These are representative sample records from Science.gov related to your search topic.
For comprehensive and current results, perform a real-time search at Science.gov.
1

Routine fluorescence in situ hybridization in soil  

Microsoft Academic Search

The use of fluorescence in situ hybridization (FISH) to identify and enumerate soil bacteria has long been hampered by the autofluorescence of soil particles masking the bacterial signals and because the need of counting hundreds of bacteria in order to achieve statistically reliable data is time consuming. Recently, it was demonstrated that Nycodenz facilitates FISH in soil by concentrating bacteria

J. Bertaux; U. Gloger; M. Schmid; A. Hartmann; S. Scheu

2007-01-01

2

Molecular cytogenetics using fluorescence in situ hybridization  

SciTech Connect

Fluorescence in situ hybridization (FISH) with chromosome-specific probes enables several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of specific genetic sequences in single metaphase or interphase cells. in this approach, termed molecular cytogenetics, the genetic loci to be analyzed are made microscopically visible in single cells using in situ hybridization with nucleic acid probes specific to these loci. To accomplish this, the DNA in the target cells is made single stranded by thermal denaturation and incubated with single-stranded, chemically modified probe under conditions where the probe will anneal only with DNA sequences to which it has high DNA sequence homology. The bound probe is then made visible by treatment with a fluorescent reagent such as fluorescein that binds to the chemical modification carried by the probe. The DNA to which the probe does not bind is made visible by staining with a dye such as propidium iodide that fluoresces at a wavelength different from that of the reagent used for probe visualization. We show in this report that probes are now available that make this technique useful for biological dosimetry, prenatal diagnosis and cancer biology. 31 refs., 3 figs.

Gray, J.W.; Kuo, Wen-Lin; Lucas, J.; Pinkel, D.; Weier, H-U.; Yu, Loh-Chung.

1990-12-07

3

21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.  

Code of Federal Regulations, 2011 CFR

...Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 ...fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification . An automated FISH enumeration system is a device...

2011-04-01

4

21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.  

...Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 ...fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification. An automated FISH enumeration system is a device...

2014-04-01

5

21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.  

Code of Federal Regulations, 2013 CFR

...Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 ...fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification . An automated FISH enumeration system is a device...

2013-04-01

6

21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.  

Code of Federal Regulations, 2012 CFR

...Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 ...fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification . An automated FISH enumeration system is a device...

2012-04-01

7

21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.  

Code of Federal Regulations, 2010 CFR

...Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 ...fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification . An automated FISH enumeration system is a device...

2010-04-01

8

Fluorescence In Situ Hybridization in Studying the Human Genome  

Microsoft Academic Search

Physical chromosome mapping by fluorescence in situ hybridization (FISH) is among the major lines of research on the human genome (as well as genomes of numerous other organisms). To localize particular genes or anonymous DNA sequences on individual chromosomes or chromosome regions, FISH was developed in the late 1980s and early 1990s, when the International Human Genome Project and the

A. V. Zelenin

2004-01-01

9

FISH-ing for Genes: Modeling Fluorescence "in situ" Hybridization  

ERIC Educational Resources Information Center

Teaching methods of genetic analysis such as fluorescence in situ hybridization (FISH) can be an important part of instructional units in biology, microbiology, and biotechnology. Experience, however, indicates that these topics are difficult for many students. The authors of this article describe how they created an activity that effectively…

Baker, William P.; Jones, Carleton Buck

2006-01-01

10

PGD for dystrophin gene deletions using fluorescence in situ hybridization  

Microsoft Academic Search

Duchenne muscular dystrophy and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene (Xp21). In two-thirds of DMD\\/BMD cases, the mutation is a large deletion of one or several exons. We have established PGD for DMD\\/BMD using interphase fluorescence in situ hybridization (FISH) analysis on single nuclei from blastomeres for the detection of deletions of

H. Malmgren; I. White; S. Johansson; L. Levkov; E. Iwarsson; M. Fridström; Elisabeth Blennow

2006-01-01

11

Sexing of Dog Sperm by Fluorescence In Situ Hybridization  

PubMed Central

Abstract Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples. PMID:23059640

OI, Maya; YAMADA, Keisuke; HAYAKAWA, Hiroyuki; SUZUKI, Hiroshi

2012-01-01

12

Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization  

PubMed Central

We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the ?-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of ?-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the ?-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats. PMID:15466568

Sekar, Raju; Fuchs, Bernhard M.; Amann, Rudolf; Pernthaler, Jakob

2004-01-01

13

Pallister-Killian syndrome detected by fluorescence in situ hybridization  

SciTech Connect

The Pallister-Killian syndrome is a rare cytogenetic condition first described in 1977 by Pallister et al. in 3 adults; the first affected child was reported in 1981. This syndrome (also known as Pallister mosaic aneuploidy syndrome or isochromosome 12p mosaicism) is characterized by postnatal growth retardation, seizures, hypotonia, deafness, profound mental retardation, minimal speech development, and a distinctive facial appearance (high prominent forehead, ocular hypertelorism, sparse anterior scalp hair, prominent lower lip, large ears with thick protruding lobules, cupid-bow shaped upper lip, and a long philtrum). A chromosome 12 abnormality (tetrasomy 12p) has been reported in skin biopsies from these patients but this chromosome anomaly is usually not found (or in only a small proportion, e.g., <0.5%, of blood cells) in peripheral blood. We report on an additional patient with Pallister-Killian syndrome confirmed with fluorescence in situ hybridization (FISH) using an alpha satellite DNA probe for chromosome 12. This report further illustrates the application of FISH in identifying the source of chromosomal markers of unknown origin in infants with multiple congenital anomalies specifically before the natural history of a condition allows for definitive diagnosis based on clinical findings. 9 refs., 2 figs.

Butler, M.G.; Dev, V.G. [Genetics Associates, Nashville, TN (United States)

1995-07-03

14

Detection of dengue group viruses by fluorescence in situ hybridization  

PubMed Central

Background Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. Methods Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. Results The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. Conclusion Based on the FISH technique, we propose a way to design and use oligonucleotide probes to detect arboviruses. Results showed that this method was successfully implemented to specifically detect DENV in a mosquito cell line, as well as in mosquito salivary glands for the DENV-2 serotype. In addition, we emphasize that FISH could be an alternative method to detect arboviruses in host tissues, also offering to circumvent the discontinuity of antibodies used in immunofluorescent assays. PMID:23110979

2012-01-01

15

Mapping Small DNA Sequences by Fluorescence in situ Hybridization Directly on Banded Metaphase Chromosomes  

Microsoft Academic Search

A procedure for mapping small DNA probes directly on banded human chromosomes by fluorescence in situ hybridization has been developed. This procedure allows for the simultaneous visualization of banded chromosomes and hybridization signal without overlaying two separate photo-graphic images. This method is simple and rapid, requires only a typical fluorescence microscope, has proven successful with DNA probes as small as

Yao-Shan Fan; Lisa M. Davis; Thomas B. Shows

1990-01-01

16

Single-mRNA counting using fluorescent in situ hybridization in budding yeast  

Microsoft Academic Search

Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for

Tatjana Trcek; Jeffrey A Chao; Daniel R Larson; Hye Yoon Park; Daniel Zenklusen; Shailesh M Shenoy; Robert H Singer

2012-01-01

17

Influence of growth rate and starvation on fluorescent in situ hybridization of Rhodopseudomonas palustris  

Microsoft Academic Search

In situ hybridization with a fluorescently labeled 16S rRNA-targeted probe was examined using Rhodopseudomonas palustris as a model organism, which had been grown at different rates and under different conditions of growth and starvation. The specific growth rate did not affect the percentage of hybridized cells in aerobically grown R. palustris cultures. However, significant changes in the percentage of hybridized

Yasuhiro Oda; Simen-Jan Slagman; Wim G. Meijer; Larry J. Forney; Jan C. Gottschal

2000-01-01

18

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes  

PubMed Central

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups. PMID:23007640

Timoshevskiy, Vladimir A.; Sharma, Atashi; Sharakhov, Igor V.; Sharakhova, Maria V.

2012-01-01

19

Nature of B chromosomes in the harvest mouse Reithrodontomys megalotis by fluorescence in situ hybridization (FISH)  

Microsoft Academic Search

Using fluorescence in situ hybridization, we examined the characteristics of two types of B chromosomes in harvest mice of the genus Reithrodontomys. B chromosomes were interrogated with rDNA, telomeric repeat, LINE element and centromeric heterochromatin probes. The two types of B chromosomes share the following features: (a) telomeres present on the ends of both arms; (b) hybridization to LINE probes;

John A. Peppers; Lara E. Wiggins; Robert J. Baker

1997-01-01

20

Enumeration of viable E. coli in rivers and wastewaters by fluorescent in situ hybridization  

Microsoft Academic Search

A combination of direct viable count (DVC) and fluorescent in situ hybridization (FISH) procedures was used to enumerate viable Escherichia coli in river waters and wastewaters. A probe specific for the 16S rRNA of E. coli labeled with the CY3 dye was used; enumeration of hybridized cells was performed by epifluorescence microscopy. Data showed that the method was able to

Tamara Garcia-Armisen; Pierre Servais

2004-01-01

21

Applications and technical challenges of fluorescence in situ hybridization in stem cell research  

Microsoft Academic Search

Stem cell research, maintenance, and manipulations have advanced significantly in recent years, and we now witness successful clinical applications of stem therapies. However, challenges in regard to karyotypic stability and the ploidy status of stem cell lines have been addressed only marginally. Our approach to develop technology to address these highly relevant issues is based on fluorescence in situ hybridization

Heinz-Ulli G Weier; Lisa W Chu; John P Murnane; Jingly F Weier

2004-01-01

22

Multicolor fluorescence in situ hybridization studies in multiple myeloma and monoclonal gammopathy of undetermined significance  

Microsoft Academic Search

The aim of the study was to test the multicolor fluorescence in situ hybridization technique (M- FISH) in seven multiple myeloma (MM) and eight monoclonal gammopathy (MGUS) patients. None of the eight MGUS patients had chromosomal abnormalities by conventional cytogenetics. In two of these patients structural abnormalities of chromosomes 2, 11 and 19 were found by M-FISH. However, these findings

Norma C Gutie Rrez; Jordi Camps; Jesu S M Herna Ndez; Juan L Garc?a; Esther Prat; M Bele N Gonza Lez; Rosa Miro; Jesu S F San Miguel

2003-01-01

23

Cytogenetic, fluorescence in situ hybridization, and immunohistochemistry studies in a malignant pleural solitary fibrous tumor  

Microsoft Academic Search

Pleural solitary fibrous tumor is a normally benign fibroblastic neoplasm; its recurrences and metastasis are associated with clinical and morphological characteristics of variable interpretation and efficacy of surgical treatment. Immunohistochemistry techniques have contributed decisively to the correct diagnosis of the lesion and define its prognosis. In the present case, cytogenetic and fluorescence in situ hybridization analyses revealed multiple chromosomal aberrations,

Francisco J. Torres-Olivera; Maria T. Vargas; Francisco J. Torres-Gómez; Inmaculada Trigo; Mario Díaz; Ricardo González-Cámpora

2009-01-01

24

Fluorescence in situ hybridization analysis of HER2\\/neu in brushings of normal oral mucosa  

Microsoft Academic Search

Oncogene alterations have been clearly demonstrated to be related to the carcinogenesis and progression of oral squamous cell carcinoma (OSCC). However, the analysis of these alterations for screening and early diagnostic purposes generally requires invasive techniques for surgical removal of pathological epithelium. The aim of the present study was to assess the feasibility of fluorescence in situ hybridization (FISH) analysis

Angelo Paradiso; Marta Abatangelo; Sandra Piepoli; Stefania Tommasi; Jian-Ming Xu; Maria Angela Caponio; Franco Marzullo; Carlo D'Auria; Gaetano Achille; Luciano Grammatica

2002-01-01

25

Methods for the Rapid Screening of Group A Streptococci: Fluorescent in situ Hybridization versus Immunochromatography  

Microsoft Academic Search

Objective: To evaluate the accuracy of detection for screening group A streptococci (GAS) in pediatric clinics using fluorescent in situ hybridization (FISH) and immunochromatographic assay (ICA). Subjects and Methods: A group of 630 children who attended an outpatient clinic with signs and symptoms of acute upper respiratory tract infection were enrolled in this study. Specimens were collected using a double-swab

Jin-Ya Ding; Pei Wang

2011-01-01

26

A novel three-color, clone-specific fluorescence in situ hybridization procedure for monoclonal gammopathies  

Microsoft Academic Search

We have developed a three-color cytoplasmic immunoglobulin (clg) and fluorescence in situ hybridization (FISH) technique to detect plasma cell chromosomal aneuploidy in patients with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and amyloidosis (AL). Immunofluorescent-labeled antibodies to detect light chain expression and six directly labeled ?-satellite chromosome specific enumeration probes (CEP) were used simultaneously

Gregory J. Ahmann; Syed M. Jalal; Amy L. Juneau; Eric R. Christensen; Curtis A. Hanson; Gordon W. Dewald; Philip R. Greipp

1998-01-01

27

Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for

Annelie Pernthaler; Jakob Pernthaler; Rudolf Amann

2002-01-01

28

Comparison of Fluorescence In Situ Hybridization and Chromogenic In Situ Hybridization for Low and High Throughput HER2 Genetic Testing  

PubMed Central

The purpose was to evaluate and compare 5 different HER2 genetic assays with different characteristics that could affect the performance to analyze the human epidermal growth factor 2 (HER2) gene copy number under low and high throughput conditions. The study included 108 tissue samples from breast cancer patients with HER2 immunohistochemistry (IHC) results scored as 0/1+, 2+, and 3+. HER2 genetic status was analysed using chromogenic in situ hybridization (CISH) and fluorescence in situ hybridization (FISH). Scoring results were documented through digital image analysis. The cancer region of interest was identified from a serial H&E stained slide following tissue cores were transferred to a tissue microarrays (TMA). When using TMA in a routine flow, all patients will be tested for HER2 status with IHC followed by CISH or FISH, thereby providing individual HER2 results. In conclusion, our results show that the differences between the HER2 genetic assays do not have an effect on the analytic performance and the CISH technology is superior to high throughput HER2 genetic testing due to scanning speed, while the IQ-FISH may still be a choice for fast low throughput HER2 genetic testing. PMID:24383005

Poulsen, Tim S.; Espersen, Maiken L. M.; Kofoed, Vibeke; Dabetic, Tanja; H?gdall, Estrid; Balslev, Eva

2013-01-01

29

Application of Fluorescence In Situ Hybridization (FISH) to Fish Genetics and Genome Mapping  

Microsoft Academic Search

The various applications of the technique of fluorescence in situ hybridization (FISH) to fish genetics will be reviewed for fishes being used as model organisms to study human disease, including those species for which major genome projects have been initiated. ``FISH on fish'' has been used to map highly repetitive sequences including centromere-specific sequences and sex-specific sequences, moderately repetitive sequences

Ruth B. Phillips

2001-01-01

30

FISH - (Fluoresence In Situ Hybridization)  

NSDL National Science Digital Library

Fluorescence in situ hybridization (FISH) is a process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules. This technique is useful for identifying chromosomal abnormalities and gene mapping.

Darryl Leja (National Human Genome Research Institute REV)

2005-04-04

31

Mapping pachytene chromosomes of coffee using a modified protocol for fluorescence in situ hybridization  

PubMed Central

Fluorescence in situ hybridization (FISH) is the most direct method for physically mapping DNA sequences on chromosomes. Fluorescence in situ hybridization mapping of meiotic chromosomes during the pachytene stage is an important tool in plant cytogenetics, because it provides high-resolution measurements of physical distances. Fluorescence in situ hybridization mapping of coffee pachytene chromosomes offers significant advantages compared with FISH mapping of somatic chromosomes, because pachytene chromosomes are 30 times longer and provide additional cytological markers. However, the application of this technique to pachytene chromosomes has been complicated by problems in making preparations of meiotic chromosomes and by difficulties in the application of standard FISH protocols. We have been able to overcome most of these obstacles in applying the FISH technique to the pachytene chromosomes of coffee plants. Digesting the external callose layer surrounding the pollen mother cells (PMCs) in conjunction with other procedures permitted suitable pachytene chromosomes to be obtained by increasing cell permeability, which allowed the probe sequences to enter the cells. For the first time, hybridization signals were registered on coffee pachytene chromosomes using the FISH technique with a repetitive sequence as a probe. We obtained slides on which 80 % of the PMCs had hybridization signals, resulting in FISH labelling with high efficiency. The procedure does not seem to be dependent on the genotype, because hybridization signals were detected in genetically different coffee plants. These findings enhance the possibilities for high-resolution physical mapping of coffee chromosomes. PMID:24244840

Iacia, Ana Amelia Sanchez; Pinto-Maglio, Cecilia A. F.

2013-01-01

32

Painting of Parental Chromatin in Beta Hybrids by Multi?colour Fluorescent in situ Hybridization  

PubMed Central

Sugar beet (Beta vulgaris L.) is a relatively young crop and has a narrow gene pool. In order to introduce genetic variability into the crop, interspecific hybrids, selected from crosses with wild beets of the sections Corollinae and Procumbentes, have been generated. The introgressed B. procumbens chromatin carries resistance genes to beet cyst nematode Heterodera schachtii Schm. These lines are important for breeding of nematode?resistant sugar beet, while Corollinae species are potential donors of tolerance to biotic and abiotic stresses such as drought or saline soils. We have used in situ hybridization of genomic DNA to discriminate the parental chromosomes in these interspecific hybrids. Suppression of cross?hybridization by blocking DNA was not necessary indicating that the investigated Beta genomes contain sufficient species?specific DNA enabling the unequivocal determination of the genomic composition of the hybrids. Interspecific hybrid lines with an additional chromosome (2n = 18 + 1), chromosome fragment (2n = 18 + fragment) or translocation of B. procumbens (2n = 18) were analysed by genomic in situ hybridization (GISH) at mitosis and meiosis. Species?specific satellites and ribosomal genes used in combination with genomic DNA or in rehybridization experiments served as landmark probes for chromosome identification in hybrid genomes. The detection of a B. procumbens translocation of approx. 1 Mbp demonstrated the sensitivity and resolution of GISH and showed that this approach is a powerful method in genome analysis projects of the genus Beta. PMID:12099348

DESEL, CHRISTINE; JANSEN, RITA; DEDONG, GUE; SCHMIDT, THOMAS

2002-01-01

33

Numerical chromosome abnormalities in spermatozoa of fertile and infertile men detected by fluorescence in situ hybridization  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) with single-color chromosome-specific probes was used to study the rates of disomy for chromosome 1, 16, X, and Y in sperm of fertile and infertile subjects. Diploidy rates were studied using a two-color cocktail of probes for chromosomes 17 and 18 in the same sperm samples. Two-color methodology was not available at the outset of

Norio Miharu; Robert G. Best; S. Robert Young

1994-01-01

34

Fluorescence in situ hybridization for MDM2 gene amplification as a diagnostic tool in lipomatous neoplasms  

Microsoft Academic Search

Well-differentiated liposarcoma\\/atypical lipomatous tumor and dedifferentiated liposarcoma can be difficult to distinguish from benign lipomatous neoplasms and other high-grade sarcomas, respectively. Cytogenetics in these tumors has identified ring and giant chromosomes composed of 12q13-15 amplicons including the MDM2 gene. Identifying MDM2 amplification by fluorescence in situ hybridization may prove an adjunctive tool in the diagnosis of lipomatous neoplasms. Dual color

Joshua Weaver; Erinn Downs-Kelly; John R Goldblum; Sondra Turner; Sucheta Kulkarni; Raymond R Tubbs; Brian P Rubin; Marek Skacel

2008-01-01

35

microFIND(®) approach to fluorescent in situ hybridization (FISH).  

PubMed

FISH technology has gained increasing attention in the management of cancer disease, either for predictive or prognostic indications. Molecular cytogenetics has greatly improved diagnostic capability of classical cytogenetics analysis of metaphase-based chromosome for the identification of genetic aberrations. The availability of a large number of fluorescent probes, each specific for different genetic lesions, together with a robust protocol for interphase FISH, provide the pathologist with the essential tools for an accurate evaluation of patient's disease. Hemato-oncological and many of the solid tumors have been comprehensively characterized by peculiar genetic defects and are now routinely evaluated by interphase FISH. Despite the reliability of the method, which has undergone only minor changes since the 1970s, FISH assay is still hampered by reagents cost, preventing its adoption in large-scale oncological screening. In this chapter we describe a major improvement of interphase FISH assay for cytological samples through the description of the miniaturized device microFIND(®) that offers, besides reduction of cost per assay, a completely novel vision to the FISH technology, thanks to the perspective of full automation of FISH assay using a dedicated robotic platform for microFIND(®) handling, (not presently described in the chapter). PMID:23329459

Zanardi, Andrea; Barborini, Emanuele; Carbone, Roberta

2013-01-01

36

Bacterioplankton Compositions of Lakes and Oceans: a First Comparison Based on Fluorescence In Situ Hybridization  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea, and of these, about half could be identified at the subdomain level with a set of group-specific

FRANK OLIVER GLOCKNER; BERNHARD M. FUCHS; RUDOLF AMANN

1999-01-01

37

3D-fluorescence in situ hybridization of intact, anaerobic biofilm.  

