mycoplasma encephalitis. Other etiologies included Lennox Gastaut Syndrome, non-ketotic hyperglycinemia, PCDH19 and GABRG2 genetic epilepsy, New Onset Refractory Status Epilepticus, and Febrile Infection-Related Epilepsy Syndrome. 4/10 patients' EEG features suggested focal with status epilepticus, and 6/10 suggested generalized with status epilepticus. Median hospital length was 61days and median ICU length was 27days. The median number of antiepileptic medications prior to diet initiation was 3.0 drugs, and the median after ketogenic diet treatment was 3.5 drugs. Median duration of status epilepticus prior to KD was 18days. 9/10 patients had resolution of super-refractory status epilepticus in a median of 7days after diet initiation. 8/9 patients were weaned off anesthesia within 15days of diet initiation, and within 1day of achieving ketonuria. 1/10 patients experienced side effects on the diet requiring supplementation. Most patients achieved resolution of status epilepticus on KD therapy, suggesting it could be an effective therapy that can be utilized early in the treatment of children with super refractory status epilepticus. Copyright © 2016. Published by Elsevier Ltd.

Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6.

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Evans, Ben A; Smith, Olivia L; Pickerill, Ethan S; York, Mary K; Buenconsejo, Kristen J P; Chambers, Antonio E; Bernstein, Douglas A

2018-01-01

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn 2+ -binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans . Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

Historic, clinical, and prognostic features of epileptic encephalopathies caused by CDKL5 mutations.

Science.gov (United States)

Moseley, Brian D; Dhamija, Radhika; Wirrell, Elaine C; Nickels, Katherine C

2012-02-01

Mutations within the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene are important causes of early-onset epileptic encephalopathies. We sought to determine the historic, clinical, and prognostic features of epilepsy secondary to CDKL5 mutations. We performed retrospective chart reviews of children at our institution with epilepsy and CDKL5 mutations. Six children were identified. One manifested a deletion in exons 10-15 of the CDKL5 gene, another manifested a single base-pair duplication in exon 3, and the rest manifested base-pair exchanges. The mean age of seizure onset was 1.8 months (range, 1-3 months). Although the majority (4/6, 67%) presented with partial-onset seizures, all children developed infantile spasms. All children demonstrated developmental delay and visual impairment. Although such mutations are X-linked, two children were boys. They did not present with more severe phenotypes than their female counterparts. Despite trials of antiepileptic drugs (mean, 5; range, 3-7), steroids/adrenocorticotropic hormone (4/6; 67%), and the ketogenic diet (6/6; 100%), all children manifested refractory seizures at last follow-up. Although no treatment eliminated seizures, topiramate, vigabatrin, and the ketogenic diet were most helpful at reducing seizure frequency. Copyright © 2012 Elsevier Inc. All rights reserved.

Diagnostic Devices for Isothermal Nucleic Acid Amplification

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Chia-Chen Chang

2012-06-01

Full Text Available Since the development of the polymerase chain reaction (PCR technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

Diagnostic devices for isothermal nucleic acid amplification.

Science.gov (United States)

Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

2012-01-01

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

Realization of the conceptual ideal for x-ray amplification

Energy Technology Data Exchange (ETDEWEB)

Borisov, Alex B; Racz, Ervin; Zhang Ping; McCorkindale, John C; Khan, Shahab F; Poopalasingam, Sankar; Zhao Ji; Rhodes, Charles K [Laboratory for X-Ray Microimaging and Bioinformatics, Department of Physics, University of Illinois at Chicago, Chicago, IL 60607-7059 (United States)

2008-05-28

The Xe(L) system is an amplifier with fundamentally different dynamic characteristics from all previously developed laser amplifiers; it represents the conceptual ideal through full utilization of the Kramers-Kronig relations that fundamentally couple the dispersive and absorptive components. The dispersive response of the system, through optimal governance of the power compression, rules the amplification and establishes a minimum gain for the amplifier. Accordingly, the amplification requires a minimum value of the dispersion to be surpassed; the corresponding gain follows automatically. As a leading consequence, since this minimum gain is sufficiently high, the key experimental observation is the uniform presence of saturated amplification signaled by strong spectral hole burning on all transitions exhibiting amplification, including double-vacancy lines. This cardinal signature demonstrates that the amplification is legislated by the saturated gain g{sub s}, not the corresponding small signal value g{sub 0}. The chief outcome is that explosive dispersion yields perforce explosive amplification and the efficient generation of maximally bright coherent power.

Mutational analysis in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD): Identification of five mutations in the PKD1 gene.

Science.gov (United States)

Abdelwahed, Mayssa; Hilbert, Pascale; Ahmed, Asma; Mahfoudh, Hichem; Bouomrani, Salem; Dey, Mouna; Hachicha, Jamil; Kamoun, Hassen; Keskes-Ammar, Leila; Belguith, Neïla

2018-05-31

Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most frequent genetic disorder of the kidneys, is characterized by a typical presenting symptoms include cysts development in different organs and a non-cysts manifestations. ADPKD is caused by mutations in PKD1 or PKD2 genes. In this study, we aimed to search for molecular causative defects among PKD1 and PKD2 genes. Eighteen patients were diagnosed based on renal ultrasonography and renal/extra-renal manifestations. Then, Sanger sequencing was performed for PKD1 and PKD2 genes. Multiplex Ligation dependent Probe Amplification method (MLPA) methods was performed for both PKD genes. Mutational analysis of the PKD2 gene revealed the absence of variants and no deletions or duplications of both PKD genes were detected. But three novels mutations i.e. p.S463C exon 7; c. c.11156+2T>C IVS38 and c.8161-1G>A IVS22 and two previously reported c.1522T>C exon 7 and c.412C>T exon 4 mutations in the PKD1 gene were detected. Bioinformatics tools predicted that the novel variants have a pathogenic effects on splicing machinery, pre-mRNA secondary structure and stability and protein stability. Our results highlighted molecular features of Tunisian patients with ADPKD and revealed novel variations that can be utilized in clinical diagnosis and in the evaluation of living kidney donor. To the best of our knowledge, this is the first report of Autosomal Polycystic Kidney Disease in Tunisia. Copyright © 2017. Published by Elsevier B.V.

Central Diabetes Insipidus in Refractory Antineutrophil Cytoplasmic Antibody-associated Vasculitis.

Science.gov (United States)

Ohashi, Keiji; Morishita, Michiko; Watanabe, Haruki; Sada, Ken-Ei; Katsuyama, Takayuki; Miyawaki, Yoshia; Katsuyama, Eri; Narazaki, Mariko; Tatebe, Noriko; Watanabe, Katsue; Kawabata, Tomoko; Wada, Jun

2017-11-01

We herein describe two cases of refractory antineutrophil cytoplasmic antibody-associated vasculitis (AAV) complicated with diabetes insipidus (DI) possibly related to hypertrophic pachymeningitis (HP). One patient had microscopic polyangiitis and HP, which were refractory to cyclophosphamide, azathioprine, rituximab, mycophenolate mofetil (MMF), and mizoribine. Remission was finally achieved with the use of etanercept, but DI occurred 5 years later. The other patient had granulomatosis with polyangiitis, which that was refractory to cyclophosphamide, methotrexate, MMF, and rituximab. DI subsequently developed, but was successfully treated with etanercept. Dura mater hypertrophy was macroscopically observed in the latter case.

Mutational profile of KIT and PDGFRA genes in gastrointestinal stromal tumors in Peruvian samples

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José Buleje

2015-02-01

Full Text Available Introduction: Gastrointestinal stromal tumors (GISTs are mesenchymal neoplasms usually caused by somatic mutations in the genes KIT (c-KIT or PDGFRA. Mutation characterization has become an important exam for GIST patients because it is useful in predicting the response to the inhibitors of receptor tyrosine kinase (RTK. Objectives: The aim of this study was to determine the frequency of KIT and PDGFRA mutations in 25 GIST samples collected over two years at two national reference hospitals in Peru. There were 21 samples collected from the Instituto Nacional de Enfermedades Neoplásicas (INEN, national cancer center and 4 samples collected from Hospital A. Loayza. Methods and materials: In this retrospective study, we performed polymerase chain reaction (PCR amplification and deoxyribonucleic acid (DNA sequencing of KIT (exons 9, 11, 13, and 17 and PDGFRA (exons 12 and 18 genes in 20 FFPE (formalin-fixed, paraffin-embedded and 5 frozen GIST samples. Results: We report 21 mutations, including deletions, duplications, and missense, no mutations in 2 samples, and 2 samples with no useful DNA for further analysis. Eighty-six percent of these mutations were located in exon 11 of KIT, and 14 % were located in exon 18 of PDGFRA. Conclusions: Our study identified mutations in 21 out of 25 GIST samples from 2 referential national hospitals in Peru, and the mutation proportion follows a global tendency observed from previous studies (i.e., the majority of samples presented KIT mutations followed by a minor percentage of PDGFRA mutations. This study presents the first mutation data of the KIT and PDGFRA genes from Peruvian individuals with GIST.

A Review on Recent Advances on Magnesia-Doloma Refractories by Nano-Technology.

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Salman Ghasemi-Kahrizsangi

2017-07-01

Full Text Available Due to exhibiting an excellent chemical resistance against basic environments at high temperature, good thermal shock resistance, thermodynamic stability in the presence of carbon, and a suitable abrasion resistance, MgO-CaO refractories are widely used in argon-oxygen decarburization furnaces in the metallurgy industry and cement rotary kilns. Furthermore, MgO-CaO refractories are beneficial to removing inclusions from molten steels; thus, they have been considered to be one of the effective refractory types for processing clean steel products. Also, MgO-CaO materials have become one of the attractive steelmaking refractories because of their low cost and high ore reserves.  However, in spite of these primary advantages, the application of MgO-CaO refractories has not been popular due to its tendency to hydration when exposed to the atmosphere.  In world most of MgO-CaO bricks producers used of organic components such as tar, pitch, and peck for produce MgO-CaO refractories. But during the application of these bricks in steel and cement and industrials, they released CO and CO2 gases to air and pollute the atmosphere. For this reason, recently some researcher investigate the effect of additive nanoparticles on MgO-CaO refractories performance. They reported the used of Nano-additive have acceptable results and additive nanoparticles can replace by aforementioned environment contaminating organic compounds. In this study, we reviewed all effort that done for improving the hydration resistance of MgO-CaO refractories by application of Nano-additives with an emphasis on the beneficial the use of additives nanoparticle for reduction of environmental pollution by various industries which used of MgO-CaO refractory bricks.

Frequency of BRAF V600E Mutation in the Mexican Population of Patients With Metastatic Melanoma

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Erika Ruiz-Garcia

2017-06-01

Full Text Available Purpose: The BRAF V600E mutation has been described in melanomas occurring in the Caucasian, European, and Asian populations. However, in the Mexican population, the status and clinical significance of BRAF mutation has not been researched on a large scale. Methods: Consecutive BRAF-tested Mexican patients with metastatic melanoma (n = 127 were analyzed for mutations in exon 15 of the BRAF gene in genomic DNA by real-time polymerase chain reaction technology for amplification and detection. The results were correlated with the clinical-pathologic features and the prognosis of the patients. Results: The frequency of somatic mutation V600E within the BRAF gene was 54.6% (43 of 127 patients. Nodular melanoma was the most prevalent subtype in our population, with BRAF mutations in 37.2% (16 of 55 patients. In contrast, superficial spread had a frequency of 18.6% BRAF mutation (eight of 24. Other clinicopathologic features were assessed to correlate with the mutation status. Conclusion: This study searched for the most prevalent BRAF V600E mutation type in melanoma in a heterogeneous population from Mexico. Nodular melanoma was found to be the most prevalent in metastatic presentation and the presence of BRAF V600E mutation, perhaps related to the mixed ancestry; in the north, ancestry is predominantly European and in the south, it is predominantly Asian. The outcomes of the mutation correlations were similar to those found in other populations.

An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification.

Science.gov (United States)

Xu, Jingguo; Guo, Jia; Maina, Sarah Wanjiku; Yang, Yumeng; Hu, Yimin; Li, Xuanxuan; Qiu, Jiarong; Xin, Zhihong

2018-05-15

An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H 2 O 2 . The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5-10 4  CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry. Copyright © 2018 Elsevier Inc. All rights reserved.

Refractory Research Group - U.S. DOE, Albany Research Center [Institution Profile

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Bennett, James P.

2004-09-01

The refractory research group at the Albany Research Center (ARC) has a long history of conducting materials research within the U.S. Bureau of Mines, and more recently, within the U.S. Dept. of Energy. When under the U.S. Bureau of Mines, research was driven by national needs to develop substitute materials and to conserve raw materials. This mission was accomplished by improving refractory material properties and/or by recycling refractories using critical and strategic materials. Currently, as a U.S. Dept of Energy Fossil Energy field site, research is driven primarily by the need to assist DOE in meeting its vision to develop economically and environmentally viable technologies for the production of electricity from fossil fuels. Research at ARC impacts this vision by: • Providing information on the performance characteristics of materials being specified for the current generation of power systems; • Developing cost-effective, high performance materials for inclusion in the next generation of fossil power systems; and • Solving environmental emission and waste problems related to fossil energy systems. A brief history of past refractory research within the U.S. Bureau of Mines, the current refractory research at ARC, and the equipment and capabilities used to conduct refractory research at ARC will be discussed.

Galactosemia caused by a point mutation that activates cryptic donor splice site in the galactose-1-phosphate uridyltransferase gene

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Wadelius, C.; Lagerkvist, A. (Univ. Hospital, Uppsala (Sweden) Uppsala Univ. (Sweden)); Molin, A.K.; Larsson, A. (Univ. Hospital, Uppsala (Sweden)); Von Doebeln, U. (Karolinska Institute, Stockholm (Sweden))

1993-08-01

Galactosemia affects 1/84,000 in Sweden and is manifested in infancy when the child is exposed to galactose in the diet. If untreated there is a risk of severe early symptoms and, even with a lactose-free diet, late symptoms such as mental retardation and ovarial dysfunction may develop. In classical galactosemia, galactose-1-phosphate uridyltransferase (GALT) (EC 2.7.7.12) is defective and the normal cDNA sequence of this enzyme has been characterized. Recently eight mutations leading to galactosemia were published. Heparinized venous blood was drawn from a patient with classical galactosemia. In the cDNA from the patient examined, an insertion of 54 bp was found at position 1087. Amplification of the relevant genomic region of the patient's DNA was performed. Exon-intron boundaries and intronic sequences thus determined revealed that the 54-bp insertion was located immediately downstream of exon 10. It was further found that the patient was heterozygous for a point mutation, changing a C to a T (in 5 of 9 clones) at the second base in the intron downstream of the insertion. This alteration creates a sequence which, as well as the ordinary splice site, differs in only two positions from the consensus sequence. It was found that the mutation occurred in only one of the 20 alleles from galactosemic patients and in none of the 200 alleles from normal controls. The mutation is inherited from the mother, who also was found to express the 54-bp-long insertion at the mRNA level. Sequences from the 5[prime] end of the coding region were determined after genomic amplification, revealing a sequence identical to that reported. The mutation on the paternal allele has not been identified. 9 refs., 1 fig.

Brentuximab Vedotin Treatment for Primary Refractory Hodgkin Lymphoma

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Hung-Bo Wu

2013-01-01

Full Text Available Up to 40% of patients with advanced Hodgkin lymphoma (HL become refractory or relapsed after current standard chemotherapy, among which primary refractory HL confers a particularly poor outcome. With intensive salvage chemotherapy and autologous stem cell transplantation, the long-term remission rate for these patients was only 30%, but more selective treatments with higher therapeutic index are needed. We report the experience of using a new anti-CD30 immunotoxin, brentuximab vedotin, in salvage treatment of a 30-year-old woman with primary refractory Hodgkin lymphoma. The patient presented with SVC syndrome due to the bulky mediastinal tumor and was confirmed to have classical Hodgkin lymphoma, nodular sclerosis type, stage IIIA. The tumor responded to induction chemotherapy transiently, but local progression was noted during subsequent cycles of treatment. Salvage radiotherapy to the mediastinal tumor, obtained no remission but was followed by rapid in-field progression and then lung metastasis. She declined stem cell transplantation and received salvage brentuximab vedotin (BV therapy, which induced dramatic shrinkage of tumor without significant side effects. Serial followup of PET/CT imaging confirmed a rapid and continuous complete remission for 12 months. Although durability of the remission needs further observation, this case illustrates the excellent efficacy of brentuximab vedotin in primary refractory Hodgkin lymphoma.

Association of a Novel Nonsense Mutation in KIAA1279 with Goldberg-Shprintzen Syndrome.

