scholarly journals Evaluation of BRAF Gene Status in Gliomas

2021 ◽  
pp. 1-6
Author(s):  
Awadhesh Kumar Jaiswal ◽  
Awadhesh Kumar Jaiswal ◽  
Sarita Agrawal ◽  
Sushila Jaiswal ◽  
Kuntal Kanti Das ◽  
...  
Keyword(s):  
Pilocytic Astrocytoma ◽  
Gene Fusion ◽  
Prognostic Significance ◽  
Rt Pcr ◽  
Braf Gene ◽  
Who Grade ◽  
Reverse Transcription Pcr ◽  
Significant Difference ◽  
Braf Fusion ◽  
Gene Status

Background: Development of different molecular markers has given a new insight in the glioma management. KIAA1549-BRAF gene fusion has a diagnostic and prognostic significance. Aim: The aim of this study was to determine the KIAA1549-BRAF gene fusion in glioma and their correlation with various clinical parameters. Material and Methods: Forty cases of glioma were studied for KIAA1549-BRAF gene fusion by reverse transcription-PCR (RT-PCR). Results: Overall, KIAA1549-BRAF gene fusion was found in 22% (9/40) cases of glioma. Children had higher KIAA1549-BRAF fusion (72%; 8/11) as compared to adults (10%; 3/29) and this difference was statistically significant. Cerebellar location of tumor was significantly associated with KIAA1549-BRAF fusion. KIAA1549-BRAF fusion was highest in pilocytic astrocytoma (89%), and this difference was statistically significant. Statistically significant difference was noted between KIAA1549-BRAF fusion expression and WHO grade I glioma. Conclusion: Overall, KIAA1549-BRAF gene fusion was found in 22% (9/40) cases of glioma. Childhood age, pilocytic astrocytoma histology, cerebellar location and WHO grade I tumor were significantly associated with KIAA1549-BRAF gene fusion.

Investigations on the Frequency of Norovirus Contamination of Ready-to-Eat Food Items in Istanbul, Turkey, by Using Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (5) ◽  
pp. 840-843 ◽  
Author(s):  
AYSUN YILMAZ ◽  
KAMIL BOSTAN ◽  
EDA ALTAN ◽  
KARLO MURATOGLU ◽  
NURI TURAN ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Epidemiological Studies ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Food Items ◽  
Reverse Transcription Pcr ◽  
Significant Difference ◽  
Tomato Sample ◽  
Pcr Assays

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

scholarly journals GacS-Dependent Regulation of Polyhydroxyalkanoate Synthesis in Pseudomonas putida CA-3

2013 ◽  
Vol 79 (6) ◽  
pp. 1795-1802 ◽  
Author(s):  
William J. Ryan ◽  
Niall D. O'Leary ◽  
Mark O'Mahony ◽  
Alan D. W. Dobson
Keyword(s):  
Pseudomonas Putida ◽  
Recombinant Expression ◽  
Wild Type ◽  
Rt Pcr ◽  
Posttranscriptional Control ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Medium Chain Length ◽  
Regulatory Cascade ◽  
Significant Difference

ABSTRACTTo date, limited reports are available on the regulatory systems exerting control over bacterial synthesis of the biodegradable polyester group known as polyhydroxyalkanoates (PHAs). In this study, we performed random mini-Tn5mutagenesis of thePseudomonas putidaCA-3 genome and screened transconjugants on nitrogen-limited medium for reduced PHA accumulation phenotypes. Disruption of a GacS sensor kinase in one such mutant was found to eliminate medium-chain-length PHA production inPseudomonas putidaCA-3. Recombinant expression of wild-typegacSfrom a pBBRgacS vector fully restored PHA accumulation capacity in the mutant strain. PCR-based screening of theP. putidaCA-3 genome identified gene homologues of the GacS/GacA-rsmsmall RNA (sRNA) regulatory cascade with 96% similarity to publishedP. putidagenomes. However, reverse transcription-PCR (RT-PCR) analyses revealed active transcription of thersmYandrsmZsRNAs ingacS-disruptedP. putidaCA-3, which is atypical of the commonly reported Gac/Rsm regulatory cascade. Quantitative real-time RT-PCR analyses of thephaC1synthase responsible for polymer formation inP. putidaCA-3 indicated no statistically significant difference in transcript levels between the wild-type andgacS-disrupted strains. Subsequently, SDS-PAGE protein analyses of these strains identified posttranscriptional control ofphaC1synthase as a key aspect in the regulation of PHA synthesis byP. putidaCA-3.