PubMed

FISH (fluorescence in situ hybridization) is a valuable technique to visualize and quantify localization of different microbial species within biofilms. Biofilm conformation can be altered during typical sample preparation for FISH, which can impact observations in multispecies biofilms, including the relative positions of cells. Here, we describe methods to preserve 3-D structure during FISH for visualization of an anaerobic coculture biofilm of Desulfovibrio vulgaris Hildenborough and Methanococcus maripaludis. PMID:24838887

Brileya, Kristen A; Camilleri, Laura B; Fields, Matthew W

2014-01-01

38

Efficacy of two fluorescence in situ hybridization (FISH) probes for diagnosing malignant pleural effusions.  

PubMed

It is difficult to differentiate tumor cells in pleural fluid from reactive benign mesothelium. Fluorescence in situ hybridization (FISH) can increase diagnostic accuracy. Two hundred pleural fluid samples were analyzed by using FISH probes for chromosomes 11 and 17. Histological analysis was used to diagnose cancer. Clinical, radiological, and histological data were used to exclude malignancy. Eighty-two pleural effusion samples had positive cytology, 51 were benign, and 67 were atypical, but inconclusive. The 82 positive cases were confirmed to be malignant. Among the 51 negative cytology cases, videothoracoscopy-guided pleural biopsy revealed malignancy in three; aneuploid cells were detected by FISH in all cases. In 43 of the 67 cases with inconclusive cytology, malignancy was confirmed based on histology and fluorescence in situ hybridization. One case of parapneumonic effusion with no evidence of cancer during clinical follow-up had a suspicious cytology and positive fluorescence in situ hybridization result. The remaining 23 cases had no histological, radiological, clinical, or genetic evidence of malignancy. This study demonstrated that cytogenetic analysis of fresh pleural fluid samples using only two FISH probes is a valuable ancillary method for the identification of malignant pleural effusion, particularly in cases in which oncotic cytology is inconclusive. PMID:23453645

Rosolen, Débora C B; Kulikowski, Leslie D; Bottura, Giorgio; Nascimento, Amon M; Acencio, Milena; Teixeira, Lisete; Vargas, Francisco S; Sales, Roberta K; Antonangelo, Leila

2013-06-01

39

Mapping of a rat multidrug resistance gene by fluorescence in situ hybridization  

SciTech Connect

A cDNA clone encoding the rat mdr1b (Pgy2) gene was recently isolated and characterized. This gene has a high degree of sequence identity with other Pgy genes, particularly the mouse Pgy2 gene. By means of in situ fluorescence hybridization, the rat Pgy gene was localized on chromosome 4 band q12. This regional mapping will facilitate the identification of synteny groups on rat, mouse, and human genomes and chromosomal rearrangements during mammalian evolution. 17 refs., 2 figs.

Popescu, N.C.; Silverman, J.A.; Thorgeirsson, S.S. (National Institutes of Health, Bethesda, MD (United States))

1993-01-01

40

Detection of sex chromosome aneuploidy in dog spermatozoa by triple color fluorescence in situ hybridization.  

PubMed

With the development of a direct visualization of sex chromosome in a single sperm by fluorescence in situ hybridization (FISH) technique, the frequency of aberration (aneuploidy) in spermatozoa in several mammals has been investigated. However, there is no report in the incidence of X-Y aneuploidy in the sperm population of dogs. Therefore, in this study, the aneuploidy in dog spermatozoa was examined by multicolor FISH using specific molecular probes for canine sex chromosomes and autosome. Semen from eight male Labrador retrievers was used as specimen. For decondensation of sperm nuclei, the specimen was treated with 1 M NaOH for 4 minutes at room temperature. Probes for chromosomes X, Y, and 1, labeled with SpectrumGreen, Cy3 and Cy5, respectively, were hybridized with decondensed spermatozoa. Fluorescence in situ hybridization signals in sperm heads were clearly detected in each specimen, regardless of the sperm donor. The FISH signal of at least one of the three probes was detected in all sperm heads examined. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X and Y chromosomes in spermatozoa of all the eight dogs. Mean percentage of sex chromosome aneuploidy was 0.127% (ranged between 0% and 0.316%). This percentage of canine sex chromosome aneuploidy was lower than the one reported in cattle, horses, river buffalo, and goats sperm, but higher than that observed in mice and sheep. PMID:24962971

Komaki, Haruna; Oi, Maya; Suzuki, Hiroshi

2014-09-01

41

Genomic analysis of sorghum by fluorescence in situ hybridization  

E-print Network

relationships between pericentromeric heterochromatin, centromeres, mapped markers and recombination rates. These relationships will help guide the development and use of sorghum genomics. In the fifth study, I used FISH in two ongoing gene-targeted efforts...

Kim, Jeong-Soon

2004-11-15

42

Characterization of circulating tumor cells by fluorescence in situ hybridization  

Microsoft Academic Search

Tumor cells in blood of patients with metastatic carcinomas have been associated with poor survival prospects. Further characterization of these cells may provide further insights into the metastatic process. Circulating Tumor Cells (CTC) were enumerated in 7.5 mL of blood with the CellSearch™ system. After enumeration of Cytokeratin+, CD45?, nucleated cells, the cells are fixed in the cartridge while maintaining

Joost F. Swennenhuis; Arjan G. J. Tibbe; Rianne Levink; Ronald C. J. Sipkema; Leon W. M. M. Terstappen

2009-01-01

43

Catalyzed reported deposition-fluorescence in situ hybridization protocol to evaluate phagotrophy in mixotrophic protists.  

PubMed

We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists. PMID:16269774

Medina-Sánchez, Juan M; Felip, Marisol; Casamayor, Emilio O

2005-11-01

44

Rapid identification of Stenotrophomonas maltophilia by peptide nucleic acid fluorescence in situ hybridization  

PubMed Central

The objective of this study was to develop a novel peptide nucleic acid (PNA) probe for Stenotrophomonas maltophilia identification by fluorescence in situ hybridization (FISH). The probe was evaluated using 33 human and veterinary clinical S. maltophilia isolates and 45 reference strains representing common bacterial species in the respiratory tract. The probe displayed 100% sensitivity and 100% specificity on pure cultures and allowed detection in sputum from cystic fibrosis patients. The detection limit was 104 CFU/mL in spiked tracheal aspirate and bronchoalveolar lavage from healthy horses. Altogether the study shows that this species-specific PNA FISH probe facilitates rapid detection of S. maltophilia in biological specimens. PMID:25356348

Hansen, N; Rasmussen, A K I; Fiandaca, M J; Kragh, K N; Bjarnsholt, T; H?iby, N; Stender, H; Guardabassi, L

2014-01-01

45

Characterization of supernumerary ring marker chromosomes by fluorescence in situ hybridization (FISH)  

SciTech Connect

Five cases with small supernumerary ring chromosomes are characterized at the molecular level. Routine chromosome banding analysis was insufficient for identification of the ring chromosomes, and none of them was DA/DAPI positive. Fluorescence in situ hybridization utilizing repetitive centromeric probes for all chromosomes has determined that one of these five ring chromosomes originates in each of chromosomes 4, 7, 8, 9, and 20. Chromosome painting with chromosome-specific libraries has confirmed this and excluded the involvement of additional chromosomes in the rearrangements. 30 refs., 3 figs., 2 tabs.

Blennow, E.; Nordenskjoeld, M. (Karolinska Hospital (Sweden)); Asadi, E. (Region Hospital, Oerebro (Sweden)); Anneren, G.; Berggren, E.; Nordenskjoeld, M.

1993-08-01

46

Fluorescence in situ hybridization assessment of c-myc gene amplification in breast tumor tissues.  

PubMed

This chapter details methods used for analysis of DNA copy-number changes in breast tumor tissues through the use of fluorescence in situ hybridization. The specific DNA probe described herein is the oncogene c-myc, although the tissue fluorescence in situ hybridization methodology presented is suitable for dual-color studies of most unique sequence and chromosome specific control probes. The breast tumor tissue sections are first deparaffinized in a solvent and clearing agent, and then pretreated with a protease to allow the target DNA within the breast tissue cells to be uncovered. This allows the DNA to be available for hybridization with the labeled c-myc probe. The tissue sections are then analyzed to assure that appropriate digestion of cellular material has been attained. The tissues are then denatured. The c-myc probe and control probe for the centromere of chromosome 8 are commercially available as differentially labeled and are cohybridized to the tissue and sealed beneath a cover slip in a humid chamber. They are incubated for 12-16 h. The cover slip is then removed, and the section is postwashed in 2X saline sodium citrate at 72 degrees C for 5 min and allowed to cool to room temperature in a detergent solution. The slides are then counterstained with 4',6-diamidino-2-phenylindole, and a cover slip is applied. The slides are then viewed with fluorescence microscopy using filters that allow the c-myc and chromosome 8 signals to be visualized. If possible, 50 cells are counted, and the data are expressed as number of c-myc signals/number of chromosome 8 signals. PMID:16491608

Blancato, Jan K; Williams, Mary Steele; Dickson, Robert B

2006-01-01

47

Fluorescence in situ hybridization evaluation of chromosome deletion patterns in prostate cancer.  

PubMed Central

Various nonrandom chromosomal aberrations have been identified in prostate carcinoma. These aberrations include deletions of several chromosome regions, particularly the chromosome 8 short arm. Large-scale numerical aberrations, reflected in aberrant DNA ploidy, are also found in a minority of cases. However, it is unclear whether prostate carcinomas contain aberrations of certain chromosome regions that are deleted frequently in other common types of cancer. In this study, we performed dual-color fluorescence in situ hybridization on intact nuclei from touch preparations of 16 prostate cancers. Chromosome copy number was determined using pericentromeric probes, whereas potential chromosome arm deletions were evaluated using yeast artificial chromosome (YAC) and P1 probes. Two YAC probes targeted chromosome 8 short arm regions known to be deleted frequently in prostate cancer. Other YACs and P1s were for chromosome regions, including 1p22, 3p14, 6q21, 9p21, and 22q12, that are deletion targets in a variety of cancers although not extensively studied in prostate cancer. Hybridization efficiencies and signal intensities were excellent for both repeat sequence (alpha-satellite) and single, copy (YAC and P1) fluorescence in situ hybridization probes. Of 16 prostate cancers, 11 had clonal aberrations of 1 or more of the 13 chromosome regions evaluated, and 10 cases (62.5%) had 8p deletions, including 4 cases with 8p deletion in virtually all cells and aneuploidy in only a subset of those deleted cells. Deletions at 3p14, 6q21, and 22q12 were identified in 2, 1, and 1 case, respectively, and each of those cases had a similarly sized cell population with 8p deletion. These studies confirm 8p deletion in the majority of prostate carcinomas. 8p deletions appear to be early events in prostate tumorigenesis, often antedating aneuploidy. Fluorescence in situ hybridization strategies incorporating pericentromeric and single-copy regional chromosome probes offer a powerful and efficient means for determining frequency and progression of oncogenetic events in prostate cancer. Images Figure 1 PMID:8909246

Huang, S. F.; Xiao, S.; Renshaw, A. A.; Loughlin, K. R.; Hudson, T. J.; Fletcher, J. A.

1996-01-01

48

Detection of a complex translocation using fluorescent in situ hybridization (FISH)  

SciTech Connect

The use of fluorescent in situ hybridization (FISH) allowed the detection of a complex 3-way translocation in a patient with multiple congenital malformations and mental retardation. The patient was a 10-year-old girl with mental retardation, seizures, repaired cleft palate, esotropia, epicanthal folds, broad nasal bridge, upward slanting palpebral fissures, single transverse palmar crease, brachydactyly, hypoplastic nails, ectrodactyly between the third and fourth right toes, and hypoplasia of the left third toe. Chromosome analysis performed at birth was reported as normal. We performed high resolution banding analysis which revealed an apparently balanced translocation between chromosomes 2 and 9. However, because of her multiple abnormalities, further studies were ordered. Fluorescent in situ hybridization (FISH) using chromosome painting probes revealed a karyotype of 46,XX,t(2;8;9) (2pter{yields}q31::8q21.2{yields}8qter; 8pter{yields}q21.2::2q31{yields}q34::9q34{yields}qter; 9pter{yields}q34::2q34{yields}qter). The 3-way translocation appears to be de novo, as neither parent is a translocation carrier. This case illustrates the importance of using FISH to further investigate cases of apparently balanced translocations in the presence of phenotypic abnormalities and/or mental retardation.

Rosen, B.A. [Brandeis Univ., Waltham, MA (United States); Abuelo, D.N. [Rhode Island Hospital, Providence, RI (United States); Mark, H.F. [Brown Univ. School of Medicine, Providence, RI (United States)

1994-09-01

49

Nature of B chromosomes in the harvest mouse Reithrodontomys megalotis by fluorescence in situ hybridization (FISH).  

PubMed

Using fluorescence in situ hybridization, we examined the characteristics of two types of B chromosomes in harvest mice of the genus Reithrodontomys. B chromosomes were interrogated with rDNA, telomeric repeat, LINE element and centromeric heterochromatin probes. The two types of B chromosomes share the following features: (a) telomeres present on the ends of both arms; (b) hybridization to LINE probes; (c) absence of hybridization to the ribosomal gene probes; (d) C-band-positive centromeric regions; and (e) euchromatic arms. They differ as follows: (a) the larger B element hybridizes to the centromeric heterochromatin (pMeg-1) probe whereas the smaller B element does not; (b) the amount of C-band-positive material is reduced in the smaller B chromosome relative to that present on the larger B chromosome; and (c) the smaller element is reduced in size by about a third. It is concluded that the larger B chromosome arose as a leftover centromere from centric fusion, whereas the smaller element has a different origin perhaps as an intact fragment or as an amplified region from the A chromosomes. The presence of euchromatic regions on B chromosomes may account for their survival in the karyotype. PMID:9421265

Peppers, J A; Wiggins, L E; Baker, R J

1997-11-01

50

Single-molecule fluorescence in situ hybridization: Quantitative imaging of single RNA molecules  

PubMed Central

In situ detection of RNAs is becoming increasingly important for analysis of gene expression within and between intact cells in tissues. International genomics efforts are now cataloging patterns of RNA transcription that play roles in cell function, differentiation, and disease formation, and they are demonstrating the importance of coding and noncoding RNA transcripts in these processes. However, these techniques typically provide ensemble averages of transcription across many cells. In situ hybridization-based analysis methods complement these studies by providing information about how expression levels change between cells within normal and diseased tissues, and they provide information about the localization of transcripts within cells, which is important in understanding mechanisms of gene regulation. Multi-color, single-molecule fluorescence in situ hybridization (smFISH) is particularly useful since it enables analysis of several different transcripts simultaneously. Combining smFISH with immunofluorescent protein detection provides additional information about the association between transcription level, cellular localization, and protein expression in individual cells. [BMB Reports 2013; 46(2): 65-72] PMID:23433107

Kwon, Sunjong

2013-01-01

51

Chromosome orientation fluorescence in situ hybridization (CO-FISH) to study sister chromatid segregation in vivo  

PubMed Central

Previously, assays for sister chromatid segregation patterns relied on incorporation of BrdU and indirect methods to infer segregation patterns after two cell divisions. Here we describe a method to differentially label sister chromatids of murine cells and directly assay sister chromatid segregation patterns following one cell division in vitro and in vivo by adaptation of the well-established CO-FISH (chromosome orientation fluorescent in situ hybridization) technique. 5-bromo-2?-deoxyuridine (BrdU) is incorporated into newly-formed DNA strands, followed by photolysis and exonuclease digestion to create single-stranded sister chromatids containing parental template DNA only. Such single-stranded sister chromatids are differentially labeled using unidirectional probes to major satellite sequences coupled to fluorescent markers. Differentially-labeled sister chromatids in post-mitotic cells are visualized using fluorescence microscopy and sister chromatid segregation patterns can be directly assayed after one cell division. This procedure requires four days for in vivo mouse tissues, and two days for in vitro cultured cells. PMID:20595964

Falconer, Ester; Chavez, Elizabeth; Henderson, Alexander; Lansdorp, Peter M.

2013-01-01

52

Study of clonality in myelodysplastic syndromes: Detection of trisomy 8 in bone marrow cell smears by fluorescence in situ hybridization  

Microsoft Academic Search

The lineage involvement in myelodysplastic syndromes (MDS) is still unclear. To determine the clonality and the evolution of the disorder, a retrospective study on bone marrow smears from seven MDS patients with trisomy 8 was performed using fluorescence in situ hybridization (FISH).We observed that the trisomy of chromosome 8 was selectively expressed in the myeloid-derived cells. No mature lymphocytes or

Elisabetta Abruzzese; David Buss; Robert Rainer; P. Nagesh Rao; Mark J. Pettenati

1996-01-01

53

Chromogenic in situ hybridization is a reliable alternative to fluorescence in situ hybridization for diagnostic testing of 1p and 19q loss in paraffin-embedded gliomas.  

PubMed

Recent studies imply the importance of rapid and reliable diagnostic assessment of 1p/19q status in oligodendroglial tumors. To date, fluorescence in situ hybridization (FISH) is the most commonly applied technique. FISH, however, has several technical shortcomings that are suboptimal for diagnostic applications: results must be viewed in a fluorescence microscope, results are usually evaluated by a single investigator only, and signal fading excludes physical archiving. Also, in gliomas, the distinction of diffusely infiltrating tumor cells from reactively altered normal tissue may be challenging in fluorescence microscopy. Dual-color chromogenic in situ hybridization (CISH) has started to replace FISH in some diagnostic tests performed in pathology. Here, we present the first single institute experience with a side-by-side analysis of 1p/19q FISH and CISH in a series of 42 consecutive gliomas. FISH and CISH produced identical results for 1p and 19q in 93% of cases (n?=?39/42). Discrepant results were reevaluated by repeated FISH and a polymerase chain reaction (PCR)-based microsatellite marker analysis for loss of heterozygosity. Reevaluation confirmed CISH data in all three cases. We conclude that CISH is a reliable alternative in 1p/19q testing in paraffin-embedded tissues likely to be more sensitive to detect 1p/19q status than FISH analysis. PMID:23107103

Lass, Ulrike; Hartmann, Christian; Capper, David; Herold-Mende, Christel; von Deimling, Andreas; Meiboom, Maren; Mueller, Wolf

2013-05-01

54

Organization of small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in situ hybridization and immunocytochemistry.  

PubMed Central

The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations. Images PMID:7535131

Matera, A G; Tycowski, K T; Steitz, J A; Ward, D C

1994-01-01

55

Enumeration of methanogens with a focus on fluorescence in situ hybridization.  

PubMed

Methanogens, the members of domain Archaea are potent contributors in global warming. Being confined to the strict anaerobic environment, their direct cultivation as pure culture is quite difficult. Therefore, a range of culture-independent methods have been developed to investigate their numbers, substrate uptake patterns, and identification in complex microbial communities. Unlike other approaches, fluorescence in situ hybridization (FISH) is not only used for faster quantification and accurate identification but also to reveal the physiological properties and spatiotemporal dynamics of methanogens in their natural environment. Aside from the methodological aspects and application of FISH, this review also focuses on culture-dependent and -independent techniques employed in enumerating methanogens along with associated problems. In addition, the combination of FISH with micro-autoradiography that could also be an important tool in investigating the activities of methanogens is also discussed. PMID:21475941

Kumar, Sanjay; Dagar, Sumit Singh; Mohanty, Ashok Kumar; Sirohi, Sunil Kumar; Puniya, Monica; Kuhad, Ramesh C; Sangu, K P S; Griffith, Gareth Wyn; Puniya, Anil Kumar

2011-06-01

56

Chromosome translocations measured by fluorescence in-situ hybridization: A promising biomarker  

SciTech Connect

A biomarker for exposure and risk assessment would be most useful if it employs an endpoint that is highly quantitative, is stable with time, and is relevant to human risk. Recent advances in chromosome staining using fluorescence in situ hybridization (FISH) facilitate fast and reliable measurement of reciprocal translocations, a kind of DNA damage linked to both prior exposure and risk. In contrast to other biomarkers available, the frequency of reciprocal translocations in individuals exposed to whole-body radiation is stable with time post exposure, has a rather small inter-individual variability, and can be measured accurately at the low levels. Here, the authors discuss results from their studies demonstrating that chromosome painting can be used to reconstruct radiation dose for workers exposed within the dose limits, for individuals exposed a long time ago, and even for those who have been diagnosed with leukemia but not yet undergone therapy.

Lucas, J.N.; Straume, T.