Science.gov (United States)

Salehpour, Shadab; Hashemi-Gorji, Feyzollah; Soltani, Ziba; Ghafouri-Fard, Soudeh; Miryounesi, Mohammad

2017-01-01

Goldberg-Shprintzen syndrome (OMIM 609460) (GOSHS) is an autosomal recessive multiple congenital anomaly syndrome distinguished by intellectual disability, microcephaly, and dysmorphic facial characteristics. Most affected individuals also have Hirschsprung disease and/or gyral abnormalities of the brain. This syndrome has been associated with KIAA1279 gene mutations at 10q22.1. Here we report a 16 yr old male patient referred to Center for Comprehensive Genetic Services, Tehran, Iran in 2015 with cardinal features of GOSHS in addition to refractory seizures. Whole exome sequencing in the patient revealed a novel nonsense (stop gain) homozygous mutation in KIAA1279 gene (KIAA1279: NM_015634:exon6:c.C976T:p.Q326X). Considering the wide range of phenotypic variations in GOSHS, relying on phenotypic characteristics for discrimination of GOSH from similar syndromes may lead to misdiagnosis. Consequently, molecular diagnostic tools would help in accurate diagnosis of such overlapping phenotypes.

Alternative Chemical Amplification Methods for Peroxy Radical Detection

Science.gov (United States)

Wood, E. C. D.

2014-12-01

Peroxy radicals (HO2, CH3O2, etc.) are commonly detected by the chemical amplification technique, in which ambient air is mixed with high concentrations of CO and NO, initiating a chain reaction that produces 30 - 200 NO2 molecules per sampled peroxy radical. The NO2 is then measured by one of several techniques. With the exception of CIMS-based techniques, the chemical amplification method has undergone only incremental improvements since it was first introduced in 1982. The disadvantages of the technique include the need to use high concentrations of CO and the greatly reduced sensitivity of the amplification chain length in the presence of water vapor. We present a new chemical amplification scheme in which either ethane or acetaldehyde is used in place of CO, with the NO2 product detected using Cavity Attenuated Phase Shift spectroscopy (CAPS). Under dry conditions, the amplification factor of the alternative amplifiers are approximately six times lower than the CO-based amplifier. The relative humidity "penalty" is not as severe, however, such that at typical ambient relative humidity (RH) values the amplification factor is within a factor of three of the CO-based amplifier. Combined with the NO2 sensitivity of CAPS and a dual-channel design, the detection limit of the ethane amplifier is less than 2 ppt (1 minute average, signal-to-noise ratio 2). The advantages of these alternative chemical amplification schemes are improved safety, a reduced RH correction, and increased sensitivity to organic peroxy radicals relative to HO2.

Testing of the melter lid refractory for the West Valley Demonstration Project (WVDP)

International Nuclear Information System (INIS)

Gupta, A.; Jain, V.; Mahoney, J.L.; Holman, T.M.

1991-01-01

Monofrax H and Mulfrax 202 refractory were tested for potential application as the melter lid refractory for the WVDP. Resistance to spalling and corrosion by the slurry and offgas salts were primary criteria for selection. Test specimens were subjected to thermal cycling between 450 and 1,100C for five weeks. Visual examination indicated some corrosion but no spalling. SEM/EDS analysis was performed to determine the glass/refractory interface corrosion mechanism. The refractory selection basis will be discussed

Identification of novel mutations in the α-galactosidase A gene in patients with Fabry disease: pitfalls of mutation analyses in patients with low α-galactosidase A activity.

Science.gov (United States)

Yoshimitsu, Makoto; Higuchi, Koji; Miyata, Masaaki; Devine, Sean; Mattman, Andre; Sirrs, Sandra; Medin, Jeffrey A; Tei, Chuwa; Takenaka, Toshihiro

2011-05-01

Fabry disease is an X-linked lysosomal storage disorder caused by mutations of the α-galactosidase A (GLA) gene, and the disease is a relatively prevalent cause of left ventricular hypertrophy followed by conduction abnormalities and arrhythmias. Mutation analysis of the GLA gene is a valuable tool for accurate diagnosis of affected families. In this study, we carried out molecular studies of 10 unrelated families diagnosed with Fabry disease. Genetic analysis of the GLA gene using conventional genomic sequencing was performed in 9 hemizygous males and 6 heterozygous females. In patients with no mutations in coding DNA sequence, multiplex ligation-dependent probe amplification (MLPA) and/or cDNA sequencing were performed. We identified a novel exon 2 deletion (IVS1_IVS2) in a heterozygous female by MLPA, which was undetectable by conventional sequencing methods. In addition, the g.9331G>A mutation that has previously been found only in patients with cardiac Fabry disease was found in 3 unrelated, newly-diagnosed, cardiac Fabry patients by sequencing GLA genomic DNA and cDNA. Two other novel mutations, g.8319A>G and 832delA were also found in addition to 4 previously reported mutations (R112C, C142Y, M296I, and G373D) in 6 other families. We could identify GLA gene mutations in all hemizygotes and heterozygotes from 10 families with Fabry disease. Mutations in 4 out of 10 families could not be identified by classical genomic analysis, which focuses on exons and the flanking region. Instead, these data suggest that MLPA analysis and cDNA sequence should be considered in genetic testing surveys of patients with Fabry disease. Copyright © 2011 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

Effectiveness of botulinum toxin A in treatment of refractory erythromelalgia

Directory of Open Access Journals (Sweden)

Kuan-Hsiang Lin

2013-05-01

Full Text Available Erythromelalgia is characterized by intense burning pain, erythema, and heat in affected areas after precipitating factors such as warm temperature or stress. It is refractory to treatment in some situations. We describe a woman with adenosquamous cell carcinoma of the lung and medically refractory erythromelalgia. The symptoms of erythromelalgia presented as refractory to any medical treatment. Due to the unresponsive nature of her condition, botulinum toxin type A (onabotulinumtoxin A was injected over both of her cheeks, periodically for six cycles. Her symptoms responded dramatically to subcutaneous and intradermal injection of botulinum toxin type A. Repetitive injection demonstrated consistent and reproducible responses, and the efficacy was maintained for approximately 1 month. No adverse effects or complications were noted. Botulinum toxin type A might be safe and effective as an alternative treatment for refractory erythromelalgia, but further large-scale studies are required.

Rolling circle amplification of metazoan mitochondrialgenomes

Energy Technology Data Exchange (ETDEWEB)

Simison, W. Brian; Lindberg, D.R.; Boore, J.L.

2005-07-31

Here we report the successful use of rolling circle amplification (RCA) for the amplification of complete metazoan mt genomes to make a product that is amenable to high-throughput genome sequencing techniques. The benefits of RCA over PCR are many and with further development and refinement of RCA, the sequencing of organellar genomics will require far less time and effort than current long PCR approaches.

Effects of nitrogen ion irradiation on endoglucanase activity and gene mutation of Bacillus subtilis Bac01

International Nuclear Information System (INIS)

Lv Jie; Mao Peihong; Jin Xiang; Yu Long; Ying Hanjie

2009-01-01

Bacillus subtilis Bac01 was mutated by 15 keV N + ions of 1.5xl0 16 cm -2 . The mutant strain Bac11 with high yield of endoglucanase was isolated using carboxymethylcellulose sodium and congo red indicative plates. It exhibited higher endoglucanase activity (381.89IU) than the original strain Bac01 (93.33IU). Two 1,500 bp endoglucanase gene fragments were obtained with PCR amplification from B. subtilis Bac01 and mutant strain Bac11. BLAST comparison result indicated that 10 nucleotides mutated. Bioinformatics methods were used to analyze the two predicted amino acid sequences, and it was found that 5 amino acid residues changed, being all in the cellulose-binding domain of endoglucanase. (authors)

Theoretical and practical aspects about corrosion of refractories used in steel metallurgy: part 3: characterization of commercial refractories; Aspectos teoricos e praticos sobre a corrosao de refratarios utilizados na metalurgia: caracterizacao de refratarios comerciais

Energy Technology Data Exchange (ETDEWEB)

Braganca, S.R., E-mail: saulorb@ufrgs.br [Universidade Federal do Rio Grande Sul (DEMAT/UFRGS), RS (Brazil)

2012-07-01

In this study, it was reviewed the main aspects found in the literature about refractories corrosion, evaluating the feasibility of certain tests and relating them with experimental results. The physical properties and microstructure of commercial refractories were analyzed, considering the differences between them and the quality implications and probable life of the refractory. Thus, it was studied the various types of refractories used as lining on steel ladle. Magnesia-carbon and doloma-carbon refractories were analyzed, highlighting the differences between them. The examined refractory showed characteristics favoring high resistance to corrosion process, presenting a series of properties to be selected in accordance with industry practice. (author)

Interventional embolization for refractory hyperthyroidism

International Nuclear Information System (INIS)

Jiang Guomin; Zhang Yunping; Chen Yaxian; Zhao Jinwei; Yang Weihuai; Chen Jinluo

2001-01-01

Objective: To evaluate the clinical effects of the thyroid artery embolization for refractory hyperthyroidism. Methods: Super selective catheterization and interventional embolization of thyroid superior and inferior arteries were performed with PVA microspheres and Gelfoam particles. Changes of function and size of thyroid were investigated after embolization. Results: The procedures were succeeded in all 21 patients. Followed up by 3-12 months, serum level of thyroid hormones dropped significantly (median FT 3 from 17.1 pmol/L to 7.44 pmol/L, median FT 4 from 51.1 pmol/L to 23.3 pmol/L, P< 0.01). The size of thyroid glands decreased remarkably. Symptoms of hyperthyroidism were controlled in 4 patients and were effectively controlled through low dose antithyroid medication in 17 patients. No serious complications occurred. Conclusions: Thyroid artery embolization is a new safe and effective method for refractory hyperthyroidism

The use of high resolution melting analysis to detect Fabry mutations in heterozygous females via dry bloodspots.

Science.gov (United States)

Tai, Chang-Long; Liu, Mei-Ying; Yu, Hsiao-Chi; Chiang, Chiang-Chuan; Chiang, Hung; Suen, Jeng-Hung; Kao, Shu-Min; Huang, Yu-Hsiu; Wu, Tina Jui-Ting; Yang, Chia-Feng; Tsai, Fang-Chih; Lin, Ching-Yuang; Chang, Jan-Gowth; Chen, Hong-Duo; Niu, Dau-Ming

2012-02-18

As an X-linked genetic disorder, Fabry disease was first thought to affect males only, and females were generally considered to be asymptomatic carriers. However, recent research suggests that female carriers of Fabry disease may still develop vital organ damage causing severe morbidity and mortality. In the previous newborn screening, from 299,007 newborns, we identified a total of 20 different Fabry mutations and 121 newborns with Fabry mutations. However, we found that most female carriers are not detected by enzyme assays. A streamlined method for high resolution melting (HRM) analysis was designed to screen for GLA gene mutations using a same PCR and melting program. Primer sets were designed to cover the 7 exons and the Chinese common intronic mutation, IVS4+919G>A of GLA gene. The HRM analysis was successful in identifying heterozygous and hemizygous patients with the 20 surveyed mutations. We were also successful in using this method to test dry blood spots of newborns afflicted with Fabry mutations without having to determine DNA concentration before PCR amplification. The results of this study show that HRM could be a reliable and sensitive method for use in the rapid screening of females for GLA mutations. Copyright © 2011 Elsevier B.V. All rights reserved.

Efficacy of infliximab in refractory posterior uveitis in Behcet's disease patients

OpenAIRE

Ayman K. El Garf; Amira A. Shahin; Sherif A. Shawky; Mohammed A. Azim; Dina A. Effat; Sherry K. Abdelrahman

2018-01-01

Aim of the work: Ocular manifestations are the main cause of morbidity in Behcet's disease (BD). Infliximab (IFX), a chimeric monoclonal antibody directed against tumor necrosis factor-alpha, may be efficient in refractory uveitis due to BD. The aim of this study was to assess the efficacy and safety of IFX in the treatment of patients with BD-associated refractory posterior uveitis (PU). Patient and Methods: Twenty patients with refractory Behcet's PU received IFX therapy as intravenous infu...

Progression inference for somatic mutations in cancer

Directory of Open Access Journals (Sweden)

Leif E. Peterson

2017-04-01

Full Text Available Computational methods were employed to determine progression inference of genomic alterations in commonly occurring cancers. Using cross-sectional TCGA data, we computed evolutionary trajectories involving selectivity relationships among pairs of gene-specific genomic alterations such as somatic mutations, deletions, amplifications, downregulation, and upregulation among the top 20 driver genes associated with each cancer. Results indicate that the majority of hierarchies involved TP53, PIK3CA, ERBB2, APC, KRAS, EGFR, IDH1, VHL, etc. Research into the order and accumulation of genomic alterations among cancer driver genes will ever-increase as the costs of nextgen sequencing subside, and personalized/precision medicine incorporates whole-genome scans into the diagnosis and treatment of cancer. Keywords: Oncology, Cancer research, Genetics, Computational biology

Lidar using the backscatter amplification effect

Science.gov (United States)

Razenkov, Igor A.; Banakh, Victor A.

2018-04-01

Experimental data proving the possibility of lidar measurement of the refractive turbulence strength based on the effect of backscatter amplification (BSA) are reported. It is shown that the values of the amplification factor correlate with the variance of random jitter of optical image of an incoherent light source depending on the value of the structure constant of the air refractive index turbulent fluctuations averaged over the probing path. This paper presents the results of measurements of the BSA factor in comparison with the simultaneous measurements of the BSA peak, which is very narrow and only occurs on the laser beam axis. It is constructed the range-time images of the derivative of the amplification factor gives a comprehensive picture of the location of turbulent zones and their temporal dynamics.

Ketogenic diet in 3 cases of childhood refractory status epilepticus

DEFF Research Database (Denmark)

Sort, Rune; Born, Alfred P; Pedersen, Karen N.

2013-01-01

Refractory status epilepticus (RSE) in children is associated with a significant risk of death or neurological morbidity. Recently attention has been drawn to the ketogenic diet (KD) as an acute treatment, as it has shown promise in controlling seizures in otherwise refractory status epilepticus...

Perspectives on environmental protection of refractory metals

International Nuclear Information System (INIS)

Perking, R.A.

1992-01-01

Alloys of refractory metals which combine high strength to weight ratios with useful low-temperature toughness and ductility have been designed to meet the requirements for the next generation of high-temperature aerospace structural materials with one exception: long term resistance to oxidation. It is considered unlikely that refractory metal alloys can be modified to possess useful resistance to oxidation as bulk materials or that coating can be designed to provide high reliability fail-safe protection for the structural alloys developed to date. Recent developments indicate that bulk alloys of W and Mo can be designed to possess short term resistance to oxidation, providing a base material which can be coated to extend useful life with fail-safe protection in the event of random coating failures. Current research on silicide coatings indicates that significant improvements in coating reliability and performance also are feasible. The technical basis for these conclusions is presented in this paper and the direction of future work that could lead to environmentally stable refractory metal alloy/coating systems is discussed

HER2 overexpression and amplification as a potential therapeutic target in colorectal cancer: analysis of 3256 patients enrolled in the QUASAR, FOCUS and PICCOLO colorectal cancer trials.

Science.gov (United States)

Richman, Susan D; Southward, Katie; Chambers, Philip; Cross, Debra; Barrett, Jennifer; Hemmings, Gemma; Taylor, Morag; Wood, Henry; Hutchins, Gordon; Foster, Joseph M; Oumie, Assa; Spink, Karen G; Brown, Sarah R; Jones, Marc; Kerr, David; Handley, Kelly; Gray, Richard; Seymour, Matthew; Quirke, Philip

2016-03-01

HER2 overexpression/amplification is linked to trastuzumab response in breast/gastric cancers. One suggested anti-EGFR resistance mechanism in colorectal cancer (CRC) is aberrant MEK-AKT pathway activation through HER2 up-regulation. We assessed HER2-amplification/overexpression in stage II-III and IV CRC patients, assessing relationships to KRAS/BRAF and outcome. Pathological material was obtained from 1914 patients in the QUASAR stage II-III trial and 1342 patients in stage IV trials (FOCUS and PICCOLO). Tissue microarrays were created for HER2 immunohistochemistry. HER2-amplification was assessed using FISH and copy number variation. KRAS/BRAF mutation status was assessed by pyrosequencing. Progression-free survival (PFS) and overall survival (OS) data were obtained for FOCUS/PICCOLO and recurrence and mortality for QUASAR; 29/1342 (2.2%) stage IV and 25/1914 (1.3%) stage II-III tumours showed HER2 protein overexpression. Of the HER2-overexpressing cases, 27/28 (96.4%) stage IV tumours and 20/24 (83.3%) stage II-III tumours demonstrated HER2 amplification by FISH; 41/47 (87.2%) also showed copy number gains. HER2-overexpression was associated with KRAS/BRAF wild-type (WT) status at all stages: in 5.2% WT versus 1.0% mutated tumours (p < 0.0001) in stage IV and 2.1% versus 0.2% in stage II-III tumours (p = 0.01), respectively. HER2 was not associated with OS or PFS. At stage II-III, there was no significant correlation between HER2 overexpression and 5FU/FA response. A higher proportion of HER2-overexpressing cases experienced recurrence, but the difference was not significant. HER2-amplification/overexpression is identifiable by immunohistochemistry, occurring infrequently in stage II-III CRC, rising in stage IV and further in KRAS/BRAF WT tumours. The value of HER2-targeted therapy in patients with HER2-amplified CRC must be tested in a clinical trial. © 2015 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society

Super Refractory Status Epilepticus

African Journals Online (AJOL)

user

et al did retrospective cohort study from 1 January st. 1994 to 31 March 1998 at Presbyterian Medical. Centre in Columbia, to determine the frequency, risk factors and impact on the outcome of RSE. They found out that 69% of seizures recurred after. Key Words: Super refractory status epilepticus, Zambia. Medical Journal of ...