scholarly journals LGG-54. DETECTION OF THE KIAA1549-BRAF FUSION GENE IN CELLS FORMING MICROVASCULAR PROLIFERATIONS IN PILOCYTIC ASTROCYTOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii376-iii377
Author(s):  
Shinji Yamashita ◽  
Hideo Takeshima ◽  
Kiyotaka Saito ◽  
Takashi Watanabe ◽  
Hajime Ohta ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Pilocytic Astrocytoma ◽  
Fusion Gene ◽  
Digital Pcr ◽  
Who Grade ◽  
Mesenchymal Transition ◽  
Microvascular Proliferation ◽  
Vascular Markers ◽  
Braf Fusion ◽  
Cellular Components

Abstract Microvascular proliferation (MVP), an aberrant vascular structure is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVPs in PA contained tumor-derived cells and/or phenotypically distinct tumor cells expressing vascular markers. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP contained tumor derived cell and/or phenotypically distinct tumor cells expressing vascular markers.

Prognostic Significance Of CRLF2 Over-Expression In Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1368-1368
Author(s):  
Mio Yano ◽  
Toshihiko Imamura ◽  
Daisuke Asai ◽  
Akiko Moriya Saito ◽  
So-ichi Suenobu ◽  
...  
Keyword(s):  
Prognostic Factor ◽  
Prognostic Significance ◽  
Fusion Transcript ◽  
Genetic Alterations ◽  
Expression Level ◽  
Rt Pcr ◽  
Poor Prognostic Factor ◽  
Over Expression ◽  
Significant Difference ◽  
The Impact

Abstract Background Previous studies have reported that IKZF1 deletion was an adverse prognostic factor in pediatric B-cell-precursor ALL (BCP-ALL). However, the prognostic significance of CRLF2 over-expression (OE) is controversial. In order to assess the prognostic value of CRLF2 OE and genetic alterations involving CRLF2, we conducted genetic analysis of CRLF2 in pediatric BCP-ALL treated according to Japan Association of Childhood Leukemia Study (JACLS) ALL02 cohort. Methods We examined diagnostic bone marrow or peripheral blood samples of 167 pediatric BCP-ALL patients treated on the JACLS ALL02 protocol. The analyzed cohort included 68 patients classified in NCI-SR and 99 patients in NCI-HR. The deletion of IKZF1 and gain of CRLF2 were determined by MLPA. CRLF2 expression level was determined by quantitative RT-PCR and OE was defined as 10 times more than mean expression level of 167 cases. The P2RY8-CRLF2 fusion was detected by RT-PCR. To identify fusion transcript related to CRLF2 OE, we performed messenger RNA sequencing (mRNA-seq) on six of 17 patients with CRLF2 OE without CRLF2 gain and P2RY8-CRLF2 fusion. Results CRLF2 OE was identified in 30 (18%) of 167 patients, which was similar to that reported previously. IKZF1 deletion was found in 25(15%) of 167 patients, which was found more in CRLF2 OE patients than non-CRLF2 OE patients (30% vs 11%, p<0.05). P2RY8-CRLF2 fusion was identified in only 3 (1.7%) of 167 patients, and all of them were classified in CRLF2 OE. In detail, only one of three P2RY8-CRLF2 positive patients had IKZF1 deletion. One of two P2RY8-CRLF2 positive patients without IKZF1 deletion harbored MLL-AF4 fusion. CRLF2 gain was identified in 18(11%) of 167 patients. Eleven of these 18 patients were classified in CRLF2 OE, suggesting CRLF2 gain was significantly related to CRLF2 OE (37% vs 5%, p<0.01). Interestingly, none of CRLF2 OE patients with CRLF2 gain did have IKZF1 deletion. We identified one patient with novel fusion transcript caused by the 32kb deletion from CRLF2 intron 1. This patient was classified in CRLF2 OE with IKZF1 deletion, suggesting that the novel fusion transcript was related to CRLF2 OE. However, we could not identify any fusion transcripts related to CRLF2, such as IgH@-CRLF2, in remaining five patients by mRNA-seq. Interestingly, we identified three EBF1-PDGFRb positive patients in CRLF2 OE patients with IKZF1 deletion who did not have genetic alterations including CRLF2. In survival analysis, significant difference on the 5-year event-free survival (EFS) and overall survival (OS) between patients with and without CRLF2 OE was not observed (71% vs 75%, log rank p=0.68, 96% vs 82%, log rank p=0.11). In addition, type of genetic alterations related to CRLF2, such as CRLF2 gain and P2RY8-CRLF2 fusion, did not show the impact on EFS and OS in CRLF2 OE patients. However, significant difference on 5-year EFS between CRLF2 OE patients with and without IKZF1deletion was observed (44% vs 83%, log rank p=0.02). In multivariate analysis of 167 patients, only IKZF1 deletion was significant predictors of inferior OS (Hazard ratio: 2.427, 95%CI:1.037-5.679, p=0.04). Discussions The current analysis revealed that CRLF2 OE was caused by several different mechanisms, such as CRLF2 gain, P2RY8-CRLF2 fusion and rare fusion transcript related to CRLF2. However, regardless of type of genetic alterations of CRLF2, concomitant IKZF1 deletion holds the major impact on poor prognosis in CRLF2 OE patients. Especially, CRLF2 gain and IKZF1 deletion were mutually exclusive. Thus, CRLF2 OE caused by CRLF2 gain was not poor prognostic factor for high risk BCP-ALL. The prognostic significance of CRLF2 should be evaluated in consideration of IKZF1 status. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Comparison of Two High-Throughput Reverse Transcription-PCR Systems for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Arryn R. Craney ◽  
Priya D. Velu ◽  
Michael J. Satlin ◽  
Kathy A. Fauntleroy ◽  
Katrina Callan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
High Throughput ◽  
Reverse Transcription ◽  
Clinical Performance ◽  
Threshold Values ◽  
Limited Data ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Significant Difference ◽  
Pcr Assays