1995-10-01

57

The design of a microscopic system for typical fluorescent in-situ hybridization applications  

NASA Astrophysics Data System (ADS)

Fluorescence in situ hybridization (FISH) is a modern molecular biology technique used for the detection of genetic abnormalities in terms of the number and structure of chromosomes and genes. The FISH technique is typically employed for prenatal diagnosis of congenital dementia in the Obstetrics and Genecology department. It is also routinely used to pick up qualifying breast cancer patients that are known to be highly curable by the prescription of Her2 targeted therapy. During the microscopic observation phase, the technician needs to count typically green probe dots and red probe dots contained in a single nucleus and calculate their ratio. This procedure need to be done to over hundreds of nuclei. Successful implementation of FISH tests critically depends on a suitable fluorescent microscope which is primarily imported from overseas due to the complexity of such a system beyond the maturity of the domestic optoelectrical industry. In this paper, the typical requirements of a fluorescent microscope that is suitable for FISH applications are first reviewed. The focus of this paper is on the system design and computational methods of an automatic florescent microscopy with high magnification APO objectives, a fast spinning automatic filter wheel, an automatic shutter, a cooled CCD camera used as a photo-detector, and a software platform for image acquisition, registration, pseudo-color generation, multi-channel fusing and multi-focus fusion. Preliminary results from FISH experiments indicate that this system satisfies routine FISH microscopic observation tasks.

Yi, Dingrong; Xie, Shaochuan

2013-12-01

58

An integrated microfluidic chip for chromosome enumeration using fluorescence in situ hybridization.  

PubMed

Fluorescence in situ hybridization (FISH) is a powerful technique for probing the genetic content of individual cells at the chromosomal scale. Conventional FISH techniques provide a sensitive diagnostic tool for the detection of chromosomal alterations on a cell-by-cell basis; however, the cost-per-test in terms of reagent and highly qualified labour has prevented its wide-spread utilization in clinical settings. Here, we address the inefficient use of labour with the first integrated and automated on-chip FISH implementation, one that requires only minutes of setup time from the technician. Our microfluidic chip has lowered the reagent use by 20-fold, decreased the labour time by 10-fold, and substantially reduced the amount of support equipment needed. We believe this cost-effective platform will make sensitive FISH techniques more accessible for routine clinical usage. PMID:19023479

Sieben, Vincent J; Debes-Marun, Carina S; Pilarski, Linda M; Backhouse, Christopher J

2008-12-01

59

A 30-Mb metric fluorescence in situ hybridization map of human chromosome 19q  

SciTech Connect

A high-resolution metric physical map of chromosome 19q has been constructed by fluorescence in situ hybridization. The map locates 136 cosmid reference points that span 30 Mb. The reference points are sequentially ordered from centromere to telomere, and the distance between neighboring cosmids is known from 240 partially overlapping, redundant estimates of genomic distances in kilobases separating pairs of cosmids. The average spacing between cosmid reference points is 220 kb, with over 75% of intervals less than 300 kb. Eighty-four genes and polymorphic markers have been assigned to mapped cosmids. The information on order and genomic distances separating pairs of cosmids, both key elements for building physical maps, has furthered the construction and integration of the genetic and physical maps of chromosome 19. 24 refs., 3 figs.

Gordon, L.A.; Bergmann, A.; Christensen, M.; Danganan, L. [Lawrence Livermore National Lab., CA (United States)] [and others] [Lawrence Livermore National Lab., CA (United States); and others

1995-11-20

60

Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH).  

PubMed

Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as "mad cow disease"), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application. PMID:25034259

Lecrenier, M C; Ledoux, Q; Berben, G; Fumière, O; Saegerman, C; Baeten, V; Veys, P

2014-01-01

61

Analysis of single-cell gene transcription by RNA fluorescent in situ hybridization (FISH).  

PubMed

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE(1). Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System(2) (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types. PMID:23070076

Ronander, Elena; Bengtsson, Dominique C; Joergensen, Louise; Jensen, Anja T R; Arnot, David E

2012-01-01

62

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)  

PubMed Central

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1. Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types. PMID:23070076

Ronander, Elena; Bengtsson, Dominique C.; Joergensen, Louise; Jensen, Anja T. R.; Arnot, David E.

2012-01-01

63

Assignment of Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase (ETF-QO) to Human Chromosome 4q33 by Fluorescence in Situ Hybridization and Somatic Cell Hybridization  

Microsoft Academic Search

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a nuclear-encoded protein located in the inner mitochondrial membrane. Inherited defects of ETF-QO cause glutaric acidemia type II. We here describe the localization of the ETF-QO gene to human chromosome 4q33 by somatic cell hybridization and fluorescence in situ hybridization.

Elaine B. Spector; William K. Seltzer; Stephen I. Goodman

1999-01-01

64

Chromosomal Numerical Aberrations Detected by Fluorescence in situ Hybridization on Bladder Washings from Patients with Bladder Cancer  

Microsoft Academic Search

Objective: Previous studies on touch biopsy specimens have determined numerical or structural changes involving many different chromosomes in bladder cancer. The aim of this study was to evaluate the use of fluorescence in situ hybridization (FISH) assay in bladder washings as an objective technique to detect chromosomal numerical aberrations in bladder cancer. The main advantages of bladder washings are that

Alessandro Marano; Yi Pan; Chunde Li; Arcangelo Pagliarulo; Göran Elmberger; Bernhard Tribukait; Peter Ekman; Ulf Bergerheim

2000-01-01

65

Fluorescent in situ hybridization for sex chromosome determination before and after fertilization in mice  

PubMed Central

In mice, the relative numbers of male and female pups per litter not only can vary but can probably change over the course of pregnancy in response to numerous environmental and physiological factors. As such, a technique is required to determine gender at several developmental stages. Here we describe a robust and accurate fluorescent in situ hybridization (FISH) procedure for determining chromosomal sex that can be applied with minimal modification to sperm, pre-and post-implantation conceptuses and recovered dead post-natal pups. Sperm was prepared for FISH analysis y using a modified microwave decondensation–denaturation technique. Preimplantation conceptuses (0.5 dpc) were cultured to the morula stage before sexing. They were then acid-treated to remove the zona pellucida. Tissue homogenates from postimplantational conceptuses (8.5 dpc) and stillborn pups were fixed to pre-etched slides. Specimens were hybridized with identical, commercially available DNA probes for the X (FITC) and Y (Cy3) chromosomes. Sperm ratios met the expected value of 0.5 when determined by using XY FISH. Preimplantation conceptuses pre-treated with pepsin yielded distinct fluorescence of X and Y chromosomes in morulae, whereas microwave decondensation resulted in loss of conceptuses from the slide. Both 4.0 and 8.5 dpc conceptuses displayed mean sex ratios of 0.5. Post-natal FISH analysis allowed gender identification of pups that could not be sexed due to developmental abnormalities or partial cannibalism. FISH analysis of sperm and of multiple conceptuses or post-natal tissue provided a cost-effective, accurate alternative to PCR-based sex determination. PMID:17215034

Whyte, J.J.; Roberts, R.M.; Rosenfeld, C.S.

2007-01-01

66

Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes  

Microsoft Academic Search

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different

HENRIK STENDER; CLETUS KURTZMAN; JENS J. HYLDIG-NIELSEN; D. Sorensen; ADAM BROOMER; KENNETH OLIVEIRA; HEATHER PERRY-O' KEEFE; ANDREW SAGE; BARBARA YOUNG; JAMES COULL

2001-01-01

67

DNA Mimics for the Rapid Identification of Microorganisms by Fluorescence in situ Hybridization (FISH)  

PubMed Central

Fluorescence in situ hybridization (FISH) is a well-established technique that is used for a variety of purposes, ranging from pathogen detection in clinical diagnostics to the determination of chromosomal stability in stem cell research. The key step of FISH involves the detection of a nucleic acid region and as such, DNA molecules have typically been used to probe for the sequences of interest. However, since the turn of the century, an increasing number of laboratories have started to move on to the more robust DNA mimics methods, most notably peptide and locked nucleic acids (PNA and LNA). In this review, we will cover the state-of-the-art of the different DNA mimics in regard to their application as efficient markers for the presence of individual microbial cells, and consider their potential advantages and pitfalls. Available PNA probes are then reassessed in terms of sensitivity and specificity using rRNA databases. In addition, we also attempt to predict the applicability of DNA mimics in well-known techniques attempting to detect in situ low number of copies of specific nucleic acid sequences such as catalyzed reporter deposition (CARD) and recognition of individual genes (RING) FISH. PMID:19325728

Cerqueira, Laura; Azevedo, Nuno F.; Almeida, Carina; Jardim, Tatiana; Keevil, Charles William; Vieira, Maria J.

2008-01-01

68

In Situ Detection of Freshwater Fungi in an Alpine Stream by New Taxon-Specific Fluorescence In Situ Hybridization Probes  

Microsoft Academic Search

New rRNA-targeting oligonucleotide probes permitted thefluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora

Christiane Baschien; Werner Manz; Thomas R. Neu; Ludmila Marvanova; Ulrich Szewzyk

2008-01-01

69

Clonal evolution in chronic lymphocytic leukemia studied by interphase fluorescence in-situ hybridization.  

PubMed

The results of repeated interphase fluorescence in-situ hybridization (I-FISH, FISH) examination of 97 CLL patients and correlation of these findings with IgVH hypermutation status, ZAP-70 and CD38 expression are presented. The appearance of new, FISH-detectable, genomic aberrations during disease course, described as clonal evolution (CE), was observed in 26% of patients. The most frequent newly acquired cytogenetic abnormality was 13q deletion in 64% (16/25). In contrast to earlier studies, there was no correlation found between CE and either one of single negative prognostic factors (unmutated IgVH; CD38 positivity; ZAP-70 positivity). However, the combination of all three negative factors correlated with CE highly significantly (p=0.005) and moreover, also with a shift from lower to higher FISH risk category (p=0.010). As the prognostic data were known in all patients, this study represents the complete insight on the association of CE and other risk parameters in CLL. PMID:19580349

Berkova, A; Zemanova, Z; Trneny, M; Schwarz, J; Karban, J; Cmunt, E; Pavlistova, L; Brezinova, J; Michalova, K

2009-01-01

70

Automated evaluation of Her-2/neu status in breast tissue from fluorescent in situ hybridization images.  

PubMed

The evaluation of fluorescent in situ hybridization (FISH) images is one of the most widely used methods to determine Her-2/neu status of breast samples, a valuable prognostic indicator. Conventional evaluation is a difficult task since it involves manual counting of dots in multiple images. In this paper, we present a multistage algorithm for the automated classification of FISH images from breast carcinomas. The algorithm focuses not only on the detection of FISH dots per image, but also on combining results from multiple images taken from a slice for overall case classification. The algorithm includes mainly two stages for nuclei and dot detection respectively. The dot segmentation consists of a top-hat filtering stage followed by template matching to separate real signals from noise. Nuclei segmentation includes a nonlinearity correction step, global thresholding to identify candidate regions, and a geometric rule to distinguish between holes within a nucleus and holes between nuclei. Finally, the marked watershed transform is used to segment cell nuclei with markers detected as regional maxima of the distance transform. Combining the two stages allows the measurement of FISH signals ratio per cell nucleus and the collective classification of cases as positive or negative. The system was evaluated with receiver operating characteristic analysis and the results were encouraging for the further development of this method. PMID:16190465

Raimondo, Francesco; Gavrielides, Marios A; Karayannopoulou, Georgia; Lyroudia, Kleoniki; Pitas, Ioannis; Kostopoulos, Ioannis

2005-09-01

71

Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).  

PubMed

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin-fixed, paraffin-embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples. PMID:23121603

Kiyose, Shinichiro; Igarashi, Hisaki; Nagura, Kiyoko; Kamo, Takaharu; Kawane, Kazunori; Mori, Hiroki; Ozawa, Takachika; Maeda, Matsuyoshi; Konno, Keisuke; Hoshino, Hideaki; Konno, Hiroyuki; Ogura, Hiroyuki; Shinmura, Kazuya; Hattori, Naohiko; Sugimura, Haruhiko

2012-11-01

72

Rapid and Accurate Diagnosis of Human Intestinal Spirochetosis by Fluorescence In Situ Hybridization?  

PubMed Central

Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS. PMID:19279178

Schmiedel, Dinah; Epple, Hans-Jorg; Loddenkemper, Christoph; Ignatius, Ralf; Wagner, Jutta; Hammer, Bettina; Petrich, Annett; Stein, Harald; Gobel, Ulf B.; Schneider, Thomas; Moter, Annette

2009-01-01

73

Analysis of methanogenic activity in a thermophilic-dry anaerobic reactor: Use of fluorescent in situ hybridization  

Microsoft Academic Search

Methanogenic activity in a thermophilic-dry anaerobic reactor was determined by comparing the amount of methane generated for each of the organic loading rates with the size of the total and specific methanogenic population, as determined by fluorescent in situ hybridization. A high correlation was evident between the total methanogenic activity and retention time [?0.6988Ln(x)+2.667] (R2 0.8866). The total methanogenic activity

B. Montero; J. L. García-Morales; D. Sales; R. Solera

2009-01-01

74

Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine  

PubMed Central

Extensive studies of the genetic aberrations related to human diseases conducted over the last two decades have identified recurrent genomic abnormalities as potential driving factors underlying a variety of cancers. Over the time, a series of cutting-edge high-throughput genetic tests, such as microarrays and next-generation sequencing, have been developed and incorporated into routine clinical practice. Although it is a classical low-throughput cytogenetic test, fluorescence in situ hybridization (FISH) does not show signs of fading; on the contrary, it plays an increasingly important role in detecting specific biomarkers in solid and hematologic neoplasms and has therefore become an indispensable part of the rapidly developing field of personalized medicine. In this article, we have summarized the recent advances in FISH application for both de novo discovery and routine detection of chromosomal rearrangements, amplifications, and deletions that are associated with the pathogenesis of various hematopoietic and non-hematopoietic malignancies. In addition, we have reviewed the recent developments in FISH methodology as well. PMID:24499728

2014-01-01

75

Phylogenetic analysis of multiprobe fluorescence in situ hybridization data from tumor cell populations  

PubMed Central

Motivation: Development and progression of solid tumors can be attributed to a process of mutations, which typically includes changes in the number of copies of genes or genomic regions. Although comparisons of cells within single tumors show extensive heterogeneity, recurring features of their evolutionary process may be discerned by comparing multiple regions or cells of a tumor. A useful source of data for studying likely progression of individual tumors is fluorescence in situ hybridization (FISH), which allows one to count copy numbers of several genes in hundreds of single cells. Novel algorithms for interpreting such data phylogenetically are needed, however, to reconstruct likely evolutionary trajectories from states of single cells and facilitate analysis of tumor evolution. Results: In this article, we develop phylogenetic methods to infer likely models of tumor progression using FISH copy number data and apply them to a study of FISH data from two cancer types. Statistical analyses of topological characteristics of the tree-based model provide insights into likely tumor progression pathways consistent with the prior literature. Furthermore, tree statistics from the resulting phylogenies can be used as features for prediction methods. This results in improved accuracy, relative to unstructured gene copy number data, at predicting tumor state and future metastasis. Availability: Source code for software that does FISH tree building (FISHtrees) and the data on cervical and breast cancer examined here are available at ftp://ftp.ncbi.nlm.nih.gov/pub/FISHtrees. Contact: sachowdh@andrew.cmu.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23812984

Schwartz, Russell

2013-01-01

76

Estimate of true incomplete exchanges using fluorescence in situ hybridization with telomere probes  

NASA Technical Reports Server (NTRS)

PURPOSE: To study the frequency of true incomplete exchanges in radiation-induced chromosome aberrations. MATERIALS AND METHODS: Human lymphocytes were exposed to 2 Gy and 5 Gy of gamma-rays. Chromosome aberrations were studied using the fluorescence in situ hybridization (FISH) technique with whole chromosome-specific probes, together with human telomere probes. Chromosomes 2 and 4 were chosen in the present study. RESULTS: The percentage of incomplete exchanges was 27% when telomere signals were not considered. After excluding false incomplete exchanges identified by the telomere signals, the percentage of incomplete exchanges decreased to 11%. Since telomere signals appear on about 82% of the telomeres, the percentage of true incomplete exchanges should be even lower and was estimated to be 3%. This percentage was similar for chromosomes 2 and 4 and for doses of both 2 Gy and 5 Gy. CONCLUSIONS: The percentage of true incomplete exchanges is significantly lower in gamma-irradiated human lymphocytes than the frequencies reported in the literature.

Wu, H.; George, K.; Yang, T. C.

1998-01-01

77

Interphase fluorescence in situ hybridization signal detection by computing intensity variance along the optical axis  

NASA Astrophysics Data System (ADS)

Fluorescence in situ Hybridization technology is a commonly used tool to detect chromosome aberrations, which are often pathologically significant. Since manual FISH analysis is a tedious and time-consuming procedure, reliable and robust automated image acquisition and analysis are in demand. Under high magnification objective lenses such as 60x and 100x, the depth of field will often be too small and the FISH probes may not always lie in the same focal plane. A statistical variance based automated FISH analysis method is developed in order to address this problem. On a stack of slices at consecutive image planes with a step size d, the statistical variance alone the z-axis is calculated to form a 2-D matrix. Since pixels shift dramatically to high intensity at FISH probe location, the probes will manifest high peak values in the matrix. A computer-aided detection scheme based on top-hat transform is applied to the matrix to detect FISH probe signals. This study demonstrates a simple and robust method for FISH probe detection as well as a way of 2- D representation of 3-D data.

Li, Zheng; Zheng, Bin; Ren, Liqiang; Liu, Hong

2014-02-01

78

Does polyomavirus infection interfere with bladder cancer fluorescence in situ hybridization?  

PubMed

Urine cytology is a proven and widely used screening tool for the detection of urothelial carcinoma. However, morphologic features of polyomavirus infected cells, characterized by nuclear inclusions (decoy cells) are a known source of diagnostic confusion with malignancy. Fluorescence in situ hybridization (FISH) is now routinely used to support the cytological diagnosis of urothelial carcinoma and monitor for recurrence. We sought to determine whether polyomavirus infection could result in positive FISH results (aneuploidy). This study deals with retrospective study of 100 polyomavirus-infected urine samples from patients with no history of urothelial carcinoma or organ transplantation. All cases were stained with Papanicolaou and acid hematoxylin stain. One slide from each sample was de-stained and FISH was performed using chromosome enumeration probes 3, 7, 17, and locus-specific probe 9p21. Adequate cells for FISH analysis (25 cells) were present in 81 cases; 19 cases were insufficient due to loss of cells during de-staining and FISH preparation process. All polyomavirus-infected cells (decoy cells) exhibited a normal chromosome pattern. Four cases were FISH positive, but there were no positive decoy cells. Decoy cells did not exhibit aneuploidy by FISH. The presence of decoy cells does not exclude the possibility of concurrent urothelial carcinoma. Acid hematoxylin stain appeared to supplement the Papanicolou stain in identifying and confirming the presence of polyomavirus infection. PMID:24006232

Hossain, Deloar; Hull, David; Kalantarpour, Fatemeh; Maitlen, Rebecca; Qian, Junqi; Bostwick, David G

2014-03-01

79

Metaphase FISH on a Chip: Miniaturized Microfluidic Device for Fluorescence in situ Hybridization  

PubMed Central

Fluorescence in situ Hybridization (FISH) is a major cytogenetic technique for clinical genetic diagnosis of both inherited and acquired chromosomal abnormalities. Although FISH techniques have evolved and are often used together with other cytogenetic methods like CGH, PRINS and PNA-FISH, the process continues to be a manual, labour intensive, expensive and time consuming technique, often taking over 3 5 days, even in dedicated labs. We have developed a novel microFISH device to perform metaphase FISH on a chip which overcomes many shortcomings of the current laboratory protocols. This work also introduces a novel splashing device for preparing metaphase spreads on a microscope glass slide, followed by a rapid adhesive tape-based bonding protocol leading to rapid fabrication of the microFISH device. The microFISH device allows for an optimized metaphase FISH protocol on a chip with over a 20-fold reduction in the reagent volume. This is the first demonstration of metaphase FISH on a microfluidic device and offers a possibility of automation and significant cost reduction of many routine diagnostic tests of genetic anomalies. PMID:22163442

Vedarethinam, Indumathi; Shah, Pranjul; Dimaki, Maria; Tumer, Zeynep; Tommerup, Niels; Svendsen, Winnie E.

2010-01-01

80

Microfluidic fluorescence in situ hybridization and flow cytometry (µFlowFISH)  

PubMed Central

We describe an integrated microfluidic device (µFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(VI) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The µFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed; and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp. strain RCH2 that are involved in Cr(VI) reduction and immobilization. Combined labeling and detection efficiencies of 74–97% were observed in experiments with simple mixtures of cultured cells confirmed specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of µFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford Site. We were able to monitor the numbers of Pseudomonas sp. with only 100–200 cells loaded into the microchip. The µFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome. PMID:21755095

Liu, Peng; Meagher, Robert J.; Light, Yooli Kim; Yilmaz, Suzan; Chakraborty, Romy; Arkin, Adam P.; Hazen, Terry C.; Singh, Anup K.