Process technology - rare and refractory metals

International Nuclear Information System (INIS)

Gupta, C.K.; Bose, D.K.

1989-01-01

India has fairly rich resreves of rare and refractory metals. Abundant sources of ilmenite, rutile, zircon and rare earths are found in the placer deposits of the southern and eastern coasts of the country. Columbite-tantalite occur in mica and the mining belts of Bihar and cassiterite deposits are found in Bastar (Madhya Pradesh). Vanadium as a minor associate occurs in bauxites and in the vast deposits of titaniferrous magnetites. Over the years, research and development and pilot plant works in many research organisations in India have built up a sound technological base in the country for process metallurgy of many refractory and rare earth metals starting from their indigenous sources. The present paper provides a comprehensive view of the developments that have taken place till now on the processing of various refractory and rare earth metals with particular reference to the extensive work carried out at the Department of Atomic Energy. The coverage includes mineral benification separation of individual elements, preparation of pure intermediates, techniques of reduction to metal and final purification. The paper also reviews some of the recent developments that have been taken place in these fields and the potential application of these metals in the foreseeable future. (author). 22 refs., 18 fi g., 7 tabs

Reuse of ultrafine mineral wool production waste in the manufacture of refractory concrete.

Science.gov (United States)

Stonys, R; Kuznetsov, D; Krasnikovs, A; Škamat, J; Baltakys, K; Antonovič, V; Černašėjus, O

2016-07-01

The paper deals with the mineral wool production waste (cupola dust - CD), presents CD characterization and aims to reuse CD in production of refractory concrete with calcium aluminate cement. The study of CD covers its chemical, phase and thermal analyses along with the morphological study and determination of particles size distribution. Zeta-potential, electrical conductivity and pH values of CD suspension are presented in the paper as well. Commercial microsilica additive in refractory concrete has been replaced with cupola dust. Compositions of refractory concrete have been prepared by incorporating 1%, 2% and 3% of CD. The bulk density, ultrasonic wave velocity, cold crushing strength and thermal shock resistance of the created refractory concrete have been determined. Based on experimental results, it has been found that cupola dust may be used for the production of refractory concrete. The environmental impact related to the CD reuse in refractory concrete production has been evaluated as well. Copyright © 2016 Elsevier Ltd. All rights reserved.

When myasthenia gravis is deemed refractory: clinical signposts and treatment strategies

Science.gov (United States)

Mantegazza, Renato; Antozzi, Carlo

2018-01-01

The prognosis for patients with myasthenia gravis (MG) has improved significantly over the past half century, including substantial reductions in mortality and morbidity. However, approximately 10% of patients fails to respond adequately to current therapies and are considered treatment refractory, or treatment intolerant, and up to 80% have disease that fails to achieve complete stable remission. Although patients with autoantibodies to muscle-specific tyrosine kinase (anti-MuSK positive) are more likely to become treatment refractory than those with autoantibodies to the acetylcholine receptor (anti-AChR positive), each of these serotypes is substantially represented in the refractory MG population. Other risk factors for becoming treatment refractory include history of thymoma or thymectomy and female sex. A modified treatment algorithm for MG is proposed: patients who have disease that fails to respond to the stepwise approach to therapy, are treatment intolerant, or who require chronic rescue measures despite ongoing therapy, should be considered treatment refractory and emerging therapies should be considered. Three emerging monoclonal antibody-based therapies are discussed: the anti-B-cell agent rituximab; the terminal complement activation inhibitor eculizumab; and belimumab, which targets B-cell activating factor. Increased understanding of molecular pathophysiology and accurate antibody subtyping in MG should lead to the use of new therapeutic agents and successful management of treatment-refractory patients. PMID:29403543

EPSPS gene amplification conferring resistance to glyphosate in windmill grass (Chloris truncata) in Australia.

Science.gov (United States)

Ngo, The D; Malone, Jenna M; Boutsalis, Peter; Gill, Gurjeet; Preston, Christopher

2018-05-01

Five glyphosate-resistant populations of Chloris truncata originally collected from New South Wales were compared with one susceptible (S) population from South Australia to confirm glyphosate resistance and elucidate possible mechanisms of resistance. Based on the amounts of glyphosate required to kill 50% of treated plants (LD 50 ), glyphosate resistance (GR) was confirmed in five populations of C. truncata (A536, A528, T27, A534 and A535.1). GR plants were 2.4-8.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. There was no difference in glyphosate absorption and translocation between GR and S plants. The EPSPS gene did not contain any point mutation that had previously been associated with resistance to glyphosate. The resistant plants (A528 and A536) contained up to 32-48 more copies of the EPSPS gene than the susceptible plants. This study has identified EPSPS gene amplification contributing to glyphosate resistance in C. truncata. In addition, a Glu-91-Ala mutation within EPSPS was identified that may contribute to glyphosate resistance in this species. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

[PAX3 gene mutation analysis for two Waardenburg syndrome type Ⅰ families and their prenatal diagnosis].

Science.gov (United States)

Bai, Y; Liu, N; Kong, X D; Yan, J; Qin, Z B; Wang, B

2016-12-07

Objective: To analyze the mutations of PAX3 gene in two Waardenburg syndrome type Ⅰ (WS1) pedigrees and make prenatal diagnosis for the high-risk 18-week-old fetus. Methods: PAX3 gene was first analyzed by Sanger sequencing and multiplex ligation-dependent probe amplification(MLPA) for detecting pathogenic mutation of the probands of the two pedigrees. The mutations were confirmed by MLPA and Sanger in parents and unrelated healthy individuals.Prenatal genetic diagnosis for the high-risk fetus was performed by amniotic fluid cell after genotyping. Results: A heterozygous PAX3 gene gross deletion (E7 deletion) was identified in all patients from WS1-01 family, and not found in 20 healthy individuals.Prenatal diagnosis in WS1-01 family indicated that the fetus was normal. Molecular studies identified a novel deletion mutation c. 1385_1386delCT within the PAX3 gene in all affected WS1-02 family members, but in none of the unaffected relatives and 200 healthy individuals. Conclusions: PAX3 gene mutation is etiological for two WS1 families. Sanger sequencing plus MLPA is effective and accurate for making gene diagnosis and prenatal diagnosis.

Refractory organic substances in the environment

National Research Council Canada - National Science Library

Frimmel, F. H

2002-01-01

... and its Quality Control in Fractions of Refractory Organic Substances and the Corresponding Original Water Samples 39 Introduction 39 Description of Analytical Methods 41 Sample Treatment 41 Fl...

Evaluation of Alternative Refractory Materials for the Main Flame Deflectors at KSC Launch Complexes

Science.gov (United States)

Calle, Luz Marina; Trejo, David; Rutkowsky, Justin

2006-01-01

The deterioration of the refractory materials used to protect the KSC launch complex steel base structures from the high temperatures during launches results in frequent and costly repairs and safety hazards. KSC-SPEC-P-0012, Specification for Refractory Concrete, is ineffective in qualifying refractory materials. This study of the specification and of alternative refractory materials recommends a complete revision of the specification and further investigation of materials that were found to withstand the environment of the Solid Rocket Booster main flame deflector better than the refractory materials in current use in terms of compressive strength, tensile strength, modulus of rupture, shrinkage, and abrasion.

Activating thyrotropin receptor mutations are present in nonadenomatous hyperfunctioning nodules of toxic or autonomous multinodular goiter.

Science.gov (United States)

Tonacchera, M; Agretti, P; Chiovato, L; Rosellini, V; Ceccarini, G; Perri, A; Viacava, P; Naccarato, A G; Miccoli, P; Pinchera, A; Vitti, P

2000-06-01

Toxic multinodular goiter, a heterogeneous disease producing hyperthyroidism, is frequently found in iodine-deficient areas. The pathogenesis of this common clinical entity is still unclear. The aim of the present study was to search for activating TSH receptor (TSHr) or Gs alpha mutations in areas of toxic or functionally autonomous multinodular goiters that appeared hyperfunctioning at thyroid scintiscan but did not clearly correspond to definite nodules at physical or ultrasonographic examination. Surgical tissue specimens from nine patients were carefully dissected, matching thyroid scintiscan and thyroid ultrasonography, to isolate hyperfunctioning and nonfunctioning areas even if they did not correspond to well-defined nodules. TSHr and Gs alpha mutations were searched for by direct sequencing after PCR amplification of genomic DNA. Only 2 adenomas were identified at microscopic examination, whereas the remaining 18 hyperfunctioning areas corresponded to hyperplastic nodules containing multiple aggregates of micromacrofollicules not surrounded by a capsule. Activating TSHr mutations were detected in 14 of these 20 hyperfunctioning areas, whereas no mutation was identified in nonfunctioning nodules or areas contained in the same gland. No Gs alpha mutation was found. In conclusion, activating TSHr mutations are present in the majority of nonadenomatous hyperfunctioning nodules scattered throughout the gland in patients with toxic or functionally autonomous multinodular goiter.

Spheromak Impedance and Current Amplification

International Nuclear Information System (INIS)

Fowler, T K; Hua, D D; Stallard, B W

2002-01-01

It is shown that high current amplification can be achieved only by injecting helicity on the timescale for reconnection, τ REC , which determines the effective impedance of the spheromak. An approximate equation for current amplification is: dI TOR 2 /dt ∼ I 2 /τ REC - I TOR 2 /τ closed where I is the gun current, I TOR is the spheromak toroidal current and τ CLOSED is the ohmic decay time of the spheromak. Achieving high current amplification, I TOR >> I, requires τ REC CLOSED . For resistive reconnection, this requires reconnection in a cold zone feeding helicity into a hot zone. Here we propose an impedance model based on these ideas in a form that can be implemented in the Corsica-based helicity transport code. The most important feature of the model is the possibility that τ REC actually increases as the spheromak temperature increases, perhaps accounting for the ''voltage sag'' observed in some experiments, and a tendency toward a constant ratio of field to current, B ∝ I, or I TOR ∼ I. Program implications are discussed

Direct amplification of casework bloodstains using the Promega PowerPlex(®) 21 PCR amplification system.

Science.gov (United States)

Gray, Kerryn; Crowle, Damian; Scott, Pam

2014-09-01

A significant number of evidence items submitted to Forensic Science Service Tasmania (FSST) are blood swabs or bloodstained items. Samples from these items routinely undergo phenol:chloroform:isoamyl alcohol organic extraction and quantitative Polymerase Chain Reaction (qPCR) testing prior to PowerPlex(®) 21 amplification. This multi-step process has significant cost and timeframe implications in a fiscal climate of tightening government budgets, pressure towards improved operating efficiencies, and an increasing emphasis on rapid techniques better supporting intelligence-led policing. Direct amplification of blood and buccal cells on cloth and Whatman FTA™ card with PowerPlex(®) 21 has already been successfully implemented for reference samples, eliminating the requirement for sample pre-treatment. Scope for expanding this method to include less pristine casework blood swabs and samples from bloodstained items was explored in an endeavour to eliminate lengthy DNA extraction, purification and qPCR steps for a wider subset of samples. Blood was deposited onto a range of substrates including those historically found to inhibit STR amplification. Samples were collected with micro-punch, micro-swab, or both. The potential for further fiscal savings via reduced volume amplifications was assessed by amplifying all samples at full and reduced volume (25 and 13μL). Overall success rate data showed 80% of samples yielded a complete profile at reduced volume, compared to 78% at full volume. Particularly high success rates were observed for the blood on fabric/textile category with 100% of micro-punch samples yielding complete profiles at reduced volume and 85% at full volume. Following the success of this trial, direct amplification of suitable casework blood samples has been implemented at reduced volume. Significant benefits have been experienced, most noticeably where results from crucial items have been provided to police investigators prior to interview of

Molecular Profiling of Refractory Adrenocortical Cancers and Predictive Biomarkers to Therapy

Directory of Open Access Journals (Sweden)

Sherri Z. Millis

2015-01-01

Full Text Available Purpose Current first-line chemotherapy for patients with metastatic adrenocortical cancer (ACC includes doxorubicin, etoposide, cisplatin, and mitotane with a reported response rate of only 23.2%. New therapeutic leads for patients with refractory tumors are needed; there is no standard second-line treatment. Methods Samples from 135 ACC tumors were analyzed by immunohistochemistry, in situ hybridization (FISH or CISH, and/or gene sequencing at a single commercial reference laboratory (Caris Life Sciences to identify markers associated with drug sensitivity and resistance. Results Overexpression of proteins related to demonstrated chemotherapy sensitivity or resistance included topoisomerase 1, progesterone receptor, and topoisomerase 2-alpha in 46%, 63%, and 42% of cases, respectively. Loss of excision repair cross-complementary group 1 (ERCC1, phosophatase and tensin homolog, O(6-methylguanine-methyltransferase, and ribonucleotide reductase M1 (RRM1 was identified in 56%, 59%, 71%, and 58% of cases, respectively. Other aberrations included overexpression of programmed death-ligand 1 or programmed cell death protein 1 tumor-infiltrating lymphocytes in >40% of cases. In all, 35% of cases had a mutation in the canonical Wnt signaling pathway (either CTNNB1 or APC and 48% had a mutation in TP53. No other genomic alterations were identified. Conclusion Biomarker alterations in ACC may be used to direct therapies, including recommendations for and potential resistance of some patients to traditional chemotherapies, which may explain the low response rate in the unselected population. Limited outcomes data support the use of mitotane and platinum therapies for patients with low levels of the proteins RRM1 and ERCC1.

Therapeutic burst-suppression coma in pediatric febrile refractory status epilepticus.

Science.gov (United States)

Lin, Jainn-Jim; Chou, Cheng-Che; Lan, Shih-Yun; Hsiao, Hsiang-Ju; Wang, Yu; Chan, Oi-Wa; Hsia, Shao-Hsuan; Wang, Huei-Shyong; Lin, Kuang-Lin

2017-09-01

Evidence for the beneficial effect of therapeutic burst-suppression coma in pediatric patients with febrile refractory status epilepticus is limited, and the clinical outcomes of this treatment strategy are largely unknown. Therefore, the aim of this study was to explore the outcomes of therapeutic burst-suppression coma in a series of children with febrile refractory status epilepticus. We retrospectively reviewed consecutive pediatric patients with febrile refractory status epilepticus admitted to our pediatric intensive care unit between January 2000 and December 2013. The clinical characteristics were analyzed. Thirty-five patients (23 boys; age range: 1-18years) were enrolled, of whom 28 (80%) developed super-refractory status epilepticus. All of the patients received the continuous administration of intravenous antiepileptic drugs for febrile refractory status epilepticus, and 26 (74.3%) achieved therapeutic burst-suppression coma. All of the patients received mechanical ventilatory support, and 26 (74.3%) received inotropic agents. Eight (22.9%) patients died within 1month. The neurologically functional outcomes at 6months were good in six (27.3%) of the 22 survivors, of whom two returned to clinical baseline. The patients with therapeutic burst-suppression coma were significantly associated with hemodynamic support than the patients with electrographic seizures control (p=0.03), and had a trend of higher 1-month mortality rate, worse 6months outcomes, and a longer duration of hospitalization. Our results suggest that therapeutic burst-suppression coma to treat febrile refractory status epilepticus may lead to an increased risk of hemodynamic instability and a trend of worse outcomes. Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Zirconium determination in refractories (gravimetric method)

International Nuclear Information System (INIS)

Capiotto, N.; Narahashi, Y.; Perish, P.G.; Souza, J.R. de

1991-01-01

A gravimetric method for zirconium determination in refractories is described. X-ray fluorescence analysis is also employed in this experiment and considerations about interfering elements are presented. (M.V.M.)

Development of various welding techniques for refractory and reactive metals and alloys

International Nuclear Information System (INIS)

Tonpe, Sunil; Saibaba, N.

2016-01-01

Nuclear Fuel Complex (NFC), Hyderabad, India with its excellent manufacturing facilities, produces nuclear fuel and structural components for nuclear reactors. NFC has taken up the challenging job of production of various critical components made out of refractory and reactive metals and alloys for nuclear and aerospace applications as an indigenization import substitute program. Refractory metals are prime candidates for many high temperature aerospace components because of refractory metal's high melting points and inherent creep resistance. The use of refractory metals is often limited because of their poor room temperature properties, inadequate oxidation resistance at elevated temperatures, difficulties associated with joining or welding etc. These advanced materials demand stringent requirement with respect to chemistry, dimensional tolerances, mechanical and metallurgical properties. This paper discusses in detail various welding techniques adopted in NFC for refractory and reactive metals and alloys such as Nb, Zr, Ti, Ta, Zircaloy, Titanium-half alloy etc. to manufacture various components and assemblies required for nuclear and aerospace applications

Applications of radioisotopes for studying refractory wear-out in Bhilai Steel Plant

International Nuclear Information System (INIS)

Dubey, R.S.; Bose, U.P.; Shipstone, A.J.