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as the cause of a worldwide pandemic. Many commercial SARS-CoV-2 reverse transcription-PCR (RT-PCR) assays have received Emergency Use Authorization from the U.S. Food and Drug Administration. However, there are limited data describing their performance, in particular the performance of high-throughput SARS-CoV-2 RT-PCR systems. We analyzed the diagnostic performance of two high-throughput systems: cobas 6800 and Panther Fusion, and their associated RT-PCR assays, with a collection of 389 nasopharyngeal specimens. The overall agreement between the platforms was 96.4% (375/389). Cohen’s kappa analysis rated the strength of agreement between the two platforms as “almost perfect” (κ = 0.922; standard error, 0.051). Furthermore, there was no significant difference between corresponding cycle threshold values generated on the two systems (P value = 0.88; Student’s t test). Taken together, these data imply that the two platforms can be considered comparable in terms of their clinical performance. We believe that this information will be useful for those who have already adopted these platforms or are seeking to implement high-throughput RT-PCR testing to stem the SARS-CoV-2 pandemic.

scholarly journals ANGI-05. ORIGIN OF MICROVASCULAR PROLIFERATION IN PILOCYTIC ASTROCYTOMA - MVP IN PILOCYTIC ASTROCYTOMA MIGHT PARTIALLY COMPRISE TUMOR-DERIVED CELLS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi31-vi31
Author(s):  
Shinji Yamashita ◽  
Hideo Takeshima ◽  
Takashi Watanabe ◽  
Kiyotaka Yokogami
Keyword(s):  
Tumor Cells ◽  
Pilocytic Astrocytoma ◽  
Fusion Gene ◽  
Digital Pcr ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Microvascular Proliferation ◽  
Braf Fusion ◽  
Cellular Components