2011-01-01

81

Microfluidic fluorescence in situ hybridization and flow cytometry (?FlowFISH).  

PubMed

We describe an integrated microfluidic device (?FlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(vi) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The ?FlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed, and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp.strain RCH2 that are involved in Cr(vi) reduction and immobilization. Combined labeling and detection efficiencies of 74-97% were observed in experiments with simple mixtures of cultured cells, confirming specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of ?FlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford site. We were able to monitor the numbers of Pseudomonas sp. with only 100-200 cells loaded into the microchip. The ?FlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome. PMID:21755095

Liu, Peng; Meagher, Robert J; Light, Yooli K; Yilmaz, Suzan; Chakraborty, Romy; Arkin, Adam P; Hazen, Terry C; Singh, Anup K

2011-08-21

82

Multitarget fluorescence in situ hybridization and melanoma antigen genes analysis in primary bladder carcinoma.  

PubMed

Conventional urine cytology has a poor prognostic performance for detecting bladder cancer, particularly for low-grade tumors. Fluorescence in situ hybridization (FISH) for chromosomes altered in bladder cancer and testing for antigens selectively expressed in tumors are promising alternatives. This study investigated the use of FISH for detecting aneuploidy of chromosomes 3, 7, 17, and 9p21 and reverse transcriptase PCR (RT-PCR) for the expression of melanoma associated antigen (MAGE) genes for the diagnosis of bladder cancer in voided urine specimens. The two techniques were compared with cystoscopic bladder biopsy results in 47 patients with urothelial cancer and 15 patients with benign prostatic hyperplasia. FISH detected cancer in 42 of 47 patients (89.4%). This was significantly higher than the detection rate 30 of 47 patients (64.3%) by MAGE RT-PCR (P < 0.001). The sensitivity of FISH increased with histologic grade and stage of the tumors, correctly identifying 77.8% of pTa and pTis, 94.1% of pT1, and 100% of Pt2-4 tumors. MAGE, however, showed a decreased sensitivity in high grade advanced tumors; it was positive in 66.7% of pTa and pTis, 70.6% of pT1, and 50% of Pt2-4 tumors. Together, the tests correctly identified urothelial cancer in 46 of 47 patients (97.9%). Combined FISH and MAGE RT-PCR testing may offer a promising alternative to conventional urine cytology in screening high-risk populations and in monitoring bladder cancer patients for recurrent tumor. PMID:16364760

Kang, Ji Un; Koo, Sun Hoe; Jeong, Tae Eun; Kwon, Kye Chul; Park, Jong Woo; Jeon, Chang Ho

2006-01-01

83

TP53 deleted cells in de novo glioblastomas using fluorescence in situ hybridization.  

PubMed

Glioblastoma (GBM) has been known to have two distinct genetic pathways of tumorigenesis. Secondary GBM shows frequent TP53 mutation, but de novo (primary) GBM is usually independent of TP53 alteration. However, the subpopulation of TP53 altered cells in the latter tumor is obscure. In order to assess TP53 deleted cells in de novo GBM quantitatively, we performed dual color fluorescence in situ hybridization (FISH) for TP53 and centromere 17 in nine cases of de novo GBM with frozen surgical materials. Single TP53 signal cells indicating TP53 deletion were recognized in 8.7-35.6% (mean, 21.3%) among the nine cases. In addition, immunohistochemistry was performed for the Ki-67 antigen (MIB-1) and p53 protein in all nine cases. Labeling indices (LI) of MIB-1 ranged from 2.8 to 46.9% (mean, 20.8%). Between the group with the more dense subpopulation of TP53 deleted cells (15% or more) by FISH and the group with less subpopulation than the former, these LI of MIB-1 demonstrated statistically significant difference (respective means, 28.2% and 6.1%; P < 0.05). Conversely, LI of p53 protein shown to be 0-50.9% (mean, 24.9%) had no correlation with the subpopulation of TP53 deleted cells by FISH. Four cases who had higher LI of p53 protein (mean, 39.7%) than the subpopulation of TP53 deleted cells (mean, 12.7%), respectively, indicated the presence of many p53 protein immunoreactive cells without TP53 deletion. These results suggest that: (i) de novo GBM also has subpopulation of TP53 deleted cells; (ii) TP53 alteration, which may not be a major event, participates in cell proliferation of de novo GBM; and (iii) de novo GBM tends to have accumulation of wild-type p53 protein. PMID:11328534

Horiguchi, H; Sano, T; Hirose, T

2001-03-01

84

Peptide Nucleic Acid Fluorescent In Situ Hybridization for Hospital-Acquired Enterococcal Bacteremia: Delivering Earlier Effective Antimicrobial Therapy  

Microsoft Academic Search

Hospital-acquired vancomycin-resistant enterococcal bacteremia has been associated with increased hospi- tal costs, length of stay, and mortality. The peptide nucleic acid fluorescent in situ hybridization (PNA FISH) test for Enterococcus faecalis and other enterococci (EFOE) is a multicolor probe that differentiates E. faecalis from other enterococcal species within 3 h directly from blood cultures demonstrating gram-positive cocci in pairs and

Graeme N. Forrest; Mary-Claire Roghmann; Latoya S. Toombs; Jennifer K. Johnson; Elizabeth Weekes; Durry P. Lincalis; Richard A. Venezia

2008-01-01

85

Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles  

Microsoft Academic Search

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C.

Susan Rigby; Gary W. Procop; Gerhard Haase; Deborah Wilson; Cletus Kurtzman; Kenneth Oliveira; Sabina Von Oy; Jens J. Hyldig-Nielsen; James Coull; Henrik Stender

2002-01-01

86

Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes  

Microsoft Academic Search

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein- labeled PNA probe that targets a species-specific sequence of the 16S rRNA of

Kenneth Oliveira; Gary W. Procop; Deborah Wilson; James Coull; Henrik Stender

2002-01-01

87

Simple Adhesive-Tape-Based Sampling of Tomato Surfaces Combined with Rapid Fluorescence In Situ Hybridization for Salmonella Detection?  

PubMed Central

A simple adhesive-tape-based method for sampling of tomato surfaces was combined with fluorescence in situ hybridization for rapid culture-independent detection of Salmonella strains. Tapes could also be placed face-down on selective agar for on-tape enrichment of captured Salmonella cells. Overlay of cell-charged tapes with small volumes of liquid enrichment media enabled subsequent detection of tape-captured Salmonella via flow cytometry. PMID:19124588

Bisha, Bledar; Brehm-Stecher, Byron F.

2009-01-01

88

Assignment of the human organic anion transporting polypeptide (OATP) gene to chromosome 12p12 by fluorescence in situ hybridization  

Microsoft Academic Search

Background\\/Aims: The organic anion transporting polypeptide (OATP) of human liver mediates the basolateral hepatocellular uptake of numerous cholephilic anions and steroidal compounds. The aim of this study was to clone the human OATP gene and to map its chromosomal localization by fluorescence in situ hybridization.Methods: A polymerase chain reaction-amplified fragment of the human OATP gene was used to isolate a

Gerd-Achim Kullak-Ublick; Ulrich Beuers; Peter J. Meier; Horst Domdey; Gustav Paumgartner

1996-01-01

89

Detection of Gallibacterium spp. in chickens by fluorescent 16S rRNA in situ hybridization.  

PubMed

Gallibacterium has recently been included as a new genus of the family Pasteurellaceae Pohl 1981, which encompasses bacteria previously reported as Pasteurella anatis, "Actinobacillus salpingitidis," and avian Pasteurella haemolytica-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus Gallibacterium. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families Pasteurellaceae, Enterobacteriaceae, and Flavobacteriaceae, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of Gallibacterium. Additionally, determination of the spatial distribution and the host cell affiliation of Gallibacterium at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of Gallibacterium infections in chickens. PMID:14605154

Bojesen, Anders Miki; Christensen, Henrik; Nielsen, Ole Lerberg; Olsen, John Elmerdahl; Bisgaard, Magne

2003-11-01

90

Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes  

PubMed Central

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity. PMID:11157265

Stender, Henrik; Kurtzman, Cletus; Hyldig-Nielsen, Jens J.; S?rensen, Ditte; Broomer, Adam; Oliveira, Kenneth; Perry-O'Keefe, Heather; Sage, Andrew; Young, Barbara; Coull, James

2001-01-01

91

Rapid-prenatal diagnosis through fluorescence in situ hybridization for preventing aneuploidy related birth defects  

PubMed Central

BACKGROUND AND OBJECTIVE: Women with high-risk pregnancies are offered prenatal diagnosis through amniocentesis for cytogenetic analysis of fetal cells. The aim of this study was to evaluate the effectiveness of the rapid fluorescence in situ hybridization (FISH) technique for detecting numerical aberrations of chromosomes 13, 21, 18, X and Y in high-risk pregnancies in an Indian scenario. MATERIALS AND METHODS: A total of 163 samples were received for a FISH and/or a full karyotype for prenatal diagnosis from high-risk pregnancies. In 116 samples both conventional culture techniques for getting karyotype through G-banding techniques were applied in conjunction to FISH test using the AneuVysion kit (Abbott Molecular, Inc.), following standard recommended protocol to compare the both the techniques in our setup. RESULTS: Out of 116 patients, we got 96 normal for the five major chromosome abnormality and seven patients were found to be abnormal (04 trisomy 21, 02 monosomy X, and 01 trisomy 13) and all the FISH results correlated with conventional cytogenetics. To summarize the results of total 163 patients for the major chromosomal abnormalities analyzed by both/or cytogenetics and FISH there were 140 (86%) normal, 9 (6%) cases were abnormal and another 4 (2.5%) cases were suspicious mosaic and 10 (6%) cases of culture failure. The diagnostic detection rate with FISH in 116 patients was 97.5%. There were no false-positive and false-negative autosomal or sex chromosomal results, within our established criteria for reporting FISH signals. CONCLUSION: Rapid FISH is a reliable and prompt method for detecting numerical chromosomal aberrations and has now been implemented as a routine diagnostic procedure for detection of fetal aneuploidy in India. PMID:23901191

Fauzdar, Ashish; Chowdhry, Mohit; Makroo, R. N.; Mishra, Manoj; Srivastava, Priyanka; Tyagi, Richa; Bhadauria, Preeti; Kaul, Anita

2013-01-01

92

Fluorescent In Situ Hybridization: A New Tool for the Direct Identification and Detection of F. psychrophilum  

PubMed Central

F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium (“Pan-Flavo”) and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum. PMID:23152887

Strepparava, Nicole; Wahli, Thomas; Segner, Helmut; Polli, Bruno; Petrini, Orlando

2012-01-01

93

QUANTITATIVE IMAGING AND STATISTICAL ANALYSIS OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) OF AUREOBASIDIUM PULLULANS. (R823845)  

EPA Science Inventory

Abstract Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides , as an outgroup, hybridized with either a universal o...

94

Prenatal diagnosis of complex rearrangement of chromosome 21: The significance of interphase and metaphase fluorescence in situ hybridization and comparative genomic hybridization  

PubMed Central

Key Clinical Message Maternal serum screening–positive patient had prenatal diagnosis with amniotic fluid, which showed inconsistent results between interphase fluorescence in situ hybridization (three signals of 21q22.13-21q22.2) and G-banding analysis (46,XY). Further analyses proved that the fetus had extremely complex rearrangements of chromosome 21, including the interstitial duplication of Down syndrome critical region. PMID:25356211

Namba, Akira; Nishiyama, Miyuki; Weiser, Joseph J; Wyatt, Phillip; Kimura, Machiko; Niizawa, Rei; Miki, Akinori; Ishihara, Osamu; Itakura, Atsuo; Kamei, Yoshimasa

2013-01-01

95

Sex chromosome differentiation in Humulus japonicus Siebold & Zuccarini, 1846 (Cannabaceae) revealed by fluorescence in situ hybridization of subtelomeric repeat  

PubMed Central

Abstract Humulus japonicus Siebold et Zucc (Japanese hop) is a dioecious species of the family Cannabaceae. The chromosome number is 2n = 16 = 14 + XX for females and 2n = 17 = 14 + XY1Y2 for male. To date, no fluorescence in situ hybridization (FISH) markers have been established for the identification of Humulus japonicus sex chromosomes. In this paper, we report a method for the mitotic and meiotic sex chromosome differentiation in Humulus japonicus by FISH for HJSR, a high copy subtelomeric repeat. The signal is present in the subtelomeric region of one arm of the X chromosome. We demonstrate that males have two Y chromosomes that differ in FISH signal with the HJSR probe. Indeed, the HJSR probe hybridizes to a subtelomeric region on both arms of chromosome Y1 but not of chromosome Y2. The orientation and position of pseudoautosomal regions (PAR1 and PAR2) were also determined. PMID:24260665

Alexandrov, Oleg S.; Divashuk, Mikhail G.; Yakovin, Nikolay A.; Karlov, Gennady I.

2012-01-01

96

A high-resolution karyotype of Brassica rapa ssp. pekinensis revealed by pachytene analysis and multicolor fluorescence in situ hybridization  

Microsoft Academic Search

A molecular cytogenetic map of Chinese cabbage ( Brassica rapa ssp. pekinensis, 2 n=20) was constructed based on the 4?-6-diamino-2-phenylindole dihydrochloride-stained mitotic metaphase and pachytene chromosomes and multicolor fluorescence in situ hybridization (McFISH), using three repetitive DNA sequences, 5S rDNA, 45S rDNA, and C11-350H. The lengths of mitotic metaphase chromosomes ranged from 1.46 ?m to 3.30 ?m. Five 45S and three 5S

Dal-Hoe Koo; Prikshit Plaha; Yong Pyo Lim; Yoonkang Hur; Jae-Wook Bang

2004-01-01

97

A simple and rapid fluorescence in situ hybridization microwave protocol for reliable dicentric chromosome analysis.  

PubMed

Fluorescence in situhybridization (FISH) is an extremely effective and sensitive approach to analyzing chromosome aberrations. Until recently, this procedure has taken multiple days to complete. The introduction of telomeric and centromeric peptide nucleic acid (PNA) probes has reduced the procedure's duration to several hours, but the protocols still call for a high temperature (80-90°C) step followed by 1-3 h of hybridization. The newest method to speed up the FISH protocol is the use of a microwave to shorten the heating element to less than a minute; however this protocol still calls for a 1-h hybridization period. We have utilized PNA centromere/telomere probes in conjunction with a microwave oven to show telomere and centromere staining in as little as 30 s. We have optimized the hybridization conditions to increase the sensitivity and effectiveness of the new protocol and can effectively stain chromosomes in 2 min and 30 s of incubation. We have found that our new approach to FISH produces extremely clear and distinct signals. Radiation-induced dicentric formation in mouse and human fibroblast cells was analyzed by two individual scorers and the observed dicentrics matched very well. PMID:23161278

Cartwright, Ian M; Genet, Matthew D; Kato, Takamitsu A

2013-03-01

98

A simple and rapid fluorescence in situ hybridization microwave protocol for reliable dicentric chromosome analysis  

PubMed Central

Fluorescence in situhybridization (FISH) is an extremely effective and sensitive approach to analyzing chromosome aberrations. Until recently, this procedure has taken multiple days to complete. The introduction of telomeric and centromeric peptide nucleic acid (PNA) probes has reduced the procedure's duration to several hours, but the protocols still call for a high temperature (80–90°C) step followed by 1–3 h of hybridization. The newest method to speed up the FISH protocol is the use of a microwave to shorten the heating element to less than a minute; however this protocol still calls for a 1-h hybridization period. We have utilized PNA centromere/telomere probes in conjunction with a microwave oven to show telomere and centromere staining in as little as 30 s. We have optimized the hybridization conditions to increase the sensitivity and effectiveness of the new protocol and can effectively stain chromosomes in 2 min and 30 s of incubation. We have found that our new approach to FISH produces extremely clear and distinct signals. Radiation-induced dicentric formation in mouse and human fibroblast cells was analyzed by two individual scorers and the observed dicentrics matched very well. PMID:23161278

Cartwright, Ian M.; Genet, Matthew D.; Kato, Takamitsu A.

2013-01-01

99

Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes)  

SciTech Connect

The aim of this study was to develop a sensitive in situ hybridization methodology using fluorescence-labeled riboprobes (FISH) that allows for the evaluation of gene expression profiles simultaneously in multiple target tissues of whole fish sections of Japanese medaka (Oryzias latipes). To date FISH methods have been limited in their application due to autofluorescence of tissues, fixatives or other components of the hybridization procedure. An optimized FISH method, based on confocal fluorescence microscopy was developed to reduce the autofluorescence signal. Because of its tissue- and gender-specific expression and relevance in studies of endocrine disruption, gonadal aromatase (CYP19a) was used as a model gene. The in situ hybridization (ISH) system was validated in a test exposure with the aromatase inhibitor fadrozole. The optimized FISH method revealed tissue-specific expression of the CYP19a gene. Furthermore, the assay could differentiate the abundance of CYP19a mRNA among cell types. Expression of CYP19a was primarily associated with early stage oocytes, and expression gradually decreased with increasing maturation. No expression of CYP19a mRNA was observed in other tissues such as brain, liver, or testes. Fadrozole (100 {mu}g/L) caused up-regulation of CYP19a expression, a trend that was confirmed by RT-PCR analysis on excised tissues. In a combination approach with gonad histology, it could be shown that the increase in CYP19a expression as measured by RT-PCR on a whole tissue basis was due to a combination of both increases in numbers of CYP19a-containing cells and an increase in the amount of CYP19a mRNA present in the cells.

Park, June-Woo [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Tompsett, Amber [Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan (Canada); Zhang, Xiaowei [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan (Canada); Newsted, John L. [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); ENTRIX, Inc., Okemos, MI 48823 (United States); Jones, Paul D.; Au, Doris [Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); Kong, Richard; Wu, Rudolf S.S. [School of Environmental Science, Nanjing University, Nanjing (China); Giesy, John P. [Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan (Canada); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); School of Environmental Science, Nanjing University, Nanjing (China); ENTRIX, Inc., Saskatoon, Saskatchewan (Canada)], E-mail: JGiesy@aol.com; Hecker, Markus [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); ENTRIX, Inc., Saskatoon, Saskatchewan (Canada)

2008-10-15

100

Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization  

SciTech Connect

Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell.

Lawrence, J.B.; Marselle, L.M.; Byron, K.S.; Johnson, C.V.; Sullivan, J.L.; Singer, R.H. (Univ. of Massachusetts Medical Center, Worcester (USA))

1990-07-01

101

Evaluation of the diagnostic value of fluorescent in situ hybridization in a rat model of bacterial pneumonia.  

PubMed

In severe nosocomial pneumonia, the pathogenic responsibility of bacteria isolated from airways is far from certain, and a lung biopsy is sometimes performed. However, detection and identification of pathogens are frequently unachieved. Here, we developed a protocol for direct visualization of bacteria within the lung tissue using fluorescent in situ hybridization (FISH) in a rat model of Acinetobacter baumannii pneumonia. The reference positive diagnosis of bacterial pneumonia was the presence of pathological signs of pneumonia associated with the proof of bacteria or bacterial PCR products into the parenchyma. By analysis of 122 sets of slices from 26 rats and using the eubacterial probe EUB-338, our results show that FISH reached a sensitivity and a diagnostic accuracy higher than that of optic microscopy (sensitivity: 96% versus 55.4% and diagnostic accuracy: 96.7% versus 66.4%), whereas both approaches had 100% specificity. FISH could be useful especially on negative biopsies from patients with suspected infectious pneumonia. PMID:23747031

Atieh, Thérèse; Audoly, Gilles; Hraiech, Sami; Lepidi, Hubert; Roch, Antoine; Rolain, Jean-Marc; Raoult, Didier; Papazian, Laurent; Brégeon, Fabienne

2013-08-01

102

Fluorescence in situ hybridization on DNA halo preparations and extended chromatin fibres.  