1979-01-01

In Bhilai Steel Plant, investigations were carried out to study the refractory wear-out of (i) hearth bottom of blast furnaces, (ii) roof of open hearth furnaces, and (iii) hot metal mixer lining, by using radioisotope tracer techniques with a view to evaluate the life of the refractory lining at various locations and to help in planning its timely hot and cold repairs. The life of the refractory lining has the effective bearing on the overall production and hence on the economy of the plant. The two radiometric methods employed for studying the erosion of the refractory lining, by using isotope inserted bricks at various positions without damaging the lining are (i) based on recording the penetration of gamma rays emitting from the radioactive isotopes inserted at definite points of the brick lining and, (ii) by detecting the radioactivity of the pig iron or steel arising due to washing away of the respective radioactive isotopes previously inserted in the lining. In hot mixers also radioisotope sources were placed in the critical location of refractory lining and the washing out of radioisotope due to refractory brick wear out was detected by radiogauging at site. It has been found that radiotracer technique with periodic radiogauging is very useful method for tracing the radioisotope source if more than one refractory brick with isotope is placed, as in the case of open hearth furnaces. The results of radioanalysis revealed that radioactivity coming alongwith hot metal steel has been far below the permissible limit of concentration i.e. 20 micro-curie per ton of metal. Further, during dismantling of the residual refractory lining of open hearth furnaces or hot metal mixers, bricks containing radioisotopes have been successfully retrieved for safe disposal. (auth.)

Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification.

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Emara, Mohamed M; Liu, Hsuan; Davis, William G; Brinton, Margo A

2008-11-01

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

Intellectual Ability in the Duchenne Muscular Dystrophy and Dystrophin Gene Mutation Location

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Rasic Milic V.

2014-12-01

Full Text Available Duchenne muscular dystrophy (DMD is the most common form of muscular dystrophy during childhood. Mutations in dystrophin (DMD gene are also recognized as a cause of cognitive impairment. We aimed to determine the association between intelligence level and mutation location in DMD genes in Serbian patients with DMD. Forty-one male patients with DMD, aged 3 to 16 years, were recruited at the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia. All patients had defined DMD gene deletions or duplications [multiplex ligation- dependent probe amplification (MLPA, polymerase chain reaction (PCR] and cognitive status assessment (Wechsler Intelligence Scale for Children, Brunet-Lezine scale, Vineland-Doll scale. In 37 patients with an estimated full scale intelligence quotient (FSIQ, six (16.22% had borderline intelligence (70mutations when boundaries were set at exons 30 and 45. However, FSIQ was statistically significantly associated with mutation location when we assumed their functional consequence on dystrophin isoforms and when mutations in the 5’-untranslated region (5’UTR of Dp140 (exons 45-50 were assigned to affect only Dp427 and Dp260. Mutations affecting Dp140 and Dp71/Dp40 have been associated with more frequent and more severe cognitive impairment. Finally, the same classification of mutations explained the greater proportion of FSIQ variability associated with cumulative loss of dystrophin isoforms. In conclusion, cumulative loss of dystrophin isoforms increases the risk of intellectual impairment in DMD and characterizing the genotype can define necessity of early cognitive interventions in DMD patients.

Prenatal diagnosis of beta-thalassaemia: experience in a developing country.

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Saxena, R; Jain, P K; Thomas, E; Verma, I C

1998-01-01

We present our experience with the amplification refractory mutation system (ARMS) for the prenatal diagnosis of beta-thalassaemia in 415 pregnancies of 360 women. Five mutations of the beta-thalassaemia gene common in Asian Indians accounted for 89.2 per cent and rare mutations for 7.2 per cent of all mutant chromosomes, while 3.3 per cent of chromosomes remained uncharacterized. Identical mutations were present in both parents in 43.2 per cent of cases, due to caste-based marriages in India. A confirmed diagnosis was given in 401 (98.3 per cent) cases, of which a complete diagnosis (whether the fetus was normal, a carrier, or homozygous) was possible in 391 (94.2 per cent) of the cases. In 15 couples, the mutation was identified in only one parent. In nine of these, the identified mutation was not present in the fetus, predicting normal/carrier status, while in five the identified mutation was present in the fetus, suggesting carrier/affected status. The abortion rate was 3.9 per cent. Pitfalls in diagnosis were failure of oligonucleotides to work, maternal contamination, and false paternity. The ARMS provides an inexpensive, robust and non-isotopic method for the prenatal diagnosis of beta-thalassaemia in India. Recommendations are outlined for establishing a prenatal diagnostic service in developing countries.

Asexual populations of the human malaria parasite, Plasmodium falciparum, use a two-step genomic strategy to acquire accurate, beneficial DNA amplifications.

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Jennifer L Guler

Full Text Available Malaria drug resistance contributes to up to a million annual deaths. Judicious deployment of new antimalarials and vaccines could benefit from an understanding of early molecular events that promote the evolution of parasites. Continuous in vitro challenge of Plasmodium falciparum parasites with a novel dihydroorotate dehydrogenase (DHODH inhibitor reproducibly selected for resistant parasites. Genome-wide analysis of independently-derived resistant clones revealed a two-step strategy to evolutionary success. Some haploid blood-stage parasites first survive antimalarial pressure through fortuitous DNA duplications that always included the DHODH gene. Independently-selected parasites had different sized amplification units but they were always flanked by distant A/T tracks. Higher level amplification and resistance was attained using a second, more efficient and more accurate, mechanism for head-to-tail expansion of the founder unit. This second homology-based process could faithfully tune DNA copy numbers in either direction, always retaining the unique DNA amplification sequence from the original A/T-mediated duplication for that parasite line. Pseudo-polyploidy at relevant genomic loci sets the stage for gaining additional mutations at the locus of interest. Overall, we reveal a population-based genomic strategy for mutagenesis that operates in human stages of P. falciparum to efficiently yield resistance-causing genetic changes at the correct locus in a successful parasite. Importantly, these founding events arise with precision; no other new amplifications are seen in the resistant haploid blood stage parasite. This minimizes the need for meiotic genetic cleansing that can only occur in sexual stage development of the parasite in mosquitoes.

Added Value of HER-2 Amplification Testing by Multiplex Ligation-Dependent Probe Amplification in Invasive Breast Cancer

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Kuijpers, Chantal C. H. J.; Moelans, Cathy B.; van Slooten, Henk-Jan; Horstman, Anja; Hinrichs, John W. J.; Al-Janabi, Shaimaa; van Diest, Paul J.; Jiwa, Mehdi

2013-01-01

Background HER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA) and chromogenic in situ hybridization (CISH). Methods As local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases). Results The validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases). Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (pCISH was 98.1% (575/586) (Kappa = 0.94). Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining. Conclusions MLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well. PMID:24324739

Added value of HER-2 amplification testing by multiplex ligation-dependent probe amplification in invasive breast cancer.

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Chantal C H J Kuijpers

Full Text Available BACKGROUND: HER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA and chromogenic in situ hybridization (CISH. METHODS: As local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases. RESULTS: The validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases. Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (p<0.0001. Overall concordance between IHC and MLPA/CISH was 98.1% (575/586 (Kappa = 0.94. Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining. CONCLUSIONS: MLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well.

A single splice site mutation in human-specific ARHGAP11B causes basal progenitor amplification

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Florio, Marta; Namba, Takashi; Pääbo, Svante; Hiller, Michael; Huttner, Wieland B.

2016-01-01

The gene ARHGAP11B promotes basal progenitor amplification and is implicated in neocortex expansion. It arose on the human evolutionary lineage by partial duplication of ARHGAP11A, which encodes a Rho guanosine triphosphatase–activating protein (RhoGAP). However, a lack of 55 nucleotides in ARHGAP11B mRNA leads to loss of RhoGAP activity by GAP domain truncation and addition of a human-specific carboxy-terminal amino acid sequence. We show that these 55 nucleotides are deleted by mRNA splicing due to a single C→G substitution that creates a novel splice donor site. We reconstructed an ancestral ARHGAP11B complementary DNA without this substitution. Ancestral ARHGAP11B exhibits RhoGAP activity but has no ability to increase basal progenitors during neocortex development. Hence, a single nucleotide substitution underlies the specific properties of ARHGAP11B that likely contributed to the evolutionary expansion of the human neocortex. PMID:27957544

Prediction of BRAF mutation status of craniopharyngioma using magnetic resonance imaging features.

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Yue, Qi; Yu, Yang; Shi, Zhifeng; Wang, Yongfei; Zhu, Wei; Du, Zunguo; Yao, Zhenwei; Chen, Liang; Mao, Ying

2017-10-06

OBJECTIVE Treatment with a BRAF mutation inhibitor might shrink otherwise refractory craniopharyngiomas and is a promising preoperative treatment to facilitate tumor resection. The aim of this study was to investigate the noninvasive diagnosis of BRAF-mutated craniopharyngiomas based on MRI characteristics. METHODS Fifty-two patients with pathologically diagnosed craniopharyngioma were included in this study. Polymerase chain reaction was performed on tumor tissue specimens to detect BRAF and CTNNB1 mutations. MRI manifestations-including tumor location, size, shape, and composition; signal intensity of cysts; enhancement pattern; pituitary stalk morphology; and encasement of the internal carotid artery-were analyzed by 2 neuroradiologists blinded to patient identity and clinical characteristics, including BRAF mutation status. Results were compared between the BRAF-mutated and wild-type (WT) groups. Characteristics that were significantly more prevalent (p < 0.05) in the BRAF-mutated craniopharyngiomas were defined as diagnostic features. The minimum number of diagnostic features needed to make a diagnosis was determined by analyzing the receiver operating characteristic (ROC) curve. RESULTS Eight of the 52 patients had BRAF-mutated craniopharyngiomas, and the remaining 44 had BRAF WT tumors. The clinical characteristics did not differ significantly between the 2 groups. Interobserver agreement for MRI data analysis was relatively reliable, with values of Cohen κ ranging from 0.65 to 0.97 (p < 0.001). A comparison of findings in the 2 patient groups showed that BRAF-mutated craniopharyngiomas tended to be suprasellar (p < 0.001), spherical (p = 0.005), predominantly solid (p = 0.003), and homogeneously enhancing (p < 0.001), and that patients with these tumors tended to have a thickened pituitary stalk (p = 0.014). When at least 3 of these 5 features were present, a tumor might be identified as BRAF mutated with a sensitivity of 1.00 and a specificity of 0

Fragmentation process of vitrified ceramic waste (VCW) aiming its incorporation in silico-aluminous refractory concrete for production of refractory bricks

International Nuclear Information System (INIS)

Gomes, L.B.; Brandalise, R.N.; Santos, V. dos; Bergmann, C.P.

2012-01-01

Ceramic industry generates large amounts of waste, usually disposed in landfills. Reuse could minimize their generation and provides sustainable solutions. However, the energy cost of grinding these waste becomes a hindrance to their reuse. This work aims to obtain particle sizes of vitrified ceramic waste (VCW) using a fast, efficient and low cost fragmentation process as well as its use in refractory concrete. The results shows a wide range of particle size of VCW, which can be used as a promising source of raw material for production of refractory concrete. (author)

Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

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Hoser, Mark J; Mansukoski, Hannu K; Morrical, Scott W; Eboigbodin, Kevin E

2014-01-01

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

Strand Invasion Based Amplification (SIBA®: a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

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Mark J Hoser

Full Text Available Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA. SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

A Clinical and Molecular Genetic Study of 50 Families with Autosomal Recessive Parkinsonism Revealed Known and Novel Gene Mutations.

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Taghavi, Shaghayegh; Chaouni, Rita; Tafakhori, Abbas; Azcona, Luis J; Firouzabadi, Saghar Ghasemi; Omrani, Mir Davood; Jamshidi, Javad; Emamalizadeh, Babak; Shahidi, Gholam Ali; Ahmadi, Mona; Habibi, Seyed Amir Hassan; Ahmadifard, Azadeh; Fazeli, Atena; Motallebi, Marzieh; Petramfar, Peyman; Askarpour, Saeed; Askarpour, Shiva; Shahmohammadibeni, Hossein Ali; Shahmohammadibeni, Neda; Eftekhari, Hajar; Shafiei Zarneh, Amir Ehtesham; Mohammadihosseinabad, Saeed; Khorrami, Mehdi; Najmi, Safa; Chitsaz, Ahmad; Shokraeian, Parasto; Ehsanbakhsh, Hossein; Rezaeidian, Jalal; Ebrahimi Rad, Reza; Madadi, Faranak; Andarva, Monavvar; Alehabib, Elham; Atakhorrami, Minoo; Mortazavi, Seyed Erfan; Azimzadeh, Zahra; Bayat, Mahdis; Besharati, Amir Mohammad; Harati-Ghavi, Mohammad Ali; Omidvari, Samareh; Dehghani-Tafti, Zahra; Mohammadi, Faraz; Mohammad Hossein Pour, Banafsheh; Noorollahi Moghaddam, Hamid; Esmaili Shandiz, Ehsan; Habibi, Arman; Taherian-Esfahani, Zahra; Darvish, Hossein; Paisán-Ruiz, Coro

2018-04-01

In this study, the role of known Parkinson's disease (PD) genes was examined in families with autosomal recessive (AR) parkinsonism to assist with the differential diagnosis of PD. Some families without mutations in known genes were also subject to whole genome sequencing with the objective to identify novel parkinsonism-related genes. Families were selected from 4000 clinical files of patients with PD or parkinsonism. AR inheritance pattern, consanguinity, and a minimum of two affected individuals per family were used as inclusion criteria. For disease gene/mutation identification, multiplex ligation-dependent probe amplification, quantitative PCR, linkage, and Sanger and whole genome sequencing assays were carried out. A total of 116 patients (50 families) were examined. Fifty-four patients (46.55%; 22 families) were found to carry pathogenic mutations in known genes while a novel gene, not previously associated with parkinsonism, was found mutated in a single family (2 patients). Pathogenic mutations, including missense, nonsense, frameshift, and exon rearrangements, were found in Parkin, PINK1, DJ-1, SYNJ1, and VAC14 genes. In conclusion, variable phenotypic expressivity was seen across all families.

MUTATIONS IN THE ARX GENE: CLINICAL, ELECTROENCEPHALOGRAPHIC AND NEUROIMAGING FEATURES IN 3 PATIENTS

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I. V. Ivanova

2017-01-01

Full Text Available The Aristaless-related homeobox (ARX gene is a member of the paired-type homeodomain transcription factor family with critical roles in embryonic development, particularly in the developing brain. Mutations in ARX gene demonstrate striking intra- and interfamilial pleiotropy together with genetic heterogeneity and lead to a broad spectrum of diseases. They give rise to 4 key phenotypic features: a different types of brain malformation, abnormal genitalia, epilepsy and intellectual disability. Authors present 3 clinical cases: a girl with duplication on the short arm of X-chromosome (Xp11.22-p22.33, which include genes ARX and CDKL5; a girl and a boy with a missense mutation in ARX gene that have not been previously described (chrX:25031522C>A, causes the substitution of an amino acid in the 197 protein position (p.Gly197Val, NM_139058.2. All patients suffer from severe epilepsy, that is refractory to antiepileptic drugs, and all of them have different degrees of psychomotor delay. The patients with missense mutation also have movement disorders: stereotypic movements in the girl and choreo athetosis and dystonia in the boy. Electroencephalographic abnormalities have been identified in all patients, and there were not significant abnormalities on magnetic resonance imaging in all cases. The described cases broaden the clinical spectrum of mutations in ARX gene.

Depression in patients with refractory temporal lobe epilepsy

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Eleonora Borges Gonçalves

2011-10-01

Full Text Available OBJECTIVE: To evaluate the comorbidity of depressive disorders in patients with refractory temporal lobe epilepsy (TLE. METHOD: We evaluated 25 consecutive patients with refractory TLE (16 women and 9 men, using semi-structured psychiatric interviews, according to the International Classification of Diseases (ICD-10, and the Beck Depression Inventory. RESULTS: Seventeen of 25 patients (68% had depressive disorder: 6 with dysthymia, three with major depressive episodes and 8 with recurrent depressive disorders. Two (8% were diagnosed with mixed anxiety and depression. Only 5 of 17 patients (29.4% were previously diagnosed with depressive disorder and received prior antidepressant treatment. Duration of epilepsy was significantly higher in patients with depressive disorder (p=0.016, but there was no relationship between depression and seizure frequency. CONCLUSION: This study confirmed that depressive disorders are common and underdiagnosed in patients with TLE refractory to AEDs. Patients with longer duration of epilepsy are at higher risk of having depression.