Abstract Microvascular proliferation (MVP), an aberrant vascular structure containing multilayered mitotically active endothelial- and smooth-muscle cells/pericytes, is a histopathological hallmark of glioblastoma multiforme (GBM). Although MVP tends to be associated with high-grade glioma, it has also been detected in WHO grade I pilocytic astrocytoma (PA). However, little is known about the mechanism underlying its formation. Using TP53 point mutations as a marker for tumor-derived cells, we earlier reported that MVP was partially converted from tumor cells via mesenchymal transition. In the current study we used the KIAA1549-BRAF fusion gene as a marker to assess whether MVP in PA contained tumor-derived cells. cDNA synthesized from frozen tissue of six PA patients operated at our institute was analyzed to detect the KIAA1549-BRAF fusion gene by reverse transcription polymerase chain reaction (RT-PCR) assay. The breakpoint in the fusion gene was characterized by long and accurate PCR (LA-PCR) and Sanger sequencing of genomic DNA. Distinct tumor cells and cellular components of MVP were obtained by laser microdissection. For the qualitative and quantitative detection of the KIAA1549-BRAF fusion gene we performed genomic and digital PCR assays. Fluorescence in situ hybridization (FISH) was used to assess gene fusion in cellular components of MVP. Samples from three PA patients harbored the KIAA1549 exon 15, BRAF exon 9 fusion gene. In two patient samples with abundant MVP, RT-PCR assay detected strong bands arising from the KIAA1549-BRAF fusion gene in both tumor cells and cellular components of MVP. Digital PCR showed that vis-à-vis tumor tissue, its relative expression in cellular components of MVP was 42% in one- and 76% in another sample. FISH revealed amplified signals in both tumor cells and cellular components of MVP indicative of tandem duplication. Our findings suggest that in patients with PA, some cellular components of MVP may be tumor-derived.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

Investigation of BRAF fusion genes as a therapeutic target in pilocytic astrocytoma

2012 ◽  
Vol 224 (06) ◽  
Author(s):  
H Cin ◽  
J Gronych ◽  
A Korshunov ◽  
DTW Jones ◽  
D Milford ◽  
...  
Keyword(s):  
Pilocytic Astrocytoma ◽  
Therapeutic Target ◽  
Fusion Genes ◽  
Braf Fusion

scholarly journals Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico

2020 ◽  
Vol 110 (1) ◽  
pp. 106-120 ◽  
Author(s):  
Avijit Roy ◽  
Andrew L. Stone ◽  
Gabriel Otero-Colina ◽  
Gang Wei ◽  
Ronald H. Brlansky ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sensu Stricto ◽  
Genome Segment ◽  
Rt Pcr ◽  
Sequence Comparisons ◽  
Orchid Fleck Virus ◽  
Reverse Transcription Pcr ◽  
Sequencing Technologies ◽  
Negative Sense

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit–specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit–specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

scholarly journals Prognostic Significance of Preoperative Neutrophil-to-Lymphocyte Ratio in Patients With Meningiomas

2020 ◽  
Vol 10 ◽  
Author(s):  
Yuki Kuranari ◽  
Ryota Tamura ◽  
Noboru Tsuda ◽  
Kenzo Kosugi ◽  
Yukina Morimoto ◽  
...  
Keyword(s):  
Prognostic Significance ◽  
Laboratory Data ◽  
Progression Free Survival ◽  
Cost Effective ◽  
Tumor Location ◽  
Neutrophil To Lymphocyte Ratio ◽  
Ki 67 ◽  
Who Grade ◽  
Lymphocyte Ratio ◽  
Subtotal Removal

BackgroundMeningiomas are the most common benign intracranial tumors. However, even WHO grade I meningiomas occasionally show local tumor recurrence. Prognostic factors for meningiomas have not been fully established. Neutrophil-to-lymphocyte ratio (NLR) has been reported as a prognostic factor for several solid tumors. The prognostic value of NLR in meningiomas has been analyzed in few studies.Materials and MethodsThis retrospective study included 160 patients who underwent surgery for meningiomas between October 2010 and September 2017. We analyzed the associations between patients’ clinical data (sex, age, primary/recurrent, WHO grade, extent of removal, tumor location, peritumoral brain edema, and preoperative laboratory data) and clinical outcomes, including recurrence and progression-free survival (PFS).ResultsForty-four meningiomas recurred within the follow-up period of 3.8 years. WHO grade II, III, subtotal removal, history of recurrence, Ki-67 labeling index ≥3.0, and preoperative NLR value ≥2.6 were significantly associated with shorter PFS (P &lt; 0.001, &lt; 0.001, 0.002, &lt; 0.001, and 0.015, respectively). Furthermore, NLR ≥ 2.6 was also significantly associated with shorter PFS in a subgroup analysis of WHO grade I meningiomas (P = 0.003). In univariate and multivariate analyses, NLR ≥2.6 remained as a significant predictive factor for shorter PFS in patients with meningioma (P = 0.014).ConclusionsNLR may be a cost-effective and novel preoperatively usable biomarker in patients with meningiomas.

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