PubMed

Although many fluorescence in situ hybridisation (FISH) protocols involve the use of intact, fixed nuclei, the resolution achieved is not always sufficient, especially for physical mapping. In light of this, several techniques are commonly used to create extended chromatin fibres or extruded loops of DNA. As a result, it is possible to visualise and distinguish regions of the genome at a resolution higher than that attained with conventional preparations for FISH. Such methodologies include fibre-FISH and the DNA halo preparation. While fibre-FISH involves the stretching of chromatin fibres across a glass slide, the DNA halo preparation is somewhat more complex; whereby DNA loops instead of chromatin fibres are generated from interphase nuclei. Furthermore, the DNA halo preparation coupled with FISH is a useful tool for examining interactions between the inextractable nuclear matrix and the cell's genome.In this chapter, we describe how to successfully generate extended chromatin fibres and extruded DNA loops. We will also provide detailed methodologies for coupling either procedure with two distinct FISH procedures; 2D-FISH, which allows for the visualisation of specific chromosomal regions, while telomere peptide nucleic acid (PNA) FISH, enables the detection of all telomeres present within human nuclei. PMID:20809301

Elcock, Lauren S; Bridger, Joanna M

2010-01-01

103

Original Articles Double-Target Fluorescence In Situ Hybridization Distinguishes Multiple Genetically Aberrant Clones in Head and Neck Squamous Cell Carcinoma  

Microsoft Academic Search

Genomic heterogeneity has been observed in several solid tumor types. To investigate this phenomenon in head and neck squamous cell carcinoma (HNSCC), we analyzed macroscopically distinct tissue samples of 12 resected tumors by a combination of fluorescence in situ hybridization (FISH) and DNA flow cytometry. Using a panel of centromeric DNA probes, numerical chromosomal aberrations were detected in 10 tumors,

Joris A. Veltman; Anton H. N. Hopman; Saskia A. van der Vlies; Fredrik J. Bot; Frans C. S. Ramaekers; Johannes J. Manni

104

Rapid Identification of Staphylococcus aureus in Blood Cultures by a Combination of Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes and Flow Cytometry  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) using peptide nucleic acid probes (PNAs) allows the identification of Staphylococcus aureus from human blood culture samples. We present data revealing that the combination of PNA FISH and flow cytometry is a possible approach for the noncultural identification of staphylococci in blood cultures.

Hanna Hartmann; Henrik Stender; Andrea Schafer; Ingo B. Autenrieth; Volkhard A. J. Kempf

2005-01-01

105

Chromosome analysis of nuclear power plant workers using fluorescence in situ hybridization and Giemsa assay  

PubMed Central

The aim of this study was to evaluate the genotoxic effects of ionizing radiation in vivo in exposed Bulgarian nuclear power plant workers by using classical cytogenetic and molecular cytogenetic analyses of peripheral lymphocytes. Chromosome analysis using fluorescence in situ hybrydization (FISH) and Giemsa techniques was undertaken on 63 workers and 45 administrative staff controls from the Bulgarian Nuclear Power Plant. Using the Giemsa method, the frequencies of cells studied with chromosome aberrations, dicentrics plus rings and chromosome fragments in the radiation workers were significantly higher compared with the control group (P = 0.044, P = 0.014, and P = 0.033, respectively). A significant association between frequencies of dicentrics plus rings and accumulated doses was registered (P < 0.01). In the present study, a FISH cocktail of whole chromosome paints for chromosomes 1, 4 and 11 was used. A significant association between frequency of translocations and accumulated doses was also observed (P < 0.001). Within the control group, a correlation was found between age and the spontaneous frequency of translocations. No correlation was found between smoking status and frequency of translocations. When compared with the control group, workers with accumulated doses up to 100 mSv showed no increase in genome translocation frequency, whereas workers with accumulated doses from 101 to 200 mSv showed a statistically significant doubling of genome translocation frequency (P = 0.009). Thus, in cases of chronic exposure and for purposes of retrospective dosimetry, the genome frequency of translocations is a more useful marker for evaluation of genotoxic effects than dicentric frequency. PMID:23536543

Hristova, Rositsa; Hadjidekova, Valeria; Grigorova, Mira; Nikolova, Teodora; Bulanova, Minka; Popova, Ljubomira; Staynova, Albena; Benova, Donka

2013-01-01

106

Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in Potable-Water Biofilms  

Microsoft Academic Search

Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybrid- ization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive

Markku J. Lehtola; Eila Torvinen; Ilkka T. Miettinen; C. William Keevil

2006-01-01

107

Evaluation of Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Identification of Clinically Relevant Mycobacteria in Clinical Specimens and Tissue Sections  

Microsoft Academic Search

With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify

Michael Lefmann; Birgitta Schweickert; Petra Buchholz; Ulf B. Gobel; Timo Ulrichs; Peter Seiler; Dirk Theegarten; Annette Moter

2006-01-01

108

Capture antibody targeted fluorescence in situ hybridization (CAT-FISH): dual labeling allows for increased specificity in complex samples.  

PubMed

Pathogen detection using biosensors is commonly limited due to the need for sensitivity and specificity in detecting targets within mixed populations. These issues were addressed through development of a dual labeling method that allows for both liquid-phase fluorescence in situ hybridization (FISH) and capture antibody targeted detection (CAT-FISH). CAT-FISH was developed using Escherichia coli O157:H7 and Staphylococcus aureus as representative bacteria, and processing techniques were evaluated with regard to FISH intensities and antibody recognition. The alternative fixative solution, methacarn, proved to be superior to standard solid-phase paraformaldehyde fixation procedures, allowing both FISH labeling and antibody recognition. CAT-FISH treated cells were successfully labeled with FISH probes, captured by immunomagnetic separation using fluorescent cytometric array beads, and detected using a cytometric array biosensor. CAT-FISH treated cells were detectable with LODs comparable to the standard antibody-based technique, (~10(3)cells/ml in PBS), and the technique was also successfully applied to two complex matrices. Although immunomagnetic capture and detection using cytometric arrays were demonstrated, CAT-FISH is readily applicable to any antibody-based fluorescence detection platform, and further optimization for sensitivity is possible via inclusion of fluorescently tagged antibodies. Since the confidence level needed for positive identification of a detected target is often paramount, CAT-FISH was developed to allow two separate levels of specificity, namely nucleic acid and protein signatures. With proper selection of FISH probes and capture antibodies, CAT-FISH may be used to provide rapid detection of target pathogens from within complex matrices with high levels of confidence. PMID:22212757

Stroot, Joyce M; Leach, Kelly M; Stroot, Peter G; Lim, Daniel V

2012-02-01

109

Genotype-phenotype correlation in satellited 1p chromosome: Importance of fluorescence in situ hybridization (FISH) applications  

SciTech Connect

Fluorescence in situ hybridization (FISH) was used to delineate the structural rearrangement of two satellited 1p chromosomes identified in a prenatal and in a postnatal case. Prenatal case: chromosome analysis of amniotic cells on a 37-year-old G2, PO, SAb1 woman, referred for advanced maternal age, revealed a 46,XX,1ps karyotype. Parental chromosome analyses showed that the satellited chromosome was paternal in origin. The satellited 1p did not stain with NOR and DA-DAPI. FISH using a probe specific for the rDNA, 18S and 28S genes also showed no hybridization to the satellited 1p. Using two different probes specific for 1p36.3 showed hybridization to both chromosomes 1p. This indicated that the terminal band was most likely present in the fetus and chromosomally balanced like the father. The pregnancy was continued and a phenotypically normal female was born. Postnatal case: Chromosome analysis of peripheral lymphocytes was performed on a 3 1/2-year old female with multiple congenital anomalies including growth retardation, developmental delay, upslanted palpebral fissures, thick eyebrows, esotropia, seizures, and mental retardation. She was born to a 14-year old, G1, PO mother, after a full-term pregnancy, by cesarian-section due to breech presentation. Her one-year-old brother is reportedly in good health. Chromosome analysis revealed a de novo 46,XX,lps. NOR and DA-DAPI stains were positive indicating the satellites are most likely chromosome 15 in origin. FISH using the rDNA probes also showed a hybridization to the 1ps. The two 1p36.3 probes showed only one signal to the normal chromosome 1, indicating a deletion which resulted in monosomy of 1p36.3. The clinical features of the child correlates with published cases of the terminal deletions of 1p.

Habibian, R.; Hajianpour, M.J.; Hajianpour, A.K. [Alfigen/The Genetics Institute, Pasadena, CA (United States)] [and others

1994-09-01

110

Detection of Ralstonia solanacearum, which causes brownrot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes  

Microsoft Academic Search

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacea- rum strains and one Ralstonia pickettii strain were PCR amplified,

B. A. WULLINGS; Beuningen van A. R; J. D. Janse; A. D. L. Akkermans

1998-01-01

111

Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium Chloride for Activity Studies by Simultaneous Use of Microautoradiography and Fluorescence In Situ Hybridization  

Microsoft Academic Search

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only

Jeppe Lund Nielsen; Marilena Aquino de Muro; Per Halkjær Nielsen

2003-01-01

112

Direct fluorescence in situ hybridization (FISH) in Escherichia coli with a target-specific quantum dot-based molecular beacon.  

PubMed

Quantum dots (QDs) are inorganic fluorescent nanocrystals with excellent properties such as tunable emission spectra and photo-bleaching resistance compared with organic dyes, which make them appropriate for applications in molecular beacons. In this work, quantum dot-based molecular beacons (QD-based MBs) were fabricated to specifically detect ?-lactamase genes located in pUC18 which were responsible for antibiotic resistance in bacteria Escherichia coli (E. coli) DH5?. QD-based MBs were constructed by conjugating mercaptoacetic acid-quantum dots (MAA-QDs) with black hole quencher 2 (BHQ2) labeled thiol DNA vial metal-thiol bonds. Two types of molecular beacons, double-strands beacons and hairpin beacons, were observed in product characterization by gel electrophoresis. Using QD-based MBs, one-step FISH in tiny bacteria DH5? was realized for the first time. QD-based MBs retained their bioactivity when hybridizing with complementary target DNA, which showed excellent advantages of eliminating background noise caused by adsorption of non-specific bioprobes and achieving clearer focus of genes in plasmids pUC18, and capability of bacterial cell penetration and signal specificity in one-step in situ hybridization. PMID:20729070

Wu, Sheng-Mei; Tian, Zhi-Quan; Zhang, Zhi-Ling; Huang, Bi-Hai; Jiang, Peng; Xie, Zhi-Xiong; Pang, Dai-Wen

2010-10-15

113

Localization of T-DNA Insertions in Petunia by Fluorescence in Situ Hybridization: Physical Evidence for Suppression of Recombination.  

PubMed Central

Using fluorescence in situ hybridization (FISH) with metaphase preparations, we localized a 4-kb single-copy T-DNA sequence in a group of petunia transformants. The selected T-DNAs previously had been shown to be linked to the phenotypic marker FI on chromosome II. Linkage analysis had revealed that recombination around the FI locus is suppressed in a wide cross relative to an inbred recombination assay. The localization of six FI-linked T-DNAs and the FI locus itself, using FISH, revealed a number of aspects of recombination in petunia: (1) the central region of chromosome II showed at least a 10-fold suppression of recombination in wide crosses relative to the distal region; (2) recombination in wide hybrids over two-thirds of the chromosome was extremely low; and (3) recombination between completely homologous chromosomes in an inbred cross also was suppressed in the central region. In addition, the T-DNAs were not evenly distributed along the chromosome, suggesting a possible preference for a distal position for T-DNA integration. Implications for such a preference are discussed. PMID:12239403

Ten Hoopen, R.; Robbins, T. P.; Fransz, P. F.; Montijn, B. M.; Oud, O.; Gerats, AGM.; Nanninga, N.

1996-01-01

114

Food selection by bacterivorous protists: insight from the analysis of the food vacuole content by means of fluorescence in situ hybridization  

Microsoft Academic Search

A modified fluorescence in situ hybridization (FISH) method was used to analyze bacterial prey composition in protistan food vacuoles in both laboratory and natural populations. Under laboratory conditions, we exposed two bacterial strains (affiliated with b- and c-Proteobacteria - Aeromonas hydrophila and Pseudomonas fluorescens, respectively) to grazing by three protists: the flagel- lates Bodo saltans and Goniomonas sp., and the

Jan Jezbera; Karel Hornak; Karel Simek

115

Assignment of the gastric inhibitory polypeptide receptor gene (GIPR) to chromosome bands 19q13.2-q13.3 by fluorescence in situ hybridization  

SciTech Connect

The gastric inhibitory polypeptide receptor gene (GIPR) was localized, using fluorescence in situ hybridization (FISH), to human chromosome bands 19q13.2-q13.3. Gastric inhibitory polypeptide (GIP) is a potent stimulator of insulin secretion and mutations in the GIPR gene may be related to non-insulin-dependent diabetes mellitus (NIDDM). 13 refs., 1 fig.

Stoffel, M.; Fernald, A.A.; Bell, G.I.; Le Beau, M.M. [Univ. of Chicago, IL (United States)] [Univ. of Chicago, IL (United States)

1995-08-10

116

Fluorescence in situ Hybridization with Human Chromosome-Specific Libraries: Detection of Trisomy 21 and Translocations of Chromosome 4  

Microsoft Academic Search

Chromosomes can be specifically stained in metaphase spreads and interphase nuclei by in situ hybridization with entire chromosome-specific DNA libraries. Unlabeled human genomic DNA is used to inhibit the hybridization of sequences in the library that bind to multiple chromosomes. The target chromosome can be made at least 20 times brighter per unit length than the others. Trisomy 21 and

D. Pinkel; J. Landegent; C. Collins; J. Fuscoe; R. Segraves; J. Lucas; J. Gray

1988-01-01

117

Development of a flow-fluorescence in situ hybridization protocol for the analysis of microbial communities in anaerobic fermentation liquor  

PubMed Central

Background The production of bio-methane from renewable raw material is of high interest because of the increasing scarcity of fossil fuels. The process of biomethanation is based on the inter- and intraspecific metabolic activity of a highly diverse and dynamic microbial community. The community structure of the microbial biocenosis varies between different biogas reactors and the knowledge about these microbial communities is still fragmentary. However, up to now no approaches are available allowing a fast and reliable access to the microbial community structure. Hence, the aim of this study was to originate a Flow-FISH protocol, namely a combination of flow cytometry and fluorescence in situ hybridization, for the analysis of the metabolically active microorganisms in biogas reactor samples. With respect to the heterogenic texture of biogas reactor samples and to collect all cells including those of cell aggregates and biofilms the development of a preceding purification procedure was indispensable. Results Six different purification procedures with in total 29 modifications were tested. The optimized purification procedure combines the use of the detergent sodium hexametaphosphate with ultrasonic treatment and a final filtration step. By this treatment, the detachment of microbial cells from particles as well as the disbandment of cell aggregates was obtained at minimized cell loss. A Flow-FISH protocol was developed avoiding dehydration and minimizing centrifugation steps. In the exemplary application of this protocol on pure cultures as well as biogas reactor samples high hybridization rates were achieved for commonly established domain specific oligonucleotide probes enabling the specific detection of metabolically active bacteria and archaea. Cross hybridization and autofluorescence effects could be excluded by the use of a nonsense probe and negative controls, respectively. Conclusions The approach described in this study enables for the first time the analysis of the metabolically active fraction of the microbial communities within biogas reactors by Flow-FISH. PMID:24304697

2013-01-01

118

Fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes to identify small phytoplankton by flow cytometry.  

PubMed Central

Because of their tiny size (0.2 to 2 microns), oceanic picophytoplanktonic cells (either cultured strains or natural communities) are difficult to identify, and some basic questions concerning their taxonomy, physiology, and ecology are still largely unanswered. The present study was designed to test the suitability of in situ hybridization with rRNA fluorescent probes detected by flow cytometry for the identification of small photosynthetic eukaryotes. Oligonucleotide probes targeted against regions of the 18S rRNAs of Chlorophyta lineage (CHLO probe) and of non-Chlorophyta (NCHLO probe) algal species were designed. The CHLO and NCHLO probes, which differed by a single nucleotide, allowed discrimination of chlorophyte from nonchlorophyte cultured strains. The sensitivity of each probe was dependent upon the size of the cells and upon their growth stage. The mean fluorescence was 8 to 80 times higher for specifically labeled than for nonspecifically labeled cells in exponential growth phase, but it decreased sharply in stationary phase. Such taxon-specific probes should increase the applicability of flow cytometry for the rapid identification of cultured pico- and nanoplanktonic strains, especially those that lack taxonomically useful morphological features. PMID:7618862

Simon, N; LeBot, N; Marie, D; Partensky, F; Vaulot, D

1995-01-01

119

Combination of adhesive-tape-based sampling and fluorescence in situ hybridization for rapid detection of Salmonella on fresh produce.  

PubMed

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55°C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (? 500 ?L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37°C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalapeño peppers. PMID:21048665

Bisha, Bledar; Brehm-Stecher, Byron F

2010-01-01

120

Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization  

SciTech Connect

Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with dithiothreitol and 3,4-diiodosalicylate salt to decondense the sperm chromatin and then were hybridized with repetitive sequence DNA probes that had been generated by PCR and differentially labeled. Hybridizations with X and Y specific probes showed the expected ratio of 50%X:50%Y bearing sperm. Sperm carrying extra fluorescence domains representing disomy for the X or Y chromosomes occurred at frequencies of {approximately} 4 per 10,000 sperm each. Cells carrying both X and Y fluorescence domains occurred at a frequency of {approximately} 6/10,000. Thus, the overall frequency of sperm that carried an extra sex chromosome was 1.4/1,000. The frequencies of sperm carrying sex chromosome aneuploidies determined by hybridization did not differ statistically from those reported from the same laboratory using the human-sperm/hamster-egg cytogenetic technique. Multi-chromosome fluorescence in situ hybridization to sperm is a promising method for assessing sex-ratio alterations in human semen and for determining the fraction of sperm carrying sex or other chromosome aneuploidies which may be transmissible to offspring. 44 refs., 1 fig., 3 tabs.

Wyrobek, A.J. [Lawrence Livermore National Lab., CA (United States); Robbins, W.A. [Lawrence Livermore National Lab., CA (United States)]|[Univ. of California, Berkeley, CA (United States); Pinkel, D.; Weier, H.U. [Univ. of California, San Francisco, CA (United States); Mehraein, Y. [Univ. of California, San Francisco, CA (United States)]|[Philipps Universitat, Marburg (Germany)

1994-10-15

121

Medical devices; hematology and pathology devices; classification of early growth response 1 gene fluorescence in-situ hybridization test system for specimen characterization. Final order.  

PubMed

The Food and Drug Administration (FDA) is classifying early growth response 1 (EGR1) gene fluorescence in-situ hybridization (FISH) test system for specimen characterization into class II (special controls). The special controls that will apply to this device are identified in this order and will be part of the codified language for the early growth response 1 (EGR1) gene fluorescence in-site hybridization (FISH) test system for specimen characterization classification. The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. PMID:25195217

2014-09-01

122

Characterization of IGH rearrangements in non-Hodgkin's B-cell lymphomas by fluorescence in situ hybridization.  

PubMed

Rearrangements involving the IGH gene have been identified in about 50% of non-Hodgkin's B-cell lymphomas (NHL) and correlated to clinical relevant subgroups. However, the detection rate varied greatly with the technique used. The incidence of IGH rearrangements was analyzed using several fluorescence in situ hybridization (FISH) techniques on metaphases obtained from 57 patients with nodal NHL. An IGH rearrangement was identified in 42 cases (73.7%). A t(14;18)(q32;q21) was found in 17 of the 20 follicular lymphomas (85%) studied and a t(11;14)(q13;q32) in 10 of the 11 mantle cell lymphomas (91%). IGH rearrangements were identified in 12 of the 26 diffuse large B-cell lymphomas (46%), including 5 t(14;18)(q32;q21) and 2 t(3;14)(q27;q32). Conventional cytogenetics was uninformative in several cases. However, the complemented analysis using Multi-FISH and/or chromosomal whole paint enabled the characterization of complex IGH translocations in follicular lymphomas and mantle cell lymphomas and the identification of all the chromosomal partners involved in the IGH rearrangement in diffuse large B-cell lymphomas. This study shows the interest of using metaphase FISH in addition to conventional cytogenetics. Following banding techniques, FISH with the IGH dual color probe could be the first approach in NHL, after which chromosome painting and M-FISH could be used to identify the chromosomal partner involved in the IGH rearrangement. PMID:16101124

Bernicot, Izabel; Douet-Guilbert, Nathalie; Le Bris, Marie-Josée; Morice, Patrick; Abgrall, Jean Francois; Berthou, Christian; Morel, Frédéric; De Braekeleer, Marc

2005-01-01

123

Quantitative fluorescence in situ hybridization measurement of telomere length in skin with/without sun exposure or actinic keratosis.  