Alkali metal-refractory metal biphase electrode for AMTEC

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Williams, Roger M. (Inventor); Bankston, Clyde P. (Inventor); Cole, Terry (Inventor); Khanna, Satish K. (Inventor); Jeffries-Nakamura, Barbara (Inventor); Wheeler, Bob L. (Inventor)

1989-01-01

An electrode having increased output with slower degradation is formed of a film applied to a beta-alumina solid electrolyte (BASE). The film comprises a refractory first metal M.sup.1 such as a platinum group metal, suitably platinum or rhodium, capable of forming a liquid or a strong surface adsorption phase with sodium at the operating temperature of an alkali metal thermoelectric converter (AMTEC) and a second refractory metal insoluble in sodium or the NaM.sup.1 liquid phase such as a Group IVB, VB or VIB metal, suitably tungsten, molybdenum, tantalum or niobium. The liquid phase or surface film provides fast transport through the electrode while the insoluble refractory metal provides a structural matrix for the electrode during operation. A trilayer structure that is stable and not subject to deadhesion comprises a first, thin layer of tungsten, an intermediate co-deposited layer of tungsten-platinum and a thin surface layer of platinum.

Modeling the amplification dynamics of human Alu retrotransposons.

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Dale J Hedges

2005-09-01

Full Text Available Retrotransposons have had a considerable impact on the overall architecture of the human genome. Currently, there are three lineages of retrotransposons (Alu, L1, and SVA that are believed to be actively replicating in humans. While estimates of their copy number, sequence diversity, and levels of insertion polymorphism can readily be obtained from existing genomic sequence data and population sampling, a detailed understanding of the temporal pattern of retrotransposon amplification remains elusive. Here we pose the question of whether, using genomic sequence and population frequency data from extant taxa, one can adequately reconstruct historical amplification patterns. To this end, we developed a computer simulation that incorporates several known aspects of primate Alu retrotransposon biology and accommodates sampling effects resulting from the methods by which mobile elements are typically discovered and characterized. By modeling a number of amplification scenarios and comparing simulation-generated expectations to empirical data gathered from existing Alu subfamilies, we were able to statistically reject a number of amplification scenarios for individual subfamilies, including that of a rapid expansion or explosion of Alu amplification at the time of human-chimpanzee divergence.

Modeling the amplification dynamics of human alu retrotransposons.

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2005-09-01

Full Text Available Retrotransposons have had a considerable impact on the overall architecture of the human genome. Currently, there are three lineages of retrotransposons (Alu, L1, and SVA that are believed to be actively replicating in humans. While estimates of their copy number, sequence diversity, and levels of insertion polymorphism can readily be obtained from existing genomic sequence data and population sampling, a detailed understanding of the temporal pattern of retrotransposon amplification remains elusive. Here we pose the question of whether, using genomic sequence and population frequency data from extant taxa, one can adequately reconstruct historical amplification patterns. To this end, we developed a computer simulation that incorporates several known aspects of primate Alu retrotransposon biology and accommodates sampling effects resulting from the methods by which mobile elements are typically discovered and characterized. By modeling a number of amplification scenarios and comparing simulation-generated expectations to empirical data gathered from existing Alu subfamilies, we were able to statistically reject a number of amplification scenarios for individual subfamilies, including that of a rapid expansion or explosion of Alu amplification at the time of human-chimpanzee divergence.

Magnetic field amplification in interstellar collisionless shock waves

International Nuclear Information System (INIS)

Chevalier, R.A.

1977-01-01

It is stated that it is commonly assumed that a simple compression of the magnetic field occurs in interstellar shock waves. Recent space observations of the Earth's bow shock have shown that turbulent amplification of the magnetic field can occur in a collisionless shock. It is shown here that radio observations of Tycho's supernova remnant indicate the presence of a shock wave with such magnetic field amplification. There is at present no theory for the microinstabilities that give rise to turbulent amplification of the magnetic field. Despite the lack of theoretical understanding the possibility of field amplification in interstellar shock waves is here considered. In Tycho's supernova remnant there is evidence for the presence of a collisionless shock, and this is discussed. On the basis of observations of the Earth's bow shock, it is expected that turbulent magnetic field amplification occurs in the shock wave of this remnant, and this is supported by radio observations of the remnant. Consideration is given as to what extent the magnetic field is amplified in the shock wave on the basis of the non-thermal radio flux. (U.K.)

Dasatinib in the treatment of imatinib refractory chronic myeloid leukemia

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Radhakrishnan Ramchandren

2009-05-01

Full Text Available Radhakrishnan Ramchandren, Charles A SchifferDivision of Hematology/Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USAAbstract: The development of imatinib for the treatment of chronic myeloid leukemia (CML has proven to be an example of medical success in the era of targeted therapy. However, imatinib resistance or intolerance occurs in a substantial number of patients. Additionally, patients who have progressed beyond the chronic phase of CML do relatively poorly with imatinib therapy. Mechanisms of imatinib resistance include BCR-ABL point mutations resulting in decreased imatinib binding, as well as mutation-independent causes of resistance such as SRC family kinase dysregulation, BCR-ABL gene amplification, drug influx/efflux mechanisms and other poorly understood processes. The options for therapy in these patients include stem cell transplantation, imatinib dose escalation as well as the use of second-generation tyrosine kinase inhibitors. Dasatinib is a second-generation multi-kinase inhibitor with several theoretical and mechanistic advantages over imatinib. Moreover, several studies have evaluated dasatinib in patients who have progressed on imatinib therapy with encouraging results. Other novel agents such as mTOR inhibitors, bosutinib and INNO 406 have also shown promise in this setting. Although treatment options have increased, the choice of second-line therapy in patients with CML is influenced by concerns surrounding the duration of response as well as toxicity. Consequently, there is no agreed upon optimal second-line agent. This paper reviews the current data and attempts to address these issues. Keywords: chronic myeloid leukemia (CML, dasatinib, imatinib, resistance (imatinib resistance, nilotinib, tyrosine kinase inhibitor

Germline PMS2 mutation screened by mismatch repair protein immunohistochemistry of colorectal cancer in Japan.

Science.gov (United States)

Sugano, Kokichi; Nakajima, Takeshi; Sekine, Shigeki; Taniguchi, Hirokazu; Saito, Shinya; Takahashi, Masahiro; Ushiama, Mineko; Sakamoto, Hiromi; Yoshida, Teruhiko

2016-11-01

Germline PMS2 gene mutations were detected by RT-PCR/direct sequencing of total RNA extracted from puromycin-treated peripheral blood lymphocytes (PBL) and multiplex ligation-dependent probe amplification (MLPA) analyses of Japanese patients with colorectal cancer (CRC) fulfilling either the revised Bethesda Guidelines or being an age at disease onset of younger than 70 years, and screened by mismatch repair protein immunohistochemistry of formalin-fixed paraffin embedded sections. Of the 501 subjects examined, 7 (1.40%) showed the downregulated expression of the PMS2 protein alone and were referred to the genetic counseling clinic. Germline PMS2 mutations were detected in 6 (85.7%), including 3 nonsense and 1 frameshift mutations by RT-PCR/direct sequencing and 2 genomic deletions by MLPA. No mutations were identified in the other MMR genes (i.e. MSH2, MLH1 and MSH6). The prevalence of the downregulated expression of the PMS2 protein alone was 1.40% among the subjects examined and IHC results predicted the presence of PMS2 germline mutations. RT-PCR from puromycin-treated PBL and MLPA may be employed as the first screening step to detect PMS2 mutations without pseudogene interference, followed by the long-range PCR/nested PCR validation using genomic DNA. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

PCR amplification on microarrays of gel immobilized oligonucleotides

Science.gov (United States)

Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

2003-11-04

The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

Plasma deposition of refractories

International Nuclear Information System (INIS)

Kudinov, V.V.; Ivanov, V.M.

1981-01-01

The problems of deposition, testing and application of plasma coating of refractory metals and oxides are considered. The process fundamentals, various manufacturing procedures and equipment for their realization are described in detail. Coating materials are given (Al, Mg, Al 2 O 3 , ZrO 2 , MgAlO 4 ) which are used in reactor engineering and their designated purposes are shown [ru

AR mutations in 28 patients with androgen insensitivity syndrome (Prader grade 0-3).

Science.gov (United States)

Wang, Yi; Gong, Chunxiu; Wang, Xiou; Qin, Miao

2017-07-01

We investigated the androgen receptor (AR) gene mutation profiles of Chinese patients exhibiting severe androgen insensitivity syndrome (AIS) phenotypes. The present study enrolled 28 patients with genetically diagnosed AIS, who presented with severe phenotypes (Prader grade 0-3). Patients and some family members were screened via amplification and sequencing of their AR exons 1-8, including the corresponding intronic flanking regions. Luteinizing (LH), follicle-stimulating (FSH), and testosterone (T) hormone levels were found to be slightly, but not significantly, higher in patients with complete androgen insensitivity syndrome (CAIS) than in patients with partial androgen insensitivity syndrome (PAIS) (P>0.05). We identified 24 different AR mutations, including 12 that were novel. Ten patients (cases 2, 3, 10, 28, 11, 12, 19, 20, 24, and 25) were found to carry five recurrent mutations (p.Y572S, p.P914S, p.S176R, p.Y782N, and p.R841H); of these, p.Y572S, p.S176R, and p.Y782N were novel. Among the mutations identified in patients with CAIS, six (66.7%) were characterized as single-nucleotide missense mutations, and six (66.7%) were found to be located in the AR ligand-binding domain (LBD). Among the mutations identified in patients with PAIS, 15 (93.8%) were found to be missense, and 11 (68.8%) were found to be located in the LBD. Patients 10 and 28 were determined to harbor the same missense mutation (p.P914S), but were diagnosed with CAIS and PAIS, respectively. Sex hormone levels were slightly, but not significantly, elevated in patients with CAIS compared to those with PAIS. Missense mutations spanning AR exons 1-8 were the predominant form of identified mutations, and these were mostly located in the AR LBD. Approximately 50% of the identified mutations were novel, and have enriched the AR gene-mutation database. Patients harboring identical mutations were in some instances found to exhibit divergent phenotypes.

Study on high gain broadband optical parametric chirped pulse amplification

International Nuclear Information System (INIS)

Zhang, S.K.; Fujita, M.; Yamanaka, C.; Yoshida, H.; Kodama, R.; Fujita, H.; Nakatsuka, M.; Izawa, Y.

2000-01-01

Optical parametric chirped pulse amplification has apparent advantages over the current schemes for high energy ultrashort pulse amplification. High gain in a single pass amplification, small B-integral, low heat deposition, high contrast ratio and, especially the extremely broad gain bandwidth with large-size crystals available bring people new hope for over multi-PW level at which the existing Nd:glass systems suffered difficulties. In this paper we present simulation and experimental studies for a high gain optical parametric chirped pulse amplification system which may be used as a preamplifier to replace the current complicated regenerative system or multi-pass Ti:sapphire amplifiers. Investigations on the amplification bandwidth and gain with BBO are performed. Analysis and discussions are also given. (author)

HER2 amplification, overexpression and score criteria in esophageal adenocarcinoma

Science.gov (United States)

Hu, Yingchuan; Bandla, Santhoshi; Godfrey, Tony E.; Tan, Dongfeng; Luketich, James D.; Pennathur, Arjun; Qiu, Xing; Hicks, David G.; Peters, Jeffrey; Zhou, Zhongren

2011-01-01

The HER2 oncogene was recently reported to be amplified and overexpressed in esophageal adenocarcinoma. However, the relationship of HER2 amplification in esophageal adenocarcinoma with prognosis has not been well defined. The scoring systems for clinically evaluating HER2 in esophageal adenocarcinoma are not established. The aims of the study were to establish a HER2 scoring system and comprehensively investigate HER2 amplification and overexpression in esophageal adenocarcinoma and its precursor lesion. Using a tissue microarray, containing 116 cases of esophageal adenocarcinoma, 34 cases of BE, 18 cases of low grade dysplasia and 15 cases of high grade dysplasia, HER2 amplification and overexpression were analyzed by HercepTest and CISH methods. The amplification frequency in an independent series of 116 esophageal adenocarcinoma samples was also analyzed using Affymetrix SNP 6.0 microarrays. In our studies, we have found that HER2 amplification does not associate with poor prognosis in total 232 esophageal adenocarcinoma patients by CISH and high density microarrays. We further confirm the similar frequency of HER2 amplification by CISH (18.10%; 21/116) and SNP 6.0 microarrays (16.4%, 19/116) in esophageal adenocarcinoma. HER2 protein overexpression was observed in 12.1 % (14/116) of esophageal adenocarcinoma and 6.67% (1/15) of HGD. No HER2 amplification or overexpression was identified in BE or LGD. All HER2 protein overexpression cases showed HER2 gene amplification. Gene amplification was found to be more frequent by CISH than protein overexpression in esophageal adenocarcinoma (18.10% vs 12.9%). A modified two-step model for esophageal adenocarcinoma HER-2 testing is recommend for clinical esophageal adenocarcinoma HER-2 trial. PMID:21460800

[Identification of novel compound heterozygous mutations of USH2A gene in a family with Usher syndrome type II].

Science.gov (United States)

Jiang, Haiou; Ge, Chuanqin; Wang, Yiwang; Tang, Genyun; Quan, Qingli

2015-06-01

To identify potential mutations in a Chinese family with Usher syndrome type II. Genomic DNA was obtained from two affected and four unaffected members of the family and subjected to amplification of the entire coding sequence and splicing sites of USH2A gene. Mutation detection was conducted by direct sequencing of the PCR products. A total of 100 normal unrelated individuals were used as controls. The patients were identified to be a compound heterozygote for two mutations: c.8272G>T (p.E2758X) in exon 42 from his mother and c.12376-12378ACT>TAA(p.T4126X) in exon 63 of the USH2A gene from his father. Both mutations were not found in either of the two unaffected family members or 100 unrelated controls, and had completely co-segregated with the disease phenotype in the family. Neither mutation has been reported in the HGMD database. The novel compound heterozygous mutations c.8272G>T and c.12376-12378ACT>TAA within the USH2A gene may be responsible for the disease. This result may provide new clues for molecular diagnosis of this disease.

Barrier Coatings for Refractory Metals and Superalloys

Energy Technology Data Exchange (ETDEWEB)

SM Sabol; BT Randall; JD Edington; CJ Larkin; BJ Close

2006-02-23

In the closed working fluid loop of the proposed Prometheus space nuclear power plant (SNPP), there is the potential for reaction of core and plant structural materials with gas phase impurities and gas phase transport of interstitial elements between superalloy and refractory metal alloy components during service. Primary concerns are surface oxidation, interstitial embrittlement of refractory metals and decarburization of superalloys. In parallel with kinetic investigations, this letter evaluates the ability of potential coatings to prevent or impede communication between reactor and plant components. Key coating requirements are identified and current technology coating materials are reviewed relative to these requirements. Candidate coatings are identified for future evaluation based on current knowledge of design parameters and anticipated environment. Coatings were identified for superalloys and refractory metals to provide diffusion barriers to interstitial transport and act as reactive barriers to potential oxidation. Due to their high stability at low oxygen potential, alumina formers are most promising for oxidation protection given the anticipated coolant gas chemistry. A sublayer of iridium is recommended to provide inherent diffusion resistance to interstitials. Based on specific base metal selection, a thin film substrate--coating interdiffusion barrier layer may be necessary to meet mission life.

Barrier Coatings for Refractory Metals and Superalloys

International Nuclear Information System (INIS)

SM Sabol; BT Randall; JD Edington; CJ Larkin; BJ Close

2006-01-01

In the closed working fluid loop of the proposed Prometheus space nuclear power plant (SNPP), there is the potential for reaction of core and plant structural materials with gas phase impurities and gas phase transport of interstitial elements between superalloy and refractory metal alloy components during service. Primary concerns are surface oxidation, interstitial embrittlement of refractory metals and decarburization of superalloys. In parallel with kinetic investigations, this letter evaluates the ability of potential coatings to prevent or impede communication between reactor and plant components. Key coating requirements are identified and current technology coating materials are reviewed relative to these requirements. Candidate coatings are identified for future evaluation based on current knowledge of design parameters and anticipated environment. Coatings were identified for superalloys and refractory metals to provide diffusion barriers to interstitial transport and act as reactive barriers to potential oxidation. Due to their high stability at low oxygen potential, alumina formers are most promising for oxidation protection given the anticipated coolant gas chemistry. A sublayer of iridium is recommended to provide inherent diffusion resistance to interstitials. Based on specific base metal selection, a thin film substrate--coating interdiffusion barrier layer may be necessary to meet mission life

Profiling of oligosaccharides and p53 gene mutation in Filipino breast tumors

International Nuclear Information System (INIS)

Deocaris, Custer C.; De Vera, Azucena C.; Magno, Jose Donato A.; Cruz, Michael Joseph B.; Prodigalidad, Abelardo-Alan T.; Jacinto, Sonia D.