PubMed

Chromosomal and genomic instability due to telomere dysfunction is known to play an important role in carcinogenesis. To study telomere shortening in the epidermis surrounding actinic keratosis, we measured telomere lengths of basal, parabasal, and suprabasal cells in epidermis with actinic keratosis (actinic keratosis group, n = 18) and without actinic keratosis (sun-protected, n = 15, and sun-exposed, n = 13 groups) and in actinic keratosis itself as well as in dermal fibroblasts in the 3 groups, using quantitative fluorescence in situ hybridization. Among the 3 cell types, telomeres of basal cells were not always the longest, suggesting that tissue stem cells are not necessarily located among basal cells. Telomeres of basal cells in the sun-exposed group were shorter than those in the sun-protected group. Telomeres in the background of actinic keratosis and in actinic keratosis itself and those of fibroblasts in actinic keratosis were significantly shorter than those in the controls. Our findings demonstrate that sun exposure induces telomere shortening and that actinic keratosis arises from epidermis with shorter telomeres despite the absence of any histologic atypia. PMID:24411948

Ikeda, Hiroyuki; Aida, Junko; Hatamochi, Atsushi; Hamasaki, Yoichiro; Izumiyama-Shimomura, Naotaka; Nakamura, Ken-Ichi; Ishikawa, Naoshi; Poon, Steven S; Fujiwara, Mutsunori; Tomita, Ken-Ichiro; Hiraishi, Naoki; Kuroiwa, Mie; Matsuura, Masaaki; Sanada, Yukihiro; Kawano, Youichi; Arai, Tomio; Takubo, Kaiyo

2014-03-01

124

Aneuploidy detection for chromosomes 1, X and Y by fluorescence in situ hybridization in human sperm from oligoasthenoteratozoospermic patients  

SciTech Connect

Oligoasthenoteratozoospermic males (n=15) were investigated for infertility as compared with proven fertile donors. The oligoasthenoteratozoospermic population showed a mean sperm concentration of 9.7 x 10{sup 6}/ml (Range 4.2-19.7), mean motility of 38.5% (Range 10.6-76.8) and morphology (measured by the percentage of normal forms evaluated by strict criteria) with a mean of 3.49% (Range 1.5-5.0). Fluorescence in situ hybridization (FISH) using satellite DNA probes specific for chromosomes 1 (puc 1.77), X (alpha satellite), and Y (satellite-III at Yqh) was performed on human interphase sperm nuclei. DNA probes were either directly labelled with rhodamine-dUTP, FITC-dUTP, or biotinylated by nick translation. Hybridization and signal detection were done by routine laboratory protocols. Microscopic analysis was performed using a cooled CCD camera attached to an epi-fluorescent microscope. After hybridization, fertile donors yielded a frequency of 0.96% (n=12) nullisomic, 98.5% (n=1231) monosomic and 0.96% (n=12) disomic for chromosome 1, whereas oligoasthenoteratozoospermic males yielded a frequency of 16% (n=600) nullisomic, 74.5% (n=2792) monosomic and 9.9% (n=370) disomic. In addition, fertile donors yielded a frequency of 45.7% (n=633) monosomic and 0.7% (n=11) disomic for chromosome X, whereas oligoasthenoteratozoospermic males yielded a frequency of 38.7% (n=760) monosomic and 0.8% (n=13) disomic. Chromosome Y frequencies for fertile donors showed 44.6% (n=614) monosomic and 0.6% (n=2) disomic, whereas oligoasthenoteratozoospermic males yielded a frequency of 33.2% (n=701) monosomic and 0.8% (n=15) disomic. This suggests that the frequency of nullisomy for chromosome 1 is significantly higher (p<0.001) in sperm from oligoasthenoteratozoospermic makes versus sperm from our fertile donors. We conclude that FISH is a powerful tool to determine the frequency of aneuploidy in sperm from oligoasthenoteratozoospermic patients.

Pang, M.G.; Zackowski, J.L.; Acosta, A.A. [Eastern Virginia Medical School, Norfolk, VA (United States)] [and others

1994-09-01

125

Temporal and spatial distribution of Bacillus and Clostridium histolyticum in swine manure composting by fluorescent in situ hybridization (FISH).  

PubMed

The temporal and spatial distribution of the genus Bacillus and Clostridium histolyticum group in swine manure composting was determined by fluorescent in situ hybridization using fluorescently labeled 16S rRNA-targeted oligonucleotide probes LGC353b and Chis150, respectively. The temporal distribution of total bacteria, Bacillus and C. histolyticum, detected in each layer of the composting pile was noticeable in that the number of them detected at the high-temperature stage was higher than that of the cooling stage. The number detected at the cooling stage was higher than that of the temperature-rising stage. The number of the total bacteria distributed in three locations achieved balance at the stage of cooling. The spatial distribution of the genus Bacillus cells was that the number and the relative abundance of Bacillus cells detected in the middle layer of composting pile were the lowest at each stage of composting. However, the minimum value of the relative abundance exceeded 8%. Compared with Bacillus spp., the C. histolyticum group displayed higher relative abundance in the same layer at different stages of composting except in the top layer at the stage of high temperature. However, the characteristic of the spatial distribution was not noticeable. The detected limits of the genus Bacillus and C. histolyticum group were both found to be the high cell density of 10(6) cells g(-1) (wet weight). These results indicated that the genus Bacillus and C. histolyticum group were the predominant bacteria in the swine manure composting process and may play important role in this complex environment. PMID:21881890

Yi, Jing; Zheng, Rong; Li, Fenge; Chao, Zhe; Deng, Chang Yan; Wu, Jian

2012-03-01

126

A Novel 4-color Fluorescence in Situ Hybridization Assay for Detection of TMPRSS2 and ERG Rearrangements in Prostate Cancer  

PubMed Central

Purpose Since the identification of TMPRSS2/ERG rearrangement as the most common fusion event in prostate cancer, various methods have been developed to detect this rearrangement and to study its prognostic significance. We hereby report a novel 4-color fluorescence in situ hybridization (FISH) assay that not only detects the typical TMPRSS2:ERG fusion but also alternative rearrangements of either the TMPRSS2 or ERG gene. Experimental design We validated this assay on fresh, frozen, or formalin-fixed paraffin-embedded prostate cancer specimens including cell lines, primary prostate cancer, xenograft tissues derived from metastatic prostate cancer, and metastatic tissues from castration-resistant prostate cancer (CRPC) patients. Results When compared with RT-PCR or Gen-Probe method as the technical reference, the 4-color FISH assay demonstrated an analytical sensitivity of 94.5% (95% Confidence Interval [CI] 0.80-0.99) and specificity of 100% (95% CI 0.89-1.00) for detecting TMPRSS2:ERG fusion. TMPRSS2:ERG fusion was detected at 41% and 43% in primary prostate cancer (n = 59) and CRPC tumors (n = 82), respectively. Alternative rearrangements other than the typical TMPRSS2:ERG fusion were confirmed by karyotype analysis and shown present in 7% primary cancer and 13% CRPC tumors. Successful karyotype analysis is reported for the first time on four of the xenograft samples, complementing the FISH results. Conclusions This 4-color FISH assay provides sensitive detection of TMPRSS2 and ERG gene rearrangements in prostate cancer. PMID:23352841

Qu, Xiaoyu; Randhawa, Grace; Friedman, Cynthia; O'Hara-Larrivee, Siobhan; Kroeger, Kathleen; Dumpit, Ruth; True, Larry; Vakar-Lopez, Funda; Porter, Christopher; Vessella, Robert; Nelson, Peter; Fang, Min

2013-01-01

127

Fluorescence in situ hybridization (FISH) using non-commercial probes in the diagnosis of clinically suspected microdeletion syndromes  

PubMed Central

Background & objectives: Microdeletion syndromes are characterized by small (<5 Mb) chromosomal deletions in which one or more genes are involved. These are frequently associated with multiple congenital anomalies. The phenotype is the result of haploinsufficiency of genes in the critical interval. Fluorescence in situ hybridization (FISH) technique is commonly used for precise genetic diagnosis of microdeletion syndromes. This study was conducted to assess the role of FISH in the diagnosis of suspected microdeletion syndrome. Methods: FISH was carried out on 301 clinically suspected microdeletion syndrome cases for the confirmation of clinical diagnosis using non-commercial probes. Of these, 177 cases were referred for 22q11.2 microdeletion, 42 cases were referred for William syndrome, 38 cases were referred for Prader Willi/Angelman and 44 cases were referred for other suspected microdeletion syndromes. Results: FISH was confirmatory in 23 cases only (7.6%). There were 17 cases of 22q11.2 microdeletion, four cases of Prader Willi syndrome and two cases of William syndrome. Interpretation & conclusion: We conclude that FISH should not be the method of choice for clinically suspected microdeletion syndromes. We propose to follow strict clinical criteria for FISH testing or preferably to follow better methods (genotype first approach). Whole genome screening may be used as first line of test and FISH may be used for confirmation of screening result, screening of family members and prenatal diagnosis. PMID:24056568

Halder, Ashutosh; Jain, Manish; Chaudhary, Isha; Gupta, Neerja; Kabra, Madhulika

2013-01-01

128

Detection of aneuploid human sperm by fluorescence in situ hybridization: Evidence for a donor difference in frequency of sperm disomic for chromosomes 1 and Y  

SciTech Connect

Fluorescence in situ hybridization with repetitive-sequence DNA probes was used to detect human sperm disomic for chromosomes 1 and Y in three healthy men. Data on these same men had been obtained previously, using the human-sperm/hamster-egg cytogenetic technique, providing a cytogenetic reference for validating sperm hybridization measurements. Air-dried smears were prepared from semen samples and treated with DTT and lithium diiodosalicylate to expand sperm chromatin. Hybridization with fluorescently tagged DNA probes for chromosomes 1 (pUC177) or Y (pY3.4) yielded average frequencies of sperm with two fluorescent domains of 14.2[+-]2.4/10,000 and 5.6[+-]1.6/10,000 sperm, respectively. These frequencies did not differ statistically from frequencies of hyperploidy observed for these chromosomes with the hamster technique. In addition, frequencies of disomic sperm from one donor were elevated [approximately]2.5-fold above those of other donors, for both chromosomes 1 (P = .045) and Y (P = .01), consistent with a trend found with the hamster technique. The authors conclude that fluorescence in situ hybridization to sperm chromosomes provides a valid and promising measure of the frequency of disomic human sperm. 43 refs., 1 fig., 4 tabs.

Robbins, W.A. (Univ. of California, Berkeley (United States) Lawrence Livermore National Lab., CA (United States)); Segraves, R.; Pinkel, D. (Univ. of California, San Francisco (United States)); Wyrobek, A.J. (Lawrence Livermore National Lab., CA (United States))

1993-04-01

129

Multitarget Fluorescence In Situ Hybridization Assay Detects Transitional Cell Carcinoma in the Majority of Patients with Bladder Cancer and Atypical or Negative Urine Cytology  

Microsoft Academic Search

PurposeThe multitarget fluorescence in situ hybridization (FISH) probe set UroVysion (Vysis, Downers Grove, Illinois), containing probes to chromosomes 3, 7 and 17, and to the 9p21 band, has been recently shown to have high sensitivity and specificity for detecting transitional cell carcinoma. In this study we retrospectively tested 120 urine samples from patients with atypical, suspicious and negative cytology for

MAREK SKACEL; MONA FAHMY; JENNIFER A. BRAINARD; JAMES D. PETTAY; CHARLES V. BISCOTTI; LOUIS S. LIOU; GARY W. PROCOP; J. STEPHEN JONES; JAMES ULCHAKER; CRAIG D. ZIPPE; RAYMOND R. TUBBS

2003-01-01

130

Evaluation of seaFAST, a Rapid Fluorescent In Situ Hybridization Test, for Detection of Helicobacter pylori and Resistance to Clarithromycin in Paraffin-Embedded Biopsy Sections  

Microsoft Academic Search

A commercially available rapid fluorescent in situ hybridization (FISH) test, (seaFAST H. pylori Combi-Kit; SeaPro Theranostics International, Lelystad, The Netherlands) was used to simultaneously detect the presence of Helicobacter pylori and determine clarithromycin susceptibility in paraffin-embedded biopsy sections. The FISH method was found to be 97% sensitive, 94% specific for the detection of H. pylori and comparable to agar dilution

Julie M. Morris; Alisa L. Reasonover; Michael G. Bruce; Dana L. Bruden; Brian J. McMahon; Frank D. Sacco; Douglas E. Berg; Alan J. Parkinson

2005-01-01

131

Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Mycobacterium Cultures  

Microsoft Academic Search

TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the Mycobacterium tuberculosis complex (MTC) and nontubercu- lous mycobacteria (NTM) in acid-fast bacillus-positive (AFB1) cultures is described. The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears

HENRIK STENDER; KAARE LUND; KENNETH H. PETERSEN; OLE F. RASMUSSEN; POONPILAS HONGMANEE; HÅKAN MIORNER; SVEN E. GODTFREDSEN

1999-01-01

132

X chromosome evolution in the suni and eland antelope: detection of homologous regions by fluorescence in situ hybridization and G-banding  

Microsoft Academic Search

Comparative cytogenetics and fluorescence in situ hybridization (FISH) were used to track structural rearrangements in the X chromosomes of two African antelope species, the eland (Taurotragus oryx; tribe Tragelaphini) and the suni (Neotragus moschatus; tribe Neotragini). Using two microdissected cattle chromosome painting probes (one specific for Xp-containing sequences corresponding to Xp24?p12 of the cattle X, and one specific for Xq-containing

T. J. Robinson; W. R. Harrison; A. Ponce de León; F. F. B. Elder

1997-01-01

133

[Rapid enrichment and cultivation of denitrifying phosphate-removal bacteria and its identification by fluorescence in situ hybridization technology].  

PubMed

The present work focused on a rapid enrichment and cultivation of denitrifying phosphate-removal bacteria (DPB) in a membrane bio-reactor(MBR) by using A2/O anaerobic sludge from a wastewater treatment plant as seed, as well as providing an identification method. In the experiments, sodium acetate was used as the carbon source and a certain amount of nitrate was added to the MBR in the anoxic stage. Results showed that, with the efficient trap of the hollow-fiber membrane module, the proportion of DPB in all the phosphate-accumulating organisms (PAOs) increased from 24% to 93% within 35 days after two-stage's cultivation including anaerobic/aerobic and anaerobic/anoxic, during which the removal efficiency of nitrogen and phosphorus reached more than 90%. The activated sludge was identified by combining a regular method and the fluorescence in situ hybridization (FISH) technique, which demonstrated that Pseudomonas sp. and Rhodocyclus sp. were the dominant bacteria in the used bioreactor. PMID:24028025

Liu, Li; Tang, Bing; Huang, Shao-Song; Fu, Feng-Lian; Zhang, Qi-Qin; Li, Jian-Bin; Luo, Jian-Zhong

2013-07-01

134

Discriminating Multi-Species Populations in Biofilms with Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA FISH)  

PubMed Central

Background Our current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data. Methodology/Principal Findings We employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ?56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm2) for 48 h biofilm: E. coli 2,1×108 (±2,4×107); L. monocytogenes 6,8×107 (±9,4×106); and S. enterica 1,4×106 (±4,1×105)]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica. Significance While PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment. PMID:21479268

Almeida, Carina; Azevedo, Nuno F.; Santos, Silvio; Keevil, Charles W.; Vieira, Maria J.

2011-01-01

135

Evaluation of fluorescence in situ hybridization to detect encapsulated Bacillus pumilus SAFR-032 spores released from poly(methylmethacrylate).  

PubMed

Bacillus pumilus SAFR-032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H(2)O(2), desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR-032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa-FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant-resistant B. pumilus SAFR-032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa-FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase-K, lysozyme, mutanolysin and Triton X-100 facilitated efficient spore detection by Alexa-FISH microscopy. Neither of the Alexa-probes tested gave rise to considerable levels of Lucite- or solvent-associated background autofluorescence, demonstrating the immense potential of Alexa-FISH for rapid quantification of encapsulated B. pumilus SAFR-032 spores released from poly(methylmethacrylate). PMID:22145981

Mohapatra, Bidyut R; La Duc, Myron T

2012-01-01

136

Chromosomal localization of 5S and 18S-5.8S-25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) was employed on mitotic metaphase chromosome preparations of five Asian Pinus species: Pinus tabuliformis, Pinus yunnanensis, Pinus densata, Pinus massoniana and Pinus merkusii, using simultaneously DNA probes of the 18S rRNA gene and the 5S rRNA gene including the non-transcribed spacer sequences. The number and location of 18S rDNA sites varied markedly (5-10 pairs of

Z.-L. Liu; D. Zhang; D.-Y. Hong; X.-R. Wang

2003-01-01

137

Utility of fluorescence in situ hybridization to detect MDM2 amplification in liposarcomas and their morphological mimics  

PubMed Central

The atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WDLS) and the de-differentiated liposarcoma (DDLS) represent the most common category of liposarcomas. ALT/WDLSs and DDLSs are often difficult to distinguish from other tumors with similar morphological characteristics. In this study, we investigated whether the detection of amplified or overexpressed murine double-minute 2 (MDM2) can be a useful diagnostic ancillary aid. We used fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) to detect MDM2 amplification and protein overexpression, respectively, in 49 WDLSs, 5 DDLSs, 23 myxoid liposarcomas, 25 benign lipomatous tumors, and 75 spindle and pleomorphic sarcomas. MDM2 amplification was detected in 48 of 49 WDLSs, 5 of 5 DDLSs, 2 of 9 malignant peripheral nerve sheath tumors, and 2 of 10 myxofibrosarcomas. We did not detect MDM2 amplification in any of the benign lipomatous tumors. FISH-mediated detection of MDM2 amplification was the most valuable diagnostic aid for ALT/WDLS, as determined by using the Fisher exact test to compare two different diagnoses of 19 biopsies. On the contrary, unequivocal nuclear overexpression of MDM2 was found in only 10 of 50 ALT/WDLSs. The sensitivity and specificity of MDM2 amplification in distinguishing a DDLS from spindle and pleomorphic sarcomas were 100% and 95%, respectively, while those of MDM2 overexpression were 100% and 87%, respectively. In conclusion, our results indicate that FISH-mediated detection of MDM2 amplification is the most useful adjunct in the diagnosis of both ALT/WDLS and DDLS. However, IHC-mediated detection of MDM2 protein is useful only for the diagnosis of DDLS. PMID:23826411

Kimura, Hiroaki; Dobashi, Yoh; Nojima, Takayuki; Nakamura, Hiroyuki; Yamamoto, Norio; Tsuchiya, Hiroyuki; Ikeda, Hiroko; Sawada-Kitamura, Seiko; Oyama, Takeru; Ooi, Akishi

2013-01-01

138

Molecular analysis of hematopoietic colonies derived from chronic myeloid leukemia patients: interphase fluorescence in situ hybridization compared with RT-PCR  

Microsoft Academic Search

In this study we compared interphase fluorescence in situ hybridization (I-FISH) with reverse transcription polymerase chain reaction (RT-PCR) for the molecular analysis of hematopoietic colonies derived from patients with chronic myeloid leukemia (CML). Molecular analysis of individual colonies is often performed to monitor purging efficacy in CML. We harvested individual colony-forming unit granulocyte–macrophage (CFU-GM) colonies. One half was analyzed with

SFT Thijsen; GJ Schuurhuis; JW van Oostveen; AP Theijsmeijer; MMAC Langenhuijsen; GJ Ossenkoppele

1997-01-01

139

Multiplex fluorescence in situ hybridization identifies novel rearrangements of chromosomes 6, 15, and 18 in primary uveal melanoma.  

PubMed

Uveal melanomas are the commonest ocular tumour of adults and are characterized by reproducible alterations of chromosomes 1, 3, 6 and 8. These alterations are of prognostic relevance and have also be shown to correlate to high risk and low risk metastatic categories of uveal melanoma as defined by micro-array analysis. It is, however, possible that a catalogue of relevant genetic alterations, involving gene rearrangement rather than amplification, have as yet eluded identification. To address this point we examined 14 primary uveal melanomas, using 24 colour multiplex fluorescence in situ hybridization (M-FISH). All tumours were karyotyped following G-Banding, and M-FISH was performed to confirm and clarify the identity of abnormal chromosomes. M-FISH data were obtained from all tumours and was able to establish the nature of most abnormalities not fully characterized by cytogenetics. Abnormalities of chromosome 6 were far more frequent than previously indicated, in approximately 70% of cases, indicating they have been substantially underrepresented in past studies of uveal melanoma. Spindle melanomas were found to have novel rearrangements affecting in particular chromosomes 6, 15 and 18, suggesting that juxtaposition of genes through translocational events may play a role in the development of some uveal melanomas. In conclusion, this study is the largest of primary uveal melanoma analysed by M-FISH and indicates that alterations of chromosome 6 have previously been underestimated. Furthermore spindle melanomas are prone to rearrangements affecting chromosomes 6, 15 and 18, which may relate to early changes in uveal melanoma development or associate with those melanomas of a more differentiated status. PMID:16684523

Sisley, Karen; Tattersall, Nicola; Dyson, Michael; Smith, Kath; Mudhar, Hardeep S; Rennie, Ian G

2006-09-01

140

Feasibility of Transferring Fluorescent In Situ Hybridization Probes to an 18S rRNA Gene Phylochip and Mapping of Signal Intensities  

Microsoft Academic Search

DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and trans- ferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve

Katja Metfies; Linda K. Medlin

2008-01-01

141

Multicenter Evaluation of a New Shortened Peptide Nucleic Acid Fluorescence In Situ Hybridization Procedure for Species Identification of Select Gram-Negative Bacilli from Blood Cultures?  

PubMed Central

A shortened protocol for two peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assays for the detection of Gram-negative bacilli from positive blood cultures was evaluated in a multicenter trial. There was 100% concordance between the two protocols for each assay (368 of 368 and 370 of 370 results) and 99.7% (367 of 368 and 369 of 370 results) agreement with routine laboratory techniques. PMID:20357212

Morgan, Margie; Marlowe, Elizabeth; Della-Latta, Phyllis; Salimnia, Hossein; Novak-Weekley, Susan; Wu, Fann; Crystal, Benjamin S.

2010-01-01

142

Fluorescence in situ hybridization for detection of MAML2 rearrangements in oncocytic mucoepidermoid carcinomas: utility as a diagnostic test.  

PubMed

Oncocytic mucoepidermoid carcinoma poses diagnostic challenge because of its histologic overlap with other oncocytic salivary gland lesions, including Warthin tumor. Although the prognostic value of the t(11;19) MECT1-MAML2 fusion gene has been established in mucoepidermoid carcinoma, its diagnostic use in discriminating oncocytic mucoepidermoid carcinoma from histologic mimics is unexplored. We evaluated the translocation status in 14 cases of oncocytic mucoepidermoid carcinoma using a MAML2-11q21 break-apart probe spanning the entire chromosome region of the MAML2 gene and correlated these findings with clinicopathologic parameters including age, sex, stage, predominant growth pattern, grade, and p63 immunostaining pattern. All oncocytic mucoepidermoid carcinomas were parotid tumors with a mean patient age of 54.6 years (range, 9-85) and a female to male ratio of 5:2. Grade distribution was as follows: low grade, 9; intermediate grade, 2; and high grade, 3. The histologic patterns observed were as follows: solid, 4; cystic, 8 (of these, 5 had Warthin-like lymphoid stroma); and mixed, 2. Solid oncocytic mucoepidermoid carcinomas showed a diffuse p63 staining pattern, whereas cystic oncocytic mucoepidermoid carcinomas showed staining of the outer layer of intermediate cells ranging from a bilayer to areas of complex multilayering and plaque-like proliferation. Ten (71%) of the 14 cases showed a MAML2 rearrangement by fluorescence in situ hybridization. No correlation was seen between rearrangement status and histologic grade, growth pattern, or p63 staining pattern. However, we demonstrate that the presence of MAML2 rearrangement can be used as supportive evidence to distinguish oncocytic mucoepidermoid carcinoma from other oncocytic lesions. PMID:21777943

García, Joaquin J; Hunt, Jennifer L; Weinreb, Ilan; McHugh, Jonathan B; Barnes, E Leon; Cieply, Kathleen; Dacic, Sanja; Seethala, Raja R

2011-12-01

143

Localization of human ARF2 and NCK genes and 13 other Notl-linking clones to chromosome 3 by fluorescence in situ hybridization  

Microsoft Academic Search

Two human genes containing Notl sites, ADP-ribosylation factor (ARF2) and melanoma NCK protein, were mapped by fluorescence in situ hybridization to 3p21.2? p21.1 and 3q21, respectively. Thirteen other NotI linking clones, representing sequence tagged sites, were also mapped to different regions of human chromosome 3. Two of these clones that contain sequences 80% homologous to the rat tropoelastin gene and

N. Vorobieva; A. Protopopov; M. Protopopova; V. Kashuba; R. L. Allikmets; W. Modi; E. R. Zabarovsky; G. Klein; L. Kisselev; A. Graphodatsky

1995-01-01

144

Differentiation of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacterial Liquid Cultures by Using Peptide Nucleic Acid-Fluorescence In Situ Hybridization Probes  

Microsoft Academic Search

A blinded comparison of peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) with routine identification methods was performed on 74 consecutively positive mycobacterial liquid cultures. All Mycobac- terium tuberculosis cultures (48 of 48) and 22 of 27 (81.5%) nontuberculous cultures were correctly identified (including one mixed culture). Five isolates yielded no reaction with either probe and were identified as Mycobacterium xenopi,

F. A. DROBNIEWSKI; P. G. MORE; G. S. HARRIS

2000-01-01

145

Molecular cytogenetic characteristics of the human hepatocellular carcinoma cell line HCCLM3 with high metastatic potential: comparative genomic hybridization and multiplex fluorescence in situ hybridization.  

PubMed

The HCCLM3 cell line was established at the authors' institute from the lung metastatic lesions of BALB/c nude mice bearing human hepatocellular carcinoma (HCC) from the metastatic HCC cell line MHCC97-H. It has been shown to have a high potential for lung metastases and extensive metastases when the cells are inoculated subcutaneously or orthotopically in athymic nude mice. In the present study, the molecular cytogenetic characteristics of this cell line were evaluated with conventional G-banding, comparative genomic hybridization, and multiplex fluorescence in situ hybridization. A hyperdiploid karyotype of 53-58 chromosomes with 10 marker chromosomes was identified. The chromosomal aberrations such as i(X)(q10), der(Y)t(Y;18)(q12;p11), der(3)t(3;20) (p25;q13), der(4)t(4;8)(q31;q22)5, der(9)t(9;13)(p21;q22), der(14)t(14;22)(p13;q13), and der(15) t(15;21)(q11;q22) were described for the first time in human HCC cells. The analysis of this cell line through a combination of molecular cytogenetic techniques provides information on the possible molecular mechanisms involved in the metastatic process of HCC. PMID:15796966

Yang, Jiong; Qin, Lun-Xiu; Li, Yan; Ye, Sheng-Long; Liu, Yin-Kun; Gao, Dong-Mei; Chen, Jie; Tang, Zhao-You

2005-04-15

146

Optimization of a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the detection of bacteria and disclosure of a formamide effect.  

PubMed

Despite the fact that fluorescence in situ hybridization (FISH) is a well-established technique to identify microorganisms, there is a lack of understanding concerning the interaction of the different factors affecting the obtained fluorescence. In here, we used flow cytometry to study the influence of three essential factors in hybridization - temperature, time and formamide concentration - in an effort to optimize the performance of a Peptide Nucleic Acid (PNA) probe targeting bacteria (EUB338). The PNA-FISH optimization was performed with bacteria representing different families employing response surface methodology. Surprisingly, the optimum concentration of formamide varied according to the bacterium tested. While hybridization on the bacteria possessing the thickest peptidoglycan was more successful at nearly 50% (v/v) formamide, hybridization on all other microorganisms appeared to improve with much lower formamide concentrations. Gram staining and transmission electron microscopy allowed us to confirm that the overall effect of formamide concentration on the fluorescence intensity is a balance between a harmful effect on the bacterial cell envelope, affecting cellular integrity, and the beneficial denaturant effect in the hybridization process. We also conclude that microorganisms belonging to different families will require different hybridization parameters for the same FISH probe, meaning that an optimum universal PNA-FISH procedure is non-existent for these situations. PMID:25034435

Santos, Rita S; Guimarães, Nuno; Madureira, Pedro; Azevedo, Nuno F

2014-10-10

147

Fluorescence in situ hybridization with Alu and L1 polymerase chain reaction probes for rapid characterization of human chromosomes in hybrid cell lines  

SciTech Connect

Human-rodent hybrid cell lines have been analyzed with regard to their human DNA content by using various DNA probe sets, derived from the hybrids, for in situ hybridization to normal human metaphase chromosome spreads. Total genomic hybrid DNA was compared with probe sets of hybrid DNA that were highly enriched in human sequences. The latter probes were obtained by amplification through the polymerase chain reaction (PCR) using oligonucleotide primers directed to human specific subsequences of the interspersed repetitive sequences Alu and L1. Previously unidentified chromosomal material within hybrid lines was characterized with speed and precision. It is demonstrated that the complete human complement of hybrid lines can be rapidly assessed by comparing the data obtained with the Alu-PCR products with the results from the L1-PCR products or from the genomic hybrid DNA. This approach using interspersed repetitive sequence-PCR products is simple and fast and also provides an alternative way of generating complex DNA probe sets for the specific delineation of entire chromosomes or subchromosomal regions by the in situ hybridization.

Lichter, P.; Ward, D.C. (Yale Univ., New Haven, CT (USA)); Ledbetter, S.A.; Ledbetter, D.H. (Baylor College of Medicine, Houston, TX (USA))

1990-09-01

148

Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization  

PubMed Central

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established. PMID:22442728

Liu, Fang; Ling, Jian; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Wang, Kunbo

2012-01-01

149

Diagnosis of bladder cancer from the voided urine specimens using multi-target fluorescence in situ hybridization  

PubMed Central

The present study aimed to evaluate the diagnostic value of chromosomal analysis by fluorescence in situ hybridization (FISH) for bladder cancer in light of the histological diagnosis. Several valuable observations using FISH technologies in voided urine cells were also reported. The multi-target FISH-containing probes for the centromeres of chromosomes 3, 7 and 17 and the 9p21 locus were applied to cytospin specimens prepared from voided urine. Urine samples from 53 bladder cancer patients and 30 patients with benign alterations were used for this study. The histological observations of surgical resection specimens showed that the specificity and sensitivity for the technique were 100.0 and 88.0%, respectively. Statistical analyses showed that there was no significant correlation between FISH-positive rate and the tumor stage/grade (P<0.05). However, the proportion of tumor cells with genetic abnormalities positively correlated with the tumor stage (P<0.01). Furthermore, the number of abnormal cells in muscle-invasive pT2 was significantly higher than that in non-muscle-invasive pTa, pT1 (P<0.01). Of 50 patients with bladder cancer, polysomies of chromosomes 3, 7 and 17 were detected in 84.0, 48.0 and 78.0% of cases, respectively, and loss of the 9p21 gene was detected in 80.0% of cases. In addition, the detailed results from different urine specimens showed that FISH assay was required. FISH assay for chromosomes 3, 7 and 17 and 9p21 has a high specificity and sensitivity in the detection of bladder cancer and may reduce the necessity for cytoscopy treatment. PMID:24396440

KE, ZUNFU; LAI, YUANHUA; MA, XUDONG; LIL, SHUHUA; HUANG, WENHUA

2014-01-01

150

Fluorescence In Situ Hybridization: A Sensitive Method for Trisomy 8 Detection in Bone Marrow Specimens  

Microsoft Academic Search

RISOMY 8 is a common cytogenetic abnormality T found in the bone marrow (BM) of patients with myeloproliferative disorders (MPD), myelodysplastic syn- dromes (MDS), or acute nonlymphocytic leukemia (ANLL).1-5 Using conventional cytogenetic methods in which 20 to 30 metaphase cells are typically examined, it is possible to find 2 or more metaphases with trisomy 8 in many of these patients.

Robert B. Jenkins; Michelle M. Le Beau; William J. Kraker; Thomas J. Borell; Paul G. Stalboerger; Elizabeth M. Davis; Lolita Penland; Anthony Fernald; Rafael Espinosa; Daniel J. Schaid; Gordon W. Dewald

1992-01-01

151

Rapid Screening for Streptococcus agalactiae in Vaginal Specimens of Pregnant Women by Fluorescent In Situ Hybridization  

Microsoft Academic Search

(98.3%), whereas by means of standard culture only 38 specimens were positive (64.4%). We recommend FISH as a rapid, specific, highly sensitive screening technique for the detection of GBS in pregnant women at delivery. Group B streptococci (GBS) are the most frequent patho- gens isolated from neonates with invasive bacterial disease. The intrapartum fetal transmission may lead to invasive dis-

Laura A. Artz; Volkhard A. J. Kempf; Ingo B. Autenrieth

2003-01-01

152

Detection of Acidithiobacillus ferrooxidans in acid mine drainage environments using fluorescent in situ hybridization (FISH)  

Microsoft Academic Search

An important microorganism of acid mine drainage (AMD) and bioleaching environments is Acidithiobacillus ferrooxidans which oxidizes ferrous iron and generates ferric iron, an oxidant. Most investigations to understand microbial aspects of sulfide mineral dissolution have focused on understanding physiological, metabolic, and genetic characteristics of A. ferrooxidans. In this study, a 16S rRNA oligonucleotide probe designated S-S-T.ferr-0584-a-A-18, and labeled at the

K. K. Mahmoud; L. G. Leduc; G. D. Ferroni

2005-01-01

153

Duplication of intrachromosomal insertion segments 4q32?q35 confirmed by comparative genomic hybridization and fluorescent in situ hybridization  

PubMed Central

A 35-year-old man with infertility was referred for chromosomal analysis. In routine cytogenetic analysis, the patient was seen to have additional material of unknown origin on the terminal region of the short arm of chromosome 4. To determine the origin of the unknown material, we carried out high-resolution banding, comparative genomic hybridization (CGH), and FISH. CGH showed a gain of signal on the region of 4q32?q35. FISH using whole chromosome painting and subtelomeric region probes for chromosome 4 confirmed the aberrant chromosome as an intrachromosomal insertion duplication of 4q32?q35. Duplication often leads to some phenotypic abnormalities; however, our patient showed an almost normal phenotype except for congenital dysfunction in spermatogenesis. PMID:22384449

Kim, Jin Woo; Park, Ju Yeon; Oh, Ah Rum; Choi, Eun Young; Ryu, Hyun Mee; Kang, Inn Soo; Koong, Mi Kyoung

2011-01-01

154

Trisomy 10p resulting from an inv dup of 10p defined by fluorescence in situ hybridization  

SciTech Connect

De novo cases of trisomy for the entire short arm of chromosome 10 are infrequently reported and are most commonly the result of translocation of 10p to an acrocentric chromosome. Most reported cases of trisomy 10p are not trisomy for the complete short arm of chromosome 10, but are duplication, deficiency syndromes that result from either inheritance of an unbalanced translocation from a parent possessing a balanced reciprocal translocation, or from a recombinant chromosome derived from a parental pericentric inversion of chromosome 10. Here, we report a case of a de novo trisomy 10p that resulted from an inverted duplication of the entire short arm of chromosome 10. A 42 year old G7,P5,SAB1 woman was referred for amniocentesis because of advanced maternal age. Ultrasound examination at 17 weeks demonstrated a fetus of normal size with no apparent anatomic abnormalities. Cytogenetic evaluation demonstrated one homologue of chromosome 10 had a tandem inverted duplication of the short arm. The fetal karyotype was interpreted to be 46,XX,inv dup (10) (peter-cen::cen-p15::q11-pter). Parental karyotype are normal. Fluorescence in situ hybridization (FISH) using a chromosome 10 paint, chromosome 10 centromere, and all telomere probe, confirmed the inverted duplication involved the entire short arm of chromosome 10. Termination of pregnancy was performed at 20 weeks gestation. Autopsy revealed multiple anomalies including low-set posteriorly rotated ears, cleft of the soft palate, ocular hypertelorism, small upturned nose, agenesis of the gallbladder, sacral hemivertebrae, and abnormal flexion of the thumbs. The fetal karyotype was confirmed by cytogenetic analysis in lung and kidney. This is the second reported case of a de novo tandem duplication of 10p of which we are aware, and the first using FISH technology to characterize the abnormality.

Clement, S.J.; Easterling, T.R.; Leppig, K.A. [Univ. of Washington, Seattle, WA (United States)] [and others

1994-09-01

155

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy  

PubMed Central

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created. PMID:22041974

Klug, Barbara; Rodler, Claudia; Koller, Martin; Wimmer, Gernot; Kessler, Harald H.; Grube, Martin; Santigli, Elisabeth

2011-01-01

156

Oral biofilm analysis of palatal expanders by fluorescence in-situ hybridization and confocal laser scanning microscopy.  

PubMed

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH). We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation. FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes. Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia. General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix), Firmicutes (LGC354 A-C; hereafter LGCmix), and Bacteroidetes (Bac303). In addition, specific probes binding to Streptococcus mutans (MUT590) and Porphyromonas gingivalis (POGI) were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle. Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created. PMID:22041974

Klug, Barbara; Rodler, Claudia; Koller, Martin; Wimmer, Gernot; Kessler, Harald H; Grube, Martin; Santigli, Elisabeth

2011-01-01

157

Fluorescence in situ hybridization for identification of Tritrichomonas foetus in formalin-fixed and paraffin-embedded histological specimens of intestinal trichomoniasis.  

PubMed

In the present study a highly species-specific oligonucleotide sequence of Tritrichomonas foetus 18S rRNA was used to design an antisense probe for identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens by means of fluorescence in situ hybridization (FISH). Using archival histological specimens from several species with light microscopic evidence of intestinal trichomoniasis, and under optimized hybridization conditions, the probe positively identified trichomonads in colonic specimens from piglets and a kitten with PCR-confirmed T. foetus infection. Neither positive hybridization of the probe or PCR amplification of T. foetus DNA was observed in histological specimens from hamster (Tritrichomonas muris), turkey, nor mouse (Entamoeba muris) intestinal protozoal infections. Sequence-specific binding of the probe was further verified by successfully out-competing the hybridization with 10 x molar excess unlabeled probe and failure of a labeled sense probe to hybridize. The FISH assay described here enables simultaneous location and molecular identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens of intestinal trichomoniasis. The methods employed are likely to also be applicable to probes designed for specific recognition of other trichomonad species, especially in mammalian tissue where red blood cell auto-fluorescence can be easily differentiated from the hybridization signal of trichomonads. PMID:20447769

Gookin, J L; Stone, M R; Yaeger, M J; Meyerholz, D K; Moisan, Peter

2010-08-27

158

Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos  

E-print Network

In C. elegans, the expression pattern of a gene provides important clues to understanding its biological function. To accurately depict endogenous transcriptional activity, a highly sensitive method is required to measure ...

Ji, Ni

159

Rapid method for measuring clastogenic fingerprints using fluorescence in situ hybridization  

DOEpatents

A method is provided for determining a clastogenic signature of a sample of chromosomes by quantifying a frequency of a first type of chromosome aberration present in the sample; quantifying a frequency of a second, different type of chromosome aberration present in the sample; and comparing the frequency of the first type of chromosome aberration to the frequency of the second type of chromosome aberration. A method is also provided for using that clastogenic signature to identify a clastogenic agent or dosage to which the cells were exposed.

Lucas, Joe N. (San Ramon, CA)

2000-01-01

160

Rapid method for measuring clastogenic fingerprints using fluorescence in situ hybridization  

SciTech Connect

A method is provided for determining a clastogenic signature of a sample of chromosomes by quantifying a frequency of a first type of chromosome aberration present in the sample; quantifying a frequency of a second, different type of chromosome aberration present in the sample; and comparing the frequency of the first type of chromosome aberration to the frequency of the second type of chromosome aberration. A method is also provided for using that clastogenic signature to identify a clastogenic agent or dosage to which the cells were exposed.

Lucas, J.N.

2000-03-28

161

Diagnosis of a constitutional five-chromosome rearrangement by fluorescent in situ hybridization (FISH)  

SciTech Connect

Complex chromosomal rearrangements are structural rearrangements involving at least three chromosomes and three or more chromosome breakpoints. Such karyotypes are often acquired during cancer multi-step development and in chromosome instability syndromes. However, extremely rare constitutional forms have been reported, most of which are incompatible with life. We present a 2-year-old female with de novo complex rearrangement consisting of five chromosomes and nine breakpoints. Clinical evaluation at two years of age revealed a weight of 5 kg, length of 66 cm, and had circumference of 38 cm, all below the 5th percentile, microcephaly, trigonocephaly, epicanthal folds, inguinal hernia, left clubfoot, hypertonicity, and developmental delay. The neurological examination revealed chorea-acanthocytosis and psychomotor delay. Cultured lymphocytes and fibroblasts revealed a karyotype consisting of five derivative chromosomes. The metaphases were further analyzed by FISH using chromosome-specific libraries and telomeric probes in order to delineate the composition of the rearranged chromosomes; FISH results demonstrated a karyotype of: 46,XX,1pter{r_arrow}1q25::1q42.1{r_arrow}1qter, 2pter{r_arrow}q32.3::1q32.3{r_arrow}2q41::2q37.3{r_arrow}2qter, 7qter{r_arrow}7q21.2::6q22.3{r_arrow}6qter::1q31{r_arrow}1q32.3::6p23{r_arrow}6q22.3, 7pter{r_arrow}7q21.1::6p23{r_arrow}6pter, 2q33{r_arrow}2q37, 1::9p21{r_arrow}9qter. This analysis demonstrates the usefulness of FISH in characterizing complex chromosome rearrangements otherwise difficult to correctly interpret using classical cytogenetics alone.

Tsien, F.; Shapira, E. [Tulane Univ. School of Medicine, New Orleans, LA (United States); Carvalho, T. [Hospital Sarah Kubitschek, Brasilia (Brazil)] [and others

1994-09-01

162

Fluorescence in situ hybridization and spectral imaging analysisof human oocytes and first polar bodies  

SciTech Connect

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18 or 21 in failed-fertilized human oocytes.While abnormalities involving chromosome 16 showed an age-dependant increase, results for the other chromosomes did not show statistically significant differences between the three age groups <35 yrs, 35-39 yrs, and >39 yrs. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Thus, for a pilot study investigating a more comprehensive analysis of oocytes and their corresponding first polar bodies (1PBs), we developed a novel 8-probe chromosome enumeration scheme using FISH and SIm.