2010-01-01

Majority of patients are diagnosed with benign tumors, however, such benign tumors can progress to an invasive disease. Since carbohydrate-mediated cell-cell adhesion and proliferative potential play crucial roles in tumorigenesis and tumor aggressive behavior, we analyzed the qualitative changes in oligosaccharide expression and analyzed for presence of mutation in the tumor suppressor p53 gene, the most mutated gene in all human cancers. Forty-three (43) breast tumors were screened for p53 mutation in exons 2-11 using polymerase chain reaction (PCR)-amplification coupled to temporal temperature gradient electrophoresis (TTGE). Paraffin-embedded tissues were stained with biotinylated-glycoproteins containing the following sugar groups: mannose (Man), lactose (Lac), fucoidan (Fuc), N-acetyl-glucosamine (GlcNac), N-acetyl-b-galactosamine (GalNAc) and hyaluronic acid (Hya). Expression of carbohydrate receptors was significantly elevated (p=0.003) in malignant compared with benign tumors, particularly at receptors for GalNAc, lac and Fuc. No change in overall glycan signatures using our panel of neoglycoconjugates was noted when grouped according to p53 mutation status in both benign and malignant cases. Although the prognostic value of carbohydrate-receptors in breast cancer has not been validated to date, our results indicate that benign and malignant tumors can be defined by their affinities to our battery of neoglyconjugates. However, result from our reverse lectin histochemistry failed to correlated glycan signature with presence of p53 mutations. (author)

Amplification of hofmeister effect by alcohols.

Science.gov (United States)

Xu, Yun; Liu, Guangming

2014-07-03

We have demonstrated that Hofmeister effect can be amplified by adding alcohols to aqueous solutions. The lower critical solution temperature behavior of poly(N-isopropylacrylamide) has been employed as the model system to study the amplification of Hofmeister effect. The alcohols can more effectively amplify the Hofmeister effect following the series methanol alcohols and following the series d-sorbitol ≈ xylitol ≈ meso-erythritol alcohols. Our study reveals that the relative extent of amplification of Hofmeister effect is determined by the stability of the water/alcohol complex, which is strongly dependent on the chemical structure of alcohols. The more stable solvent complex formed via stronger hydrogen bonds can more effectively differentiate the anions through the anion-solvent complex interactions, resulting in a stronger amplification of Hofmeister effect. This study provides an alternative method to tune the relative strength of Hofmeister effect besides salt concentration.

Prognostic Factors for Refractory Status Epilepticus

Directory of Open Access Journals (Sweden)

J. Gordon Millichap

2013-03-01

Full Text Available Researchers at the Mayo Clinic, Rochester, MN studied the outcome and identified prognostic factors for refractory status epilepticus (RSE in 54 adult patients, median age 52 years [range 18-93].

Refractory disease in autoimmune diseases

NARCIS (Netherlands)

Vasconcelos, Carlos; Kallenberg, Cees; Shoenfeld, Yehuda

Refractory disease (RD) definition has different meanings but it is dynamic, according to knowledge and the availability of new drugs. It should be differentiated from severe disease and damage definitions and it must take into account duration of adequate therapy and compliance of the patient. It

CDKL5 and ARX mutations are not responsible for early onset severe myoclonic epilepsy in infancy.

Science.gov (United States)

Nabbout, Rima; Depienne, Christel; Chipaux, Mathilde; Girard, Benoit; Souville, Isabelle; Trouillard, Oriane; Dulac, Olivier; Chelly, Jamel; Afenjar, Alexandra; Héron, Delphine; Leguern, Eric; Beldjord, Cherif; Bienvenu, Thierry; Bahi-Buisson, Nadia

2009-11-01

Severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome (DS) is a distinctive epilepsy syndrome often associated with de novo mutations in the SCN1A gene. However, 25-30% patients with SMEI/DS are negative for SCN1A mutation screening, suggesting that other molecular mechanisms may account for these disorders. Given the overlapping and heterogeneous clinical features of CDKL5- and ARX-related epilepsies and SMEI/DS, we postulated that CDKL5 mutations in females and ARX mutations gene in males may be associated with early onset seizures forms of SMEI/DS. Twenty-eight patients with early onset SMEI/DS before 6 months negative for SCN1A mutational screening were selected and screened for mutations in the ARX gene in males (n=14) or the CDKL5 gene in females (n=14). No mutations in either gene were found except one intronic variation of uncertain pathogenicity in the CDKL5 gene. All patients started seizures at mean age of 3.48 months. Thirteen patients had familial history of epilepsy or febrile seizures. Patients evolved toward refractory epilepsy with generalized tonic clonic seizures (18/28) and myoclonia (23/28) and severe neurological impairment with autistic features (13/28), ataxia (14/28) and spasticity (5/28). No patient ever exhibited infantile spasms, dystonia, or Rett-like features. Our results illustrate that mutation screening of ARX and CDKL5 is not effective in patients selected on the basis of clinical signs associated to early onset SMEI/DS. In addition, they might reflect that other phenotypic features associated with CDKL5 mutations (Rett-like features, infantile spasm) or ARX mutations (dystonia, spasticity) are more distinctive. 2009 Elsevier B.V. All rights reserved.

Chemical analysis of refractories by plasma spectrometry

International Nuclear Information System (INIS)

Coutinho, C.A.

1990-01-01

X-ray spectrometry has been, since the last two or three decades, the traditional procedure for the chemical analysis of refractories, due to its high degree of accuracy and speed to produce analytical results. An interesting alternative to X-ray fluorescence is provided by the Inductively Coupled Plasma Spectrometry technique, for those laboratories where wet chemistry facilities are already available or process control is not required at high speed, or investiment costs have to be low. This paper presents results obtained by plasma spectroscopy for the analysis of silico - aluminous refractories, showing calibration curves, precion and detection limits. Considerations and comparisons with X-ray fluorescence are also made. (author) [pt

Improvements in or relating to refractory materials

International Nuclear Information System (INIS)

Peckett, J.W.A.

1980-01-01

A process is described for the production of a refractory material which includes heating an intermediate material containing carbon to cause a thermally induced reaction involving carbon in the intermediate material, wherein the intermediate material has been produced by heating a shaped gel precipitated gel, and the carbon in the intermediate material for participating in the thermally induced reaction has been produced from a gelling agent, or a derivative thereof, incorporated in the gel during gel precipitation. As examples, the refractory material may comprise uranium/plutonium oxide, or uranium/plutonium carbide, or thorium/uranium carbide, or tungsten carbide, or tungsten carbide/cobalt metal. (author)

Development of electro fused aggregates for use in refractories for the burning zone of cement kilns

International Nuclear Information System (INIS)

Ferreira, Luis Leonardo Horne Curimbaba

2006-01-01

Electro fused aggregates are largely used in refractory production due to the better performance reached when they are employed. In this work electro fused aggregates were designed for application in refractories for the burning zone of cement kilns. Initially reaction evaluation was conducted aiming the identification of the most prone refractory systems when single refractory phases react with Portland cement phases at high temperatures. In the next step, raw materials of the best refractory systems were electro fused to generate different aggregate compositions. The electro fused aggregates properties were evaluated and the classified ones were used to produce refractory bricks for the burning zone of cement kilns. General characteristics of these bricks were measured and compared with a standard magnesia-spinel refractory. Aggregates of the system Mg O - TiO 2 - Ca O, more specifically aggregates belonged to the compatibility triangle Mg O - Mg 2 TiO 4 - CaTiO 3 , showed suitable characteristics for development of refractories for the burning zone cement kilns. (author)

Amplification of Chirality in Hydrogen-Bonded Tetrarosette Helices

NARCIS (Netherlands)

Mateos timoneda, Miguel; Crego Calama, Mercedes; Reinhoudt, David

2006-01-01

The amplification of chirality in hydrogen-bonded tetrarosette assemblies under thermodynamic equilibrium is described. The extent of the chiral amplification obtained by means of “sergeants-and-soldiers” experiments depends only on the structure of the assembly and it is independent of the

Zirconium determination in refractories (gravimetric method)

International Nuclear Information System (INIS)

Capiotto, N.; Narahashi, Y.; Perish, C.G.; Souza, J.R.

1991-01-01

The zirconium determination in refractories is described, consisting in two separation methods for eliminating the interferences. The formatted product is calcined at 1100 0 C and determined gravimetrically as Zr P z 07. (author)

Genetics of refractoriness to Plasmodium falciparum in the mosquito Anopheles stephensi

NARCIS (Netherlands)

Feldmann, A.M.; Gemert, Geert-Jan van; Vegte-Bolmer, Marga G. van de; Jansen, Ritsert C.

1998-01-01

We previously selected a line of the malaria vector mosquito Anopheles stephensi refractory (resistant) to the human malaria parasite Plasmodium falciparum, using in vitro infections with P. falciparum gametocytes. This report presents data on the genetic background of refractoriness. The results of

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae.

Science.gov (United States)

Oh, Eun Joong; Skerker, Jeffrey M; Kim, Soo Rin; Wei, Na; Turner, Timothy L; Maurer, Matthew J; Arkin, Adam P; Jin, Yong-Su

2016-06-15

Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular β-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an

Productivity Costs in Patients with Refractory Chronic Rhinosinusitis

Science.gov (United States)

Rudmik, Luke; Smith, Timothy L.; Schlosser, Rodney J.; Hwang, Peter H.; Mace, Jess C.; Soler, Zachary M.

2014-01-01

Objective Disease-specific reductions in patient productivity can lead to substantial economic losses to society. The purpose of this study was to: 1) define the annual productivity cost for a patient with refractory chronic rhinosinusitis (CRS) and 2) evaluate the relationship between degree of productivity cost and CRS-specific characteristics. Study Design Prospective, multi-institutional, observational cohort study. Methods The human capital approach was used to define productivity costs. Annual absenteeism, presenteeism, and lost leisure time was quantified to define annual lost productive time (LPT). LPT was monetized using the annual daily wage rates obtained from the 2012 US National Census and the 2013 US Department of Labor statistics. Results A total of 55 patients with refractory CRS were enrolled. The mean work days lost related to absenteeism and presenteeism was 24.6 and 38.8 days per year, respectively. A total of 21.2 household days were lost per year related to daily sinus care requirements. The overall annual productivity cost was $10,077.07 per patient with refractory CRS. Productivity costs increased with worsening disease-specific QoL (r=0.440; p=0.001). Conclusion Results from this study have demonstrated that the annual productivity cost associated with refractory CRS is $10,077.07 per patient. This substantial cost to society provides a strong incentive to optimize current treatment protocols and continue evaluating novel clinical interventions to reduce this cost. PMID:24619604

Productivity costs in patients with refractory chronic rhinosinusitis.

Science.gov (United States)

Rudmik, Luke; Smith, Timothy L; Schlosser, Rodney J; Hwang, Peter H; Mace, Jess C; Soler, Zachary M

2014-09-01

Disease-specific reductions in patient productivity can lead to substantial economic losses to society. The purpose of this study was to: 1) define the annual productivity cost for a patient with refractory chronic rhinosinusitis (CRS) and 2) evaluate the relationship between degree of productivity cost and CRS-specific characteristics. Prospective, multi-institutional, observational cohort study. The human capital approach was used to define productivity costs. Annual absenteeism, presenteeism, and lost leisure time was quantified to define annual lost productive time (LPT). LPT was monetized using the annual daily wage rates obtained from the 2012 U.S. National Census and the 2013 U.S. Department of Labor statistics. A total of 55 patients with refractory CRS were enrolled. The mean work days lost related to absenteeism and presenteeism were 24.6 and 38.8 days per year, respectively. A total of 21.2 household days were lost per year related to daily sinus care requirements. The overall annual productivity cost was $10,077.07 per patient with refractory CRS. Productivity costs increased with worsening disease-specific QoL (r = 0.440; p = 0.001). Results from this study have demonstrated that the annual productivity cost associated with refractory CRS is $10,077.07 per patient. This substantial cost to society provides a strong incentive to optimize current treatment protocols and continue evaluating novel clinical interventions to reduce this cost. © 2014 The American Laryngological, Rhinological and Otological Society, Inc.

Refractory metal based superalloys

International Nuclear Information System (INIS)

Alonso, Paula R.; Vicente, Eduardo E.; Rubiolo, Gerardo H.

1999-01-01

Refractory metals are looked as promising materials for primary circuits in fission reactors and even as fusion reactor components. Indeed, superalloys could be developed which take advantage of their high temperature properties together with the benefits of a two- phase (intermetallic compound-refractory metal matrix) coherent structure. In 1993, researchers of the Office National d'Etudes et de Recherches Aerospatiales of France reported the observation of such a coherent structure in the Ta-Ti-Zr-Al-Nb-Mo system although the exact composition is not reported. The intermetallic compound would be Ti 2 AlMo based. However, the formation of this compound and its possible coexistence with a disordered bcc phase in the ternary system Ti-Al-Mo is a controversial subject in the related literature. In this work we develop a technique to obtain homogeneous alloys samples with 50 Ti-25 Al-25 Mo composition. The resulting specimens were characterized by optical and electronic metallography (SEM), microprobe composition measurements (EPMA) and X-ray diffraction (XRD) analyses. The results show the evidence for a bcc (A2→B2) ordering reaction in the Ti-Al-Mo system in the 50 Ti-25 Al-25 Mo composition. (author)

Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

Science.gov (United States)

Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

2014-09-01

A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions.

Multiplex Ligation-Dependent Probe Amplification Analysis of GATA4 Gene Copy Number Variations in Patients with Isolated Congenital Heart Disease

Directory of Open Access Journals (Sweden)

Valentina Guida

2010-01-01

Full Text Available GATA4 mutations are found in patients with different isolated congenital heart defects (CHDs, mostly cardiac septal defects and tetralogy of Fallot. In addition, GATA4 is supposed to be the responsible gene for the CHDs in the chromosomal 8p23 deletion syndrome, which is recognized as a malformation syndrome with clinical symptoms of facial anomalies, microcephaly, mental retardation, and congenital heart defects. Thus far, no study has been carried out to investigate the role of GATA4 copy number variations (CNVs in non-syndromic CHDs. To explore the possible occurrence of GATA4 gene CNVs in isolated CHDs, we analyzed by multiplex ligation-dependent probe amplification (MLPA a cohort of 161 non-syndromic patients with cardiac anomalies previously associated with GATA4 gene mutations. The patients were mutation-negative for GATA4, NKX2.5, and FOG2 genes after screening with denaturing high performance liquid chromatography. MLPA analysis revealed that normalized MLPA signals were all found within the normal range values for all exons in all patients, excluding a major contribution of GATA4 gene CNVs in CHD pathogenesis.

Identification of de novo mutations of Duchénnè/Becker muscular dystrophies in southern Spain.

Science.gov (United States)

Garcia, Susana; de Haro, Tomás; Zafra-Ceres, Mercedes; Poyatos, Antonio; Gomez-Capilla, Jose A; Gomez-Llorente, Carolina

2014-01-01

Duchénnè/Becker muscular dystrophies (DMD/BMD) are X-linked diseases, which are caused by a de novo gene mutation in one-third of affected males. The study objectives were to determine the incidence of DMD/BMD in Andalusia (Spain) and to establish the percentage of affected males in whom a de novo gene mutation was responsible. Multiplex ligation-dependent probe amplification (MLPA) technology was applied to determine the incidence of DMD/BMD in 84 males with suspicion of the disease and 106 female relatives. Dystrophin gene exon deletion (89.5%) or duplication (10.5%) was detected in 38 of the 84 males by MLPA technology; de novo mutations account for 4 (16.7%) of the 24 mother-son pairs studied. MLPA technology is adequate for the molecular diagnosis of DMD/BMD and establishes whether the mother carries the molecular alteration responsible for the disease, a highly relevant issue for genetic counseling.

Felbamate in an adult population with severe refractory epilepsy.

LENUS (Irish Health Repository)

Kearney, H

2011-04-05

Felbamate (FBM) is efficacious in treating patients with refractory epilepsy but was withdrawn due to cases of aplastic anaemia, hepatic failure and five reported deaths. FBM is currently used in specialist centres and is only being used in one Irish centre to date. This papers aim is to review the efficacy and safety experience of FBM in Irish adult patients with refractory epilepsy. A retrospective chart review was done on patients\\' medical records. Patients were subdivided into responders and non responders based on change in seizure frequency and side effects were recorded for all. Of the 13 patients on FBM nine patients responded to FBM, four patients did not. FBM is a safe and efficacious alternative in an Irish adult population with refractory epilepsy. However close monitoring is still required given the potential fatal side effects that are possible with this anticonvulsant.

MICROWAVE MEASUREMENT OF REFRACTORY MATERIALS AT HIGH-TEMPERATURE

International Nuclear Information System (INIS)

Kharkovsky, S.; Zoughi, R.; Smith, J.; Davis, B.; Limmer, R.

2009-01-01

Knowledge of the electrical behavior of refractory materials may enable the development and optimization of microwave nondestructive techniques to detect and evaluate changes in their physical properties while the materials are in service. This paper presents the results of a limited and preliminary investigation in which two refractory materials (dense chrome and dense zircon) were subjected to increasing temperature in a furnace and in which a frequency-modulated continuous-wave radar operating in the frequency range of 8-18 GHz radar was used to evaluate their attenuation properties.