Weier, Heinz-Ulli G.; Weier, Jingly F.; Oter Renom, Maria; Zheng,Xuezhong; Colls, Pere; Nureddin, Aida; Pham, Chau D.; Chu, Lisa W.; Racowsky, Catherine; Munne, Santiago

2004-10-06

163

Partial trisomy 11q involving chromosome 1 detected by fluorescence in situ hybridization  

SciTech Connect

Partial trisomy 11q was detected in an infant delivered 3-4 weeks prematurely. The phenotype included slanted palpebral fissures, high arched palate, developmental delay, microcephaly, and cardiac defects, all of which occur in the majority of cases with this syndrome. Other features included a column-shaped skull, preauricular pit, single palmar crease, short, broad great toes, flat occiput, unilateral kidney agenesis, and strabismus. Chromosomes obtained from peripheral blood cells revealed the presence of extra material on the long arm of chromosome 1. The G-banding pattern of this extra material indicated that it might be derived from chromosome 1 or 11. Chromosomal {open_quotes}paints{close_quotes} showed that it was not chromosome 1 material, but was chromosome 11 material extending from band q21 to qter. Partial trisomy 11q arising from translocation of the 11q material to chromosome 2, 3, 4, 5, 6, 9, 10, 13, 17, 21, 22, and X has been reported previously, whereas translocation to chromosome 1 has not. The chromosome to which the 11q material is translocated does not alter the most frequent features of the partial trisomy 11q syndrome, but may influence other less common features.

McCorquodale, M.; Bereziouk, O.; McCorquodale, D.J. [Univ. of Illinois College of Medicine, Chicago, IL (United States)] [and others

1994-09-01

164

Mapping of rDNA on the chromosomes of Eleusine species by fluorescence in situ hybridization.  

PubMed

Mapping of rDNA sites on the chromosomes of four diploid and two tetraploid species of Eleusine has provided valuable information on genome relationship between the species. Presence of 18S-5.8S-26S rDNA on the largest pair of the chromosomes, location of 5S rDNA at four sites on two pairs of chromosomes and presence of 18S-5.8S-26S and 5S rDNA at same location on one pair of chromosomes have clearly differentiated E. multiflora from rest of the species of Eleusine. The two tetraploid species, E. coracana and E. africana have the same number of 18S-5.8S-26S and 5S rDNA sites and located at similar position on the chromosomes. Diploid species, E. indica, E. floccifolia and E. tristachya have the same 18S-5.8S-26S sites and location on the chromosomes which also resembled with the two pairs of 18S-5.8S-26S rDNA locations in tetraploid species, E. coracana and E. africana. The 5S rDNA sites on chromosomes of E. indica and E. floccifolia were also comparable to the 5S rDNA sites of E. africana and E. coracana. The similarity of the rDNA sites and their location on chromosomes in the three diploid and two polyploid species also supports the view that genome donors to tetraploid species may be from these diploid species. PMID:11280009

Bisht, M S; Mukai, Y

2000-12-01

165

Electron Paramagnetic Resonance and Fluorescence In Situ Hybridization-Based Investigations of Individual Doses for Persons Living at Metlino in the Upper Reaches of the Techa River  

SciTech Connect

Waterborne releases to the Techa River from the Mayak Production Association in Russia during 1949-1956 resulted in significant doses to persons living downstream; the most contaminated village was Metlino, about 7 km from the site of release. Internal and external doses have been estimated for these residents using the Techa River Dosimetry System-2000 (TRDS-2000); the primary purpose is to support epidemiological studies of the members of the Extended Techa River Cohort. Efforts to validate the calculations of external and internal dose are considered essential. One validation study of the TRDS-2000 system has been performed by the comparison of calculated doses to quartz from bricks in old buildings at Metlino with those measured by luminescence dosimetry. Two additional methods of validation considered here are electron paramagnetic resonance (EPR) measurements of teeth and fluorescence in situ hybridization (FISH) measurements of chromosome translocations in circulating lymphocytes. For electron paramagnetic resonance, 36 measurements on 26 teeth from 16 donors from Metlino were made at the GSF-National Research Center for Environment and Health (16 measurements) and the Institute of Metal Physics (20 measurements); the correlation among measurements made at the two laboratories has been found to be 0.99. Background measurements were also made on 218 teeth (63 molars, 128 premolars, and 27 incisors). Fluorescence in situ hybridization measurements were made for 31 residents of Metlino. These measurements were handicapped by the analysis of a limited number of cells; for several individuals no stable translocations were observed. Fluorescence in situ hybridization measurements were also made for 39 individuals believed to be unexposed. The EPR- and FISH-based estimates agreed well for permanent residents of Metlino: 0.67 +/- 0.21 Gy and 0.48 +/- 0.18 Gy (mean +/- standard error of the mean), respectively. Results of the two experimental methods also agreed well with the estimates derived from the use of the TRDS-2000. For all persons investigated according to each technique, the EPR-measured dose to enamel was 0.55 +/- 0.17 Gy, and the TRDS-2000 prediction for the dose to enamel for these individuals is 0.55 +/- 0.07 Gy. The fluorescence in situ hybridization-based dose, 0.38 +/- 0.10 Gy, compared well to the TRDS-2000 prediction of external dose, 0.31 +/- 0.03 Gy, to red bone marrow for these persons. Validation of external doses at the remaining villages is an active area of investigation.

Degteva, M. O.; Anspaugh, L. R.; Akleyev, A V.; Jacob, Peter; Ivanov, Denis V.; Wieser, Albrecht; Vorobiova, M I.; Shishkina, Elena A.; Shved, Valentina A.; Vozilova, Alexandra; Bayankin, Sergey N.; Napier, Bruce A.

2005-02-01

166

Detection of group B streptococci in Lim broth by use of group B streptococcus peptide nucleic acid fluorescent in situ hybridization and selective and nonselective agars.  

PubMed

The sensitivity, specificity, and positive and negative predictive values for the detection of group B streptococci from Lim enrichment broth with sheep blood agar (SBA), with selective Streptococcus agar (SSA), and by a peptide nucleic acid fluorescent in situ hybridization (PNA FISH) assay were as follows: for culture on SBA, 68.4%, 100%, 100%, and 87.9%, respectively; for culture on SSA, 85.5%, 100%, 100%, and 94.1%, respectively; and for the PNA FISH assay, 97.4%, 98.3%, 96.1%, and 98.9%, respectively. PMID:18667597

Montague, Naomi S; Cleary, Timothy J; Martinez, Octavio V; Procop, Gary W

2008-10-01

167

Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA–FISH)  

Microsoft Academic Search

Two-pass tyramide signal amplification–fluorescence in situ hybridization (two-pass TSA–FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of

Kengo Kubota; Akiyoshi Ohashi; Hiroyuki Imachi; Hideki Harada

2006-01-01

168

Multicenter Evaluation of the Candida albicans\\/Candida glabrata Peptide Nucleic Acid Fluorescent In Situ Hybridization Method for Simultaneous Dual-Color Identification of C. albicans and C. glabrata Directly from Blood Culture Bottles  

Microsoft Academic Search

We evaluated the performance of the Candida albicans\\/Candida glabrata peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method, a rapid two-color assay for detection of C. albicans and C. glabrata ,i n a multicenter study. The assay is designed for use directly from positive blood culture bottles in a FISH format. Intact, fixed cells are labeled fluorescent green (C.

Janeen R. Shepard; Rachel M. Addison; Barbara D. Alexander; Phyllis Della-Latta; Michael Gherna; Gerhard Haase; Jennifer K. Johnson; William G. Merz; Heidrun Peltroche-Llacsahuanga; Henrik Stender; Richard A. Venezia; Deborah Wilson; Gary W. Procop; Fann Wu; Mark J. Fiandaca

169

Fluorescence in situ hybridization of chromosome 17 polysomy in breast cancer using thin tissue sections causes the loss of CEP17 and HER2 signals.  

PubMed

Human epidermal growth factor receptor 2 (HER2 gene) and chromosome 17 polysomy are associated with breast cancer prognosis, chemotherapy and hormone therapy. HER2 gene analysis using fluorescence in situ hybridization (FISH) with 4-µm sections assuming a nuclear diameter of 6 µm caused the loss of genetic DNA. Using intact whole nuclei FISH (WNFISH) and thin tissue section FISH (TTFISH), 109 cases of invasive breast cancer were examined to observe correlations among HER2 gene ampli?cation, CEP17 polysomy and the HER2/CEP17 ratio. The results showed significant differences in the mean copy number of HER2 and the HER2/CEP17 ratios between the WNFISH and TTFISH groups. No significant differences were observed in HER2 amplified, equivocal and non-amplified HER2 samples. Thirty-seven cases of CEP17 polysomy and 72 cases of non?polysomy were detected by WNFISH. Twenty-nine cases of CEP17 polysomy and 72 cases of non-polysomy were detected by TTFISH. Significant differences were observed between the two methods using the McNemar test (P=0.039). In conclusion, detection of chromosome 17 polysomy in breast cancer with fluorescence in situ hybridization using thin tissue sections may cause the loss of CEP17 and HER2 signals. PMID:25119636

Jiang, Huiyong; Bai, Xiaoyan; Zhao, Tong; Zhang, Cheng; Zhang, Xuefeng

2014-11-01

170

A highly sensitive target-primed rolling circle amplification (TPRCA) method for fluorescent in situ hybridization detection of microRNA in tumor cells.  

PubMed

The ability to detect spatial and temporal microRNA (miRNA) distribution at the single-cell level is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. We report for the first time the development of a target-primed RCA (TPRCA) strategy for highly sensitive and selective in situ visualization of miRNA expression patterns at the single-cell level. This strategy uses a circular DNA as the probe for in situ hybridization (ISH) with the target miRNA molecules, and the free 3' terminus of miRNA then initiates an in situ RCA reaction to generate a long tandem repeated sequence with thousands of complementary segments. After hybridization with fluorescent detection probes, target miRNA molecules can be visualized with ultrahigh sensitivity. Because the RCA reaction can only be initiated by the free 3' end of target miRNA, the developed strategy offers the advantage over existing ISH methods in eliminating the interference from precursor miRNA or mRNA. This strategy is demonstrated to show high sensitivity and selectivity for the detection of miR-222 expression levels in human hepatoma SMMC-7721 cells and hepatocyte L02 cells. Moreover, the developed TPRCA-based ISH strategy is successfully applied to multiplexed detection using two-color fluorescent probes for two miRNAs that are differentially expressed in the two cell lines. The results reveal that the developed strategy may have great potential for in situ miRNA expression analysis for basic research and clinical diagnostics. PMID:24417222

Ge, Jia; Zhang, Liang-Liang; Liu, Si-Jia; Yu, Ru-Qin; Chu, Xia

2014-02-01

171

The analysis of ALK gene rearrangement by fluorescence in situ hybridization in non-small cell lung cancer patients  

PubMed Central

Introduction ALK gene rearrangement is observed in a small subset (3–7%) of non-small cell lung cancer (NSCLC) patients. The efficacy of crizotinib was shown in lung cancer patients harbouring ALK rearrangement. Nowadays, the analysis of ALK gene rearrangement is added to molecular examination of predictive factors. Aim of the study The frequency of ALK gene rearrangement as well as the type of its irregularity was analysed by fluorescence in situ hybridisation (FISH) in tissue samples from NSCLC patients. Material and methods The ALK gene rearrangement was analysed in 71 samples including 53 histological and 18 cytological samples. The analysis could be performed in 56 cases (78.87%), significantly more frequently in histological than in cytological materials. The encountered problem with ALK rearrangement diagnosis resulted from the scarcity of tumour cells in cytological samples, high background fluorescence noises and fragmentation of cell nuclei. Results The normal ALK copy number without gene rearrangement was observed in 26 (36.62%) patients ALK gene polysomy without gene rearrangement was observed in 25 (35.21%) samples while in 3 (4.23%) samples ALK gene amplification was found. ALK gene rearrangement was observed in 2 (2.82%) samples from males, while in the first case the rearrangement coexisted with ALK amplification. In the second case, signet-ring tumour cells were found during histopathological examination and this patient was successfully treated with crizotinib with partial remission lasting 16 months. Conclusions FISH is a useful technique for ALK gene rearrangement analysis which allows us to specify the type of gene irregularities. ALK gene examination could be performed in histological as well as cytological (cellblocks) samples, but obtaining a reliable result in cytological samples depends on the cellularity of examined materials. PMID:24592134

Krawczyk, Pawe? Adam; Ramlau, Rodryg Adam; Szumi?o, Justyna; Kozielski, Jerzy; Kalinka-Warzocha, Ewa; Bryl, Maciej; Knopik-D?browicz, Alina; Spychalski, ?ukasz; Szcz?sna, Aleksandra; Rydzik, Ewelina; Milanowski, Janusz

2013-01-01

172

Multicolor fluorescence in situ hybridization with centromeric DNA probes as a new approach to distinguish chromosome breakage from aneuploidy in interphase cells and micronuclei  

SciTech Connect

Chromosomal abnormalities are believed to contribute significantly to human reproductive failure, carcinogenesis and other pathophysiological conditions. For example, approximately 15% of recognized pregnancies terminate in spontaneous abortion, and of these approximately 30% have been shown to be chromosomally abnormal. The contribution of chromosomal abnormalities to early embryonic and fetal death appears to decrease with gestational age, suggesting that as many as 67% of the aborted embryos in early embryonic deaths are chromosomally abnormal. Furthermore, clinically significant chromosomal abnormalities can also be found to be present in approximately 0.58 to 0.67% of live births. These figures indicate that within a given year, hundreds of thousands of chromosomally abnormal babies will be born throughout the world and additional millions of chromosomally abnormal embryos will have been spontaneously aborted. For the past several years, our research has focused on utilizing new molecular cytogenetic techniques to develop assays for detecting aneuploidy-inducing agents in mammalian cells. One approach that we have sucessfully employed involves the use of fluorescence in situ hybridization with chromosome-specific DNA probes to determine the number of copies of a representative chromosome present within the nucleus following chemical exposure. DNA sequences (probes) which hybridize to blocks of repetitive centromeric DNA on specific chromosomes have been developed for most of the human chromosomes. In situ hybridization with these probes results in the staining of a compact chromosomal region which can be easily detected in interphase nuclei. The presence of 3 (or more) hybridization domains in an interphase nucleus indicates the presence of three centromeric regions and has been presumed to indicate that three copies of the entire chromosome were present in the nucleus.

Eastmond, D.A.; Rupa, D.S.; Chen, H.W.; Hasegawa, L. [Univ. of California, Riverside, CA (United States)

1993-12-31

173

In situ preparation and fluorescence quenching properties of polythiophene/ZnO nanocrystals hybrids through atom-transfer radical polymerization and hydrolysis  

NASA Astrophysics Data System (ADS)

In this paper, a new approach for in situ preparing nanocomposites of conjugated polymers (CPs) and semiconductor nanocrystals was developed. Polythiophene grafted poly(zinc methacrylate) (PTh-g-PZMA) copolymer was synthesized by atom-transfer radical polymerization (ATRP) of zinc methacrylate (ZMA) initiated from the macroinitiator poly(2,5-(3-(bromoisopropyl-carbonyl-oxymethylene) thiophene)) (PTh-Br) with pendant initiator groups. Subsequently, the polythiophene grafted poly(methacrylate)/ZnO (PTh-g-PMA/ZnO) hybrid heterojunction nanocomposites were successfully prepared by in situ hydrolysis of PTh-g-PZMA casting films in alkaline aqueous solution. The structures of PTh-Br, PTh-g-PZMA and PTh-g-PMA/ZnO were confirmed by the proton nuclear magnetic resonance ( 1H NMR) spectra, Fourier transform infrared (FTIR) spectra and X-ray photoelectron spectroscopy (XPS). The morphologies of PTh-g-PMA/ZnO films prepared for different hydrolysis time were observed in the cross-sections by scanning electron microscope (SEM). The SEM images revealed that ZnO nanocrystals were uniformly dispersed in polymers without any aggregation and the appearances of ZnO nanocrystals changed from nanoparticles to nanorods with the hydrolysis treatment time increasing. The optical properties of these nanocomposites were studied by ultraviolet-visible (UV-vis) absorption and fluorescence spectroscopy. UV-vis absorption spectroscopy showed that the adsorption band of PTh-g-PMA/ZnO hybrids was broader than that of PTh-Br, implying that the existence of ZnO nanocrystals increased the optical absorption region of hybrids. The photoluminescence (PL) spectra of the hybrids showed that fluorescence quenching occurred in PTh-g-PMA/ZnO blends and a maximum of 85% of the fluorescence intensity quenched in the PTh-g-PMA/ZnO obtained from treatment in NaOH aqueous solution for 2 h, which revealed the existence of photo-induced charge transfer between the polythiophene chains and ZnO. These results indicated that the hybrid heterojunction nanocomposites could be promising candidates for photovoltaic applications.

Peng, Xiaoming; Zhang, Lin; Chen, Yiwang; Li, Fan; Zhou, Weihua

2010-02-01

174

Non-fluorescent RNA in situ hybridization combined with antibody staining to visualize multiple gene expression patterns in the embryonic brain of Drosophila.  

PubMed

In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for non-fluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for whole mount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy. PMID:24048924

Jussen, David; Urbach, Rolf

2014-01-01

175

Combined DNA-RNA fluorescent in situ hybridization (FISH) to study X chromosome inactivation in differentiated female mouse embryonic stem cells.  

PubMed

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected. PMID:24961515

Barakat, Tahsin Stefan; Gribnau, Joost

2014-01-01

176

Dicentric chromosome aberration analysis using giemsa and centromere specific fluorescence in-situ hybridization for biological dosimetry: An inter- and intra-laboratory comparison in Indian laboratories.  

PubMed

To facilitate efficient handling of large samples, an attempt towards networking of laboratories in India for biological dosimetry was carried out. Human peripheral blood samples were exposed to (60)Co ?-radiation for ten different doses (0-5Gy) at a dose rate of 0.7 and 2Gy/min. The chromosomal aberrations (CA) were scored in Giemsa-stained and fluorescence in-situ hybridization with centromere-specific probes. No significant difference (p>0.05) was observed in the CA yield for given doses except 4 and 5Gy, between the laboratories, among the scorers and also staining methods adapted suggest the reliability and validates the inter-lab comparisons exercise for triage applications. PMID:25014548

Bhavani, M; Tamizh Selvan, G; Kaur, Harpreet; Adhikari, J S; Vijayalakshmi, J; Venkatachalam, P; Chaudhury, N K

2014-09-01

177

Comparative mapping of DNA probes derived from the V{sub k} immunoglobulin gene regions on human and great ape chromosomes by fluorescence in situ hybridization  

SciTech Connect

Fluorescence in situ hybridization (FISH) of cosmid clones of human V{sub K} gene regions to human and primate chromosomes contributed to the dating of chromosome reorganizations in evolution. A clone from the K locus at 2p11-p12 (cos 106) hybridized to the assumed homologous chromosome bands in the chimpanzees Pan troglodytes (PTR) and P. paniscus (PPA), the Gorilla gorilla (GGO), and the orangutan Pongo Pygmaeus (PPY). Human and both chimpanzees differed from gorilla and orangutan by the mapping of cos 170, a clone derived from chromosome 2cen-q11.2; the transposition of this orphon to the other side of the centromere can, therefore, be dated after the human/chimpanzee and gorilla divergence. Hybridization to homologous bands was also found with a cosmid clone containing a V{sub K}I orphon located on chromosome 1 (cos 115, main signal at 1q31-q32), although the probe is not fully unique. Also, a clone derived from the orphon V{sub K} region on chromosome 22q11 (cos 121) hybridized to the homologous bands in the great apes. This indicates that the orphons on human chromosomes 1 and 22 had been translocated early in primate evolution. 18 refs., 2 figs.

Arnold, N.; Wienberg, J.; Ermert, K. [Universitaet Muenchen (Germany)] [and others] [Universitaet Muenchen (Germany); and others

1995-03-01

178

Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry  

NASA Technical Reports Server (NTRS)

A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

Stowe, R. P.; Cubbage, M. L.; Sams, C. F.; Pierson, D. L.; Barrett, A. D.

1998-01-01

179

RNA in situ hybridization in Arabidopsis.  

PubMed

RNA in situ hybridization using digoxigenin-labeled riboprobes on tissue sections is a powerful technique for revealing microscopic spatial gene expression. Here, we describe an in situ hybridization method commonly practiced in Arabidopsis research labs. The highly stringent hybridization condition eliminates the usage of Ribonlucease A and gives highly specific signals. This also allows the use of longer probes which enhance signal strength without cross hybridization to closely related genes. In addition, using spin columns in template and riboprobe purification greatly reduces background signals. PMID:22589125

Wu, Miin-Feng; Wagner, Doris

2012-01-01

180

Detection of Ralstonia solanacearum, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes  

PubMed Central

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas. PMID:9797321

Wullings, B. A.; Van Beuningen, A. R.; Janse, J. D.; Akkermans, A. D. L.

1998-01-01

181

Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes.  

PubMed

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas. PMID:9