Microwave Measurement of Refractory Materials at High-Temperature

Science.gov (United States)

Kharkovsky, S.; Zoughi, R.; Smith, J.; Davis, B.; Limmer, R.

2009-03-01

Knowledge of the electrical behavior of refractory materials may enable the development and optimization of microwave nondestructive techniques to detect and evaluate changes in their physical properties while the materials are in service. This paper presents the results of a limited and preliminary investigation in which two refractory materials (dense chrome and dense zircon) were subjected to increasing temperature in a furnace and in which a frequency-modulated continuous-wave radar operating in the frequency range of 8-18 GHz radar was used to evaluate their attenuation properties.

The rolling circle amplification and next generation sequencing ...

African Journals Online (AJOL)

Rolling circle amplification is a simple approach of enriching populations of single-stranded DNA plant begomovirus genomes (genus, Begomovirus; family, Geminiviridae). This is an innovative approach that utilizes the robustness of the bacteriophage phi29 DNA polymerase used in circle amplification, together with deep ...

Ten patients with refractory status epilepticus in an intensive care department

NARCIS (Netherlands)

ter Maaten, JC; van Schijndel, RJM; Heimans, JJ; Schreuder, WO

1998-01-01

Status epilepticus (SE) is a serious disease, associated with a high morbidity and mortality, particularly if refractory to initial therapy. We describe the clinical manifestations and outcome in ten cases with refractory SE admitted to our medical intensive care unit. Three of these selected group

Refractory absence epilepsy associated with GLUT-1 deficiency syndrome.

LENUS (Irish Health Repository)

Byrne, Susan

2011-05-01

GLUT-1 deficiency syndrome (GLUT-1 DS) is a disorder of cerebral glucose transport associated with early infantile epilepsy and microcephaly. We report two boys who presented with refractory absence epilepsy associated with hypoglycorrhachia, both of whom have genetically confirmed GLUT-1 DS. We propose that these children serve to expand the phenotype of GLUT-1 DS and suggest that this condition should be considered as a cause of refractory absence seizures in childhood.

Treatment of Refractory Idiopathic Supraorbital Neuralgia Using Percutaneous Pulsed Radiofrequency.

Science.gov (United States)

Luo, Fang; Lu, Jingjing; Ji, Nan

2018-02-26

No ideal therapeutic method currently exists for refractory idiopathic supraorbital neuralgia patients who do not respond to conservative therapy, including medications and nerve blocks. Pulsed radiofrequency is a neuromodulation technique that does not produce sequelae of nerve damage after treatment. However, the efficacy of percutaneous pulsed radiofrequency for the treatment of refractory idiopathic supraorbital neuralgia is still not clear. The purpose of our study was to evaluate the efficacy and safety of pulsed radiofrequency treatment of the supraorbital nerve for refractory supraorbital neuralgia patients. We prospectively investigated the long-term effects of ultrasound-guided percutaneous pulsed radiofrequency in the treatment of 22 refractory idiopathic supraorbital neuralgia patients. A reduction in the verbal pain numeric rating scale score of more than 50% was used as the standard of effectiveness. The effectiveness rates at different time points within 2 years were calculated. After a single pulsed radiofrequency treatment, the effectiveness rate at 1 and 3 months was 77%, and the rates at 6 months, 1 year, and 2 years were 73%, 64%, and 50%, respectively. Except for a small portion of patients (23%) who experienced mild upper eyelid ecchymosis that gradually disappeared after approximately 2 weeks, no obvious complications were observed. In conclusion, the results of our study demonstrate that for patients with refractory idiopathic supraorbital neuralgia, percutaneous pulsed radiofrequency may be an effective and safe treatment choice. © 2018 World Institute of Pain.

Surgical disconnection of patent paraumbilical vein in refractory hepatic encephalopathy.

Science.gov (United States)

Ishikawa, Yoshinori; Yoshida, Hiroshi; Mamada, Yasuhiro; Taniai, Nobuhiko; Bando, Koichi; Mizuguchi, Yoshiaki; Kakinuma, Daisuke; Kanda, Tomohiro; Akimaru, Koho; Tajiri, Takashi

2008-06-01

Refractory hepatic encephalopathy (HE) frequently develops in patients with cirrhosis and portal-systemic shunt. Recently, patients with refractory HE associated with portal-systemic shunt have been treated with interventional radiology. We describe a promising new treatment for portal-systemic shunt, ligation of the patent paraumbilical vein (PUV) after partial splenic embolization, in patients with refractory HE. Four patients with cirrhosis (3 women and 1 man; mean age, 56 years) and refractory HE due to a patent PUV were studied. Patency of the PUV had recurred in 1 patient after primary occlusion by interventional radiological procedures. The Child-Pugh class was B in 2 patients and C in 2. Before the present treatment, all patients had been hospitalized at least 3 times because of recurrent HE. Partial splenic embolization was performed in all patients to decrease portal venous pressure before surgery. Surgical ligation of the patent PUV was performed under epidural anesthesia. The patent PUV was carefully skeletonized and doubly ligated. Esophageal varices were evaluated with upper gastrointestinal endoscopy before and after surgery. The mean follow-up duration was 15.8 months. After ligation, there were no clinically significant complications. Esophageal varices were unchanged. The serum ammonia level was higher before surgery (162.3 +/- 56.4 mug/dL, mean +/- SD) than after surgery (41.8 +/- 20.2 mug/dL; p=0.0299). No patient had symptoms of HE. Ligation of the patent PUV is an effective treatment for patients with refractory HE.

Bulk Vitrification Performance Enhancement: Refractory Lining Protection Against Molten Salt Penetration

Energy Technology Data Exchange (ETDEWEB)

Hrma, Pavel R.; Bagaasen, Larry M.; Schweiger, Michael J.; Evans, Michael B.; Smith, Benjamin T.; Arrigoni, Benjamin M.; Kim, Dong-Sang; Rodriguez, Carmen P.; Yokuda, Satoru T.; Matyas, Josef; Buchmiller, William C.; Gallegos, Autumn B.; Fluegel, Alexander

2007-08-06

Bulk vitrification (BV) is a process that heats a feed material that consists of glass-forming solids and dried low-activity waste (LAW) in a disposable refractory-lined metal box using electrical power supplied through carbon electrodes. The feed is heated to the point that the LAW decomposes and combines with the solids to generate a vitreous waste form. This study supports the BV design and operations by exploring various methods aimed at reducing the quantities of soluble Tc in the castable refractory block portion of the refractory lining, which limits the effectiveness of the final waste form.

Comparison of Refractory Performance in Black Liquor Gasifiers and a Smelt Test System

International Nuclear Information System (INIS)

Peascoe, RA

2001-01-01

Prior laboratory corrosion studies along with experience at the black liquor gasifier in New Bern, North Carolina, clearly demonstrate that serious material problems exist with the gasifier's refractory lining. Mullite-based and alumina-based refractories used at the New Bern facility suffered significant degradation even though they reportedly performed adequately in smaller scale systems. Oak Ridge National Laboratory's involvement in the failure analysis, and the initial exploration of suitable replacement materials, led to the realization that a simple and reliable, complementary method for refractory screening was needed. The development of a laboratory test system and its suitability for simulating the environment of black liquor gasifiers was undertaken. Identification and characterization of corrosion products were used to evaluate the test system as a rapid screening tool for refractory performance and as a predictor of refractory lifetime. Results from the test systems and pl ants were qualitatively similar

Novel Mutations in Sandhoff Disease: A Molecular Analysis among Iranian Cohort of Infantile Patients

Science.gov (United States)

Aryan, H; Aryani, O; Banihashemi, K; Zaman, T; Houshmand, M

2012-01-01

Background Sandhoff disease is an autosomal recessive disorder caused by β-hexosaminidase deficiency and accumulation of GM2 ganglioside resulting in progressive motor neuron manifestations and death from respiratory failure and infections in infantiles. Pathogenic mutations in HEXB gene were observed which leads to enzyme activity reduction and interruption of normal metabolic cycle of GM2 ganglioside in sandhoff patients. Methods: Six infantile index patients with typical biochemical and clinical picture of the disease were studied at the molecular level. After DNA extraction and amplification, probands and their parents, were evaluated by direct sequencing of amplicons. Results: We identified 7 different mutations among which 4 were novel. The most prevalent finding (50%) among our population was a 16 kb deletion including the promoter and exons 1–5. The other findings included c.1552delG and c.410G>A, c.362 A>G, c.550delT, c.1597C>T, c.1752delTG. Conclusion: We conclude that Cys137Tyr and R533C mutations may be pathogenic because of changing amino acid and locating at the conserved region and also they have not been observed in hundred controls. Besides, four mutations including: Cys137Tyr, c.1552delG, c.1597C>T and c.550delT fulfilled almost criteria for pathogenic mutation. PMID:23113155

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

Directory of Open Access Journals (Sweden)

Xia Li

2016-10-01

Full Text Available Bisphenol A (BPA detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA/Exonuclease III (Exo III-combined cascade amplification strategy. First, the duplex DNA probe (RP with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II-protoporphyrin IX (ZnPPIX generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification.

Science.gov (United States)

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-10-21

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of "G-quadruplex" in lantern-like structures. Finally, the continuously enriched "G-quadruplex lanterns" were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10 -17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

Social amplification of risk: a conceptual framework

International Nuclear Information System (INIS)

Kasperson, R.E.; Renn, O.; Slovic, P.; Brown, H.S.; Emel, J.; Goble, R.; Kasperson, J.X.; Ratick, S.

1988-01-01

One of the most perplexing problems in risk analysis is why some relatively minor risks or risk events, as assessed by technical experts, often elicit strong public concerns and result in substantial impacts upon society and economy. This article sets forth a conceptual framework that seeks to link systematically the technical assessment of risk with psychological, sociological, and cultural perspectives of risk perception and risk-related behavior. The main thesis is that hazards interact with psychological, social, institutional, and cultural processes in ways that may amplify or attenuate public responses to the risk or risk event. A structural description of the social amplification of risk is now possible. Amplification occurs at two stages: in the transfer of information about the risk, and in the response mechanisms of society. Signals about risk are processed by individual and social amplification stations, including the scientist who communicates the risk assessment, the news media, cultural groups, interpersonal networks, and others. Key steps of amplifications can be identified at each stage. The amplified risk leads to behavioral responses, which, in turn, result in secondary impacts. Models are presented that portray the elements and linkages in the proposed conceptual framework

Mutation inactivation of Nijmegen breakage syndrome gene (NBS1 in hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

Directory of Open Access Journals (Sweden)

Yan Wang

Full Text Available Nijmegen breakage syndrome (NBS with NBS1 germ-line mutation is a human autosomal recessive disease characterized by genomic instability and enhanced cancer predisposition. The NBS1 gene codes for a protein, Nbs1(p95/Nibrin, involved in the processing/repair of DNA double-strand breaks. Hepatocellular carcinoma (HCC is a complex and heterogeneous tumor with several genomic alterations. Recent studies have shown that heterozygous NBS1 mice exhibited a higher incidence of HCC than did wild-type mice. The objective of the present study is to assess whether NBS1 mutations play a role in the pathogenesis of human primary liver cancer, including HBV-associated HCC and intrahepatic cholangiocarcinoma (ICC. Eight missense NBS1 mutations were identified in six of 64 (9.4% HCCs and two of 18 (11.1% ICCs, whereas only one synonymous mutation was found in 89 control cases of cirrhosis and chronic hepatitis B. Analysis of the functional consequences of the identified NBS1 mutations in Mre11-binding domain showed loss of nuclear localization of Nbs1 partner Mre11, one of the hallmarks for Nbs1 deficiency, in one HCC and two ICCs with NBS1 mutations. Moreover, seven of the eight tumors with NBS1 mutations had at least one genetic alteration in the TP53 pathway, including TP53 mutation, MDM2 amplification, p14ARF homozygous deletion and promoter methylation, implying a synergistic effect of Nbs1 disruption and p53 inactivation. Our findings provide novel insight on the molecular pathogenesis of primary liver cancer characterized by mutation inactivation of NBS1, a DNA repair associated gene.

Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules

Directory of Open Access Journals (Sweden)

Rowe Leslie R

2006-04-01

Full Text Available Abstract Background Fine needle aspiration (FNA is widely utilized for evaluation of patients with thyroid nodules. However, approximately 30% are indeterminate for malignancy. Recently, a mutation in the BRAF gene has been reported to be the most common genetic event in papillary thyroid carcinoma (PTC. In this retrospective study, we assessed the utility of BRAF V600E mutation detection for refining indeterminate preoperative cytologic diagnoses in patients with PTC. Methods Archival indeterminate thyroid FNAs and corresponding formalin-fixed, paraffin-embedded (FFPE surgical samples with PTC were identified in our patient files. DNA extracted from slide scape lysates and 5 μm FFPE sections were evaluated for the BRAF V600E mutation using LightCycler PCR and fluorescent melting curve analysis (LCPCR. Amplification products that showed deviation from the wild-type genomic DNA melting peak, discordant FNA and FFPE matched pairs, and all benign control samples, underwent direct DNA sequencing. Results A total of 19 indeterminate thyroid FNAs demonstrating PTC on FFPE surgical samples were included in the study. Using BRAF mutation analysis, the preoperative diagnosis of PTC was confirmed in 3/19 (15.8% FNA samples that could not be conclusively diagnosed on cytology alone. However, 9/19 (47.4% FFPE tissue samples were positive for the V600E mutation. Of the discordant pairs, 5/6 FNAs contained less than 50% tumor cells. Conclusion When used with indeterminate FNA samples, BRAF mutation analysis may be a useful adjunct technique for confirming the diagnosis of malignancy in an otherwise equivocal case. However, overall tumor cell content of some archival FNA smear slides is a limiting factor for mutation detection.

DNA amplification is rare in normal human cells

International Nuclear Information System (INIS)

Wright, J.A.; Watt, F.M.; Hudson, D.L.; Stark, G.R.; Smith, H.S.; Hancock, M.C.

1990-01-01

Three types of normal human cells were selected in tissue culture with three drugs without observing a single amplification event from a total of 5 x 10 8 cells. No drug-resistant colonies were observed when normal foreskin keratinocytes were selected with N-(phosphonacetyl)-L-aspartate or with hydroxyurea or when normal mammary epithelial cells were selected with methotrexate. Some slightly resistant colonies with limited potential for growth were obtained when normal diploid fibroblast cells derived from fetal lung were selected with methotrexate or hydroxyurea but careful copy-number analysis of the dihydrofolate reductase and ribonucleotide reductase genes revealed no evidence of amplification. The rarity of DNA amplification in normal human cells contrasts strongly with the situation in tumors and in established cell lines, where amplification of onogenes and of genes mediating drug resistance is frequent. The results suggest that tumors and cell lines have acquired the abnormal ability to amplify DNA with high frequency

Amplification biases: possible differences among deviating gene expressions

Directory of Open Access Journals (Sweden)

Piumi Francois

2008-01-01

Full Text Available Abstract Background Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT and polymerase chain reaction (PCR, the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. Results Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3 and somatic tissues (n = 2, we proceeded to moderate amplifications starting from 1 μg of total RNA (global PCR or IVT one round. Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70% and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID. However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions and relevant (biologically validated. In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples. Conclusion Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i the sample used: brain, ovary or embryos, (ii

FGFR2 amplification is predictive of sensitivity to regorafenib in gastric and colorectal cancers in vitro.

Science.gov (United States)

Cha, Yongjun; Kim, Hwang-Phill; Lim, Yoojoo; Han, Sae-Won; Song, Sang-Hyun; Kim, Tae-You

2018-03-24

Although regorafenib has demonstrated survival benefits in patients with metastatic colorectal and gastrointestinal stromal tumors, no proven biomarker has been identified for predicting sensitivity to regorafenib. Here, we investigated preclinical activity of regorafenib in gastric and colorectal cancer cells to identify genetic alterations associated with sensitivity to regorafenib. Mutation profiles and copy number assays of regorafenib target molecules indicated that amplification of FGFR2 was the only genetic alteration associated with in vitro sensitivity to regorafenib. Regorafenib effectively inhibited phosphorylation of FGFR2 and its downstream signaling molecules in a dose-dependent manner and selectively in FGFR2 amplified cells. Regorafenib induced G1 arrest (SNU-16, KATO-III) and apoptosis (NCI-H716), however, no significant changes were seen in cell lines without FGFR2 amplification. In SNU-16 mice xenografts, regorafenib significantly inhibited tumor growth, proliferation, and FGFR signaling compared to treatment with control vehicle. Regorafenib effectively abrogates activated FGFR2 signaling in FGFR2 amplified gastric and colorectal cancer and therefore, might be considered for integration into treatment in patients with FGFR2 amplified gastric and colorectal cancers. This article is protected by copyright. All rights reserved. Molecular Oncology (2018) © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Oral tranexamic acid lightens refractory melasma.

Science.gov (United States)

Tan, Aaron Wei Min; Sen, Priya; Chua, Sze Hon; Goh, Boon Kee

2017-08-01

Melasma is a common acquired hyperpigmentary disorder, particularly among Asians and Hispanics, but its exact pathomechanism is poorly understood. Tranexamic acid has been found to lighten melasma by interfering with the interaction of melanocytes and keratinocytes by inhibiting the plasminogen/plasmin system. The aim was to evaluate the therapeutic effects of oral tranexamic acid in the treatment of melasma refractory to topical skin-lightening agents. This retrospective study analyses patients with melasma recruited from a tertiary dermatological centre in Singapore between 1 August 2009 and 31 March 2011. The patients chosen had refractory melasma treated with oral tranexamic acid 250 mg twice daily in addition to pre-existing combination topical therapy. Objective assessment using the physician's global assessment and melasma area and severity index (MASI) scores were performed based on a post-hoc analysis of photographic records by three independent physicians. A paired t-test was used to evaluate the changes in the MASI scores pre-therapy and post-treatment. Statistical significance was defined as P tranexamic acid for a mean period of 3.7 ± 0.33 months, in addition to combination topical therapy. Their mean age was 47.2 ± 1.61 years. The mean MASI scores after tranexamic acid treatment (2.7 ± 1.6) were significantly lower (P tranexamic acid can serve as a safe and useful adjunct in the treatment of refractory melasma. © 2016 The Australasian College of Dermatologists.

[Prognostic significance of MYCN amplification in children neuroblastic tumors].

Science.gov (United States)

Niu, Huilin; Xu, Tao; Wang, Fenghua; Chen, Zhengrong; Gao, Qiu; Yi, Peng; Xia, Jianqing

2015-02-01

To summarize the clinicopathologic features of neuroblastic tumors (NT), and to explore the prognostic significance of MYCN amplification in NT. The clinicopathologic data of 267 NT were reviewed. MYCN gene amplification was detected by fluorescence in situ hybridization (FISH) in 119 cases and the relationship with pathological characteristics and prognostic significance were analyzed. The study included 267 cases of children NT from patients aged from 1 day to 13 years (median 27 months). The male to female ratio was 1.43. There were 38 cases (14.2%), 43 cases (16.1%), 71 cases (26.6%), and 115 cases (43.1%) of INSS stages I, II, III and IV respectively.Favorable histology group had 157 cases (59.9%); unfavorable histology group had 110 cases (40.1%).Of the 119 NT cases with MYCN FISH performed, 18 cases (15.1%) showed amplification and the signal ratio of MYCN to CEP2 was 4.08-43.29. One hundred and one cases of non-amplified MYCN included MYCN gain in 79 cases (66.3%) and MYCN negative in 22 cases (18.5%). MYCN expression showed significant difference (P = 0.000) between ages, gender, NT type and MKI, but not INPC and clinical stage (P > 0.05).Of the 18 cases with MYCN amplification, 3 were undifferentiated, and 15 poorly differentiated; 17 had high MKI and one moderate MKI. All 18 cases were in unfavorable histology group; the overall survival rate was 3/18, with an average survival time of (17.9 ± 2.4) months.Of the 101 MYCN non-amplification cases, the overall survival rate was 68.3% (69/101), with an average survival time of (29.8 ± 1.3) months. Survival analysis showed the cases with MYCN amplification had worse prognosis (P < 0.05). NT were commonly diagnosed in early ages and easily to metastasize. Most of cases with favorable histology. The cases of MYCN amplification showed unfavorable histology, and the majority cases with high MKI; The patients with MYCN gene amplification had poor prognosis.

Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene

Energy Technology Data Exchange (ETDEWEB)

Goltsov, A.A.; Eisensmith, R.C.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (United States)); Konecki, D.S.; Lichter-Konecki, U.

1992-09-01

The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiples of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1, 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between theses alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations. 32 refs., 3 figs., 3 tabs.

Nanostructured refractory thin films for solar applications

Science.gov (United States)

Ollier, E.; Dunoyer, N.; Dellea, O.; Szambolics, H.

2014-08-01

Selective solar absorbers are key elements of all solar thermal systems. Solar thermal panels and Concentrated Solar Power (CSP) systems aim respectively at producing heat and electricity. In both cases, a surface receives the solar radiation and is designed to have the highest optical absorption (lowest optical reflectivity) of the solar radiation in the visible wavelength range where the solar intensity is the highest. It also has a low emissivity in the infrared (IR) range in order to avoid radiative thermal losses. Current solutions in the state of the art usually consist in deposited interferential thin films or in cermets [1]. Structured surfaces have been proposed and have been simulated because they are supposed to be more efficient when the solar radiation is not normal to the receiving surface and because they could potentially be fabricated with refractory materials able to sustain high operating temperatures. This work presents a new method to fabricate micro/nanostructured surfaces on molybdenum (refractory metal with a melting temperature of 2623°C). This method now allows obtaining a refractory selective surface with an excellent optical selectivity and a very high absorption in the visible range. This high absorption performance was obtained by achieving a double structuration at micro and nano scales thanks to an innovative process flow.

Low-Chrome/Chrome Free Refractories for Slagging Gasifiers

International Nuclear Information System (INIS)

Bennett, J.P.; Kwong, K.-S.; Powell, C.P.; Thomas, H.; Petty, A.V. Jr.

2007-01-01

Gasifiers are containment vessels used to react carbon-containing materials with oxygen and water, producing syngas (CO and H2) that is used in chemical and power production. It is also a potential source of H2 in a future hydrogen economy. Air cooled slagging gasifiers are one type of gasifier, operating at temperatures from 1275-1575 C and at pressures of 400 psi or higher. They typically use coal or petroleum coke as the carbon source, materials which contain ash impurities that liquefy at the gasification temperatures, producing liquid slag in quantities of 100 or more tons/day, depending on the carbon fed rate and the percent ash present in the feedstock. The molten slag is corrosive to refractory linings, causing chemical dissolution and spalling. The refractory lining is composed of chrome oxide, alumina, and zirconia; and is replaced every 3-24 months. Gasifier users would like greater on-line availability and reliability of gasifier liners, something that has impacted gasifier acceptance by industry. Research is underway at NETL to improve refractory service life and to develop a no-chrome or low-chrome oxide alternative refractory liner. Over 250 samples of no- or low-chrome oxide compositions have been evaluated for slag interactions by cup testing; with potential candidates for further studies including those with ZrO2, Al2O3, and MgO materials. The development of improved liner materials is necessary if technologies such as IGCC and DOE's Near Zero Emissions Advanced Fossil Fuel Power Plant are to be successful and move forward in the marketplace

Long-term evaluation of treatment of chronic, therapeutically refractory tinnitus by neurostimulation

NARCIS (Netherlands)

Staal, M. J.; Holm, A. F.; Mooij, J. J. A.; Albers, F. W. J.; Bartels, H.

2007-01-01

Objective: Long-term evaluation of treatment of chronic, therapeutically refractory tinnitus by means of chronic electrical stimulation of the vestibulocochlear nerve. Patients: Inclusion criteria were severe, chronic, therapeutically refractory, unilateral tinnitus and severe hearing loss at the

Super-Refractory Status Epilepticus: Report of a Case and Review of the Literature.

Science.gov (United States)

Lapenta, Leonardo; Frisullo, Giovanni; Vollono, Catello; Brunetti, Valerio; Giannantoni, Nadia Mariagrazia; Sandroni, Claudio; Di Lella, Giuseppe; Della Marca, Giacomo

2015-10-01

Super-refractory status epilepticus (SE; ie, SE continuing or recurring despite 24 hours of general anesthesia) is a severe condition with high percentage of mortality and morbidity. Usually, this condition occurs because of serious brain damage; nevertheless, some patients develop super-refractory SE without identifiable etiology. Although not uncommonly encountered in neurointensive care, scientific data on this condition are still lacking in terms of treatment and prognosis. Herein, we report a case of super-refractory SE with recovery after 50 days, despite electroencephalographic (EEG) and magnetic resonance imaging (MRI) signs traditionally related to poor prognosis. A review of the literature on super-refractory SE is also presented. © EEG and Clinical Neuroscience Society (ECNS) 2014.

A Novel Missense Mutation in Oncostatin M Receptor Beta Causing Primary Localized Cutaneous Amyloidosis

Directory of Open Access Journals (Sweden)

Marjan Saeedi

2014-01-01

Full Text Available Primary localized cutaneous amyloidosis (PLCA is a chronic skin disorder, caused by amyloid material deposition in the upper dermis. Autosomal dominant PLCA has been mapped earlier to pathogenic missense mutations in the OSMR gene, which encodes the oncostatin M receptor ß subunit (OSMRß. OSMRß is interleukin-6 family cytokine receptors and possesses two ligands, oncostatin M and interleukin-31, which both have biologic roles in inflammation and keratinocyte cell proliferation, differentiation, and apoptosis. Here, we identified a new OSMR mutation in a Kurdish family for the first time. Blood samples were taken from all the affected individuals in the family. DNA extraction was performed using salting out technique. Primers were designed for intron flanking individual exons of OSMR gene which were subjected to direct sequencing after PCR amplification for each sample. Sequencing showed a C/T substitution at position 613 in the proband. This mutation results in an L613S (leucine 613 to serine amino acid change. The identified mutation was observed in all affected family members but not in 100 ethnically matched healthy controls. Elucidating the molecular basis of familial PLCA provides new insight into mechanisms of itch in human skin and may lead to new therapeutic targets for pruritus.

Population diversity of ammonium oxidizers investigated by specific PCR amplification

Science.gov (United States)

Ward, B.B.; Voytek, M.A.; Witzel, K.-P.

1997-01-01

The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels.

Tioguanine in the treatment of refractory coeliac disease--a single centre experience.

Science.gov (United States)

Tack, G J; van Asseldonk, D P; van Wanrooij, R L J; van Bodegraven, A A; Mulder, C J

2012-08-01

Refractory coeliac disease type I is a complicated form of coeliac disease characterised by primary or secondary resistance to a gluten-free diet with persisting or reoccurring intestinal villous atrophy and symptoms of malabsorption. Besides corticosteroids, azathioprine has been advocated for the treatment of refractory coeliac disease type I. However, tioguanine (TG) might be better tolerated and more efficacious owing to a simpler metabolism towards bioactivation. To evaluate tolerability and effectiveness of the nonconventional thiopurine derivative TG in refractory coeliac disease type I. Refractory coeliac disease type I patients treated with TG between June 2001 and November 2010 with a follow-up period of at least 1 year were included. Adverse events, laboratory values, 6-thioguanine nucleotide concentrations and rates of both clinical and histological response were evaluated at baseline and during follow-up. Twelve adult refractory coeliac disease type I patients were included. The median TG treatment duration was 14 months. Ten patients tolerated TG treatment on the long term, whereas two patients withdrew treatment due to adverse events. No nodular regenerative hyperplasia of the liver was observed. During follow-up clinical and histological response was observed in 83% and 78%, respectively. Corticosteroid dependency decreased by 50%. Tioguanine appears to be a convenient drug for the treatment of refractory coeliac disease type I based on higher histological and similar clinical response rates as compared with historical conventional therapies. © 2012 Blackwell Publishing Ltd.

Refractory, Abrasive and Other Industrial Mineral Operations

Data.gov (United States)

Department of Homeland Security — This map layer includes refractory, abrasive, and other industrial minerals operations in the United States. The data represent commodities covered by the Minerals...

Is high pressure liquid chromatography an effective screening tool for characterization of molecular defects in hemoglobinopathies?

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Nikhil Moorchung

2013-01-01

Full Text Available Introduction: Hemoglobinopathies constitute entities that are generated by either abnormal hemoglobin or thalassemias. high pressure liquid chromatography (HPLC is one of the best methods for screening and detection of various hemoglobinopathies but it has intrinsic interpretive problems. The study was designed to evaluate the different mutations seen in cases of hemoglobinopathies and compare the same with screening tests. Materials and Methods: 68 patients of hemoglobinopathies were screened by HPLC. Mutation studies in the beta globin gene was performed using the polymerase chain reaction (PCR-based allele-specific Amplification Refractory Mutation System (ARMS. Molecular analysis for the sickle cell mutation was done by standard methods. Results: The IVS 1/5 mutation was the commonest mutation seen and it was seen in 26 (38.23% of the cases. This was followed by the IVS 1/1, codon 41/42, codon 8/9, del 22 mutation, codon 15 mutation and the -619 bp deletion. No mutation was seen in eight cases. There was a 100% concordance between the sickle cell trait as diagnosed by HPLC and genetic testing. Discussion and Conclusion: Our study underlies the importance of molecular testing in all cases of hemoglobinopathies. Although HPLC is a useful screening tool, molecular testing is very useful in accurately diagnosing the mutations. Molecular testing is especially applicable in cases with an abnormal hemoglobin (HbD, HbE and HbS because there may be a concomitant inheritance of a beta thalassemia mutation. Molecular testing is the gold standard when it comes to the diagnosis of hemoglobinopathies.

Hemoglobinopathies in the Çukurova Region and Neighboring Provinces.

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Yuzbasioglu Ariyurek, Sedefgul; Yildiz, Sule Menziletoglu; Yalin, Ali Erdinc; Guzelgul, Figen; Aksoy, Kiymet

2016-06-01

To contribute to the creation of a mutation map of the region, we aimed to determine the mutation spectrum of thalassemias and abnormal hemoglobins (Hbs) in the Çukurova region and surrounding provinces. In this study, a total of 8135 samples from Adana, Hatay, Mersin, Konya and Kayseri provinces between 1993 and 2014 were analyzed. Complete blood cell (CBC) counts and Hb typing were carried out using automatic cell counters, cellulose acetate membrane electrophoresis and high performance liquid chromatography (HPLC), respectively. For the molecular analyses, genomic DNA was extracted using both manual and automated DNA extraction devices. Determination of Hb mutations were done by microarray, restriction fragment length polymorphism (RFLP), amplification refractory mutation system (ARMS) and gap-polymerase chain reaction (gap-PCR) methodologies. Samples were analyzed for abnormal Hb and thalassemia mutations. Out of 8135 samples, 1382 were observed to be carrying Hb mutations. It was identified that 826 mutation carriers included abnormal Hbs with a frequency of 59.7%, 416 carriers included β-thalassemia (β-thal) mutations with a frequency of 30.7% and 136 carriers included α-thalassemia (α-thal) mutations with a frequency of 9.9%. In this study, the most frequently observed abnormal Hb in the region was Hb S [β6(A3)Glu→Val (GTG > GAG), HBB: c.20T > A], whereas the most commonly observed mutations were the IVS-I-110 (G > A) (HBB: c.93-21G > A) point mutation in β-thal and the 3.7 kb deletion in α-thal.

Evaluation of nutritional status in children with refractory epilepsy

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Testolin G

2006-04-01

Full Text Available Abstract Background children affected by refractory epilepsy could be at risk of malnutrition because of feeding difficulties (anorexia, chewing, swallowing difficulties or vomiting and chronic use of anticonvulsants, which may affect food intake and energy metabolism. Moreover, their energy requirement may be changed as their disabilities would impede normal daily activities. The aim of the present study was to evaluate nutritional status, energy metabolism and food intake in children with refractory epilepsy. Methods 17 children with refractory epilepsy (13 boys and 4 girls; mean age 9 ± 3,2 years; Body Mass Index 15,7 ± 3,6 underwent an anthropometric assessment, body composition evaluation by dual-energy X-ray absorptiometry, detailed dietetic survey and measurement of resting energy expenditure by indirect calorimetry. Weight-for-age, height-for-age (stunting and weight-for-height (wasting were estimated compared to those of a reference population of the same age. Results 40% of children were malnourished and 24% were wasted. The nutritional status was worse in the more disabled children. Dietary intake resulted unbalanced (18%, 39%, 43% of total daily energy intake derived respectively from protein, lipid and carbohydrate. Adequacy index [nutrient daily intake/recommended allowance (RDA × 100] was Conclusion many children with refractory epilepsy would benefit from individual nutritional assessment and management as part of their overall care.

Improvement of castable refractories for RH snorkel; RH shinshitsukanyo futeikeizai no kaizen

Energy Technology Data Exchange (ETDEWEB)

Nishi, K.; Obana, T.; Fijii, T.; Shimizu, I. [Harima Ceramics Corp., Hyogo (Japan)

1999-11-01

Relating to corrosion of RH submerged nozzle, resistance against iron oxide of monolithic refractories was examined. Corrosion and seepage of refractories were measured by rotating corrosion, refractories include alumina-spinel castable, which is applied to RH under tank, and alumina-magnesia castable, which is generally used for RH, submerged nozzle. Alumina-spinel castable is superior in resistance against iron oxide than alumina-magnesia castable, and the resistance decreased with increase of stainless fiber addition to castable. Alumina-spinel castable without stainless fiber was suitable for bottom end of the dipping pipe. (NEDO)

A mechanism of gene amplification driven by small DNA fragments.

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Kuntal Mukherjee

Full Text Available DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s. Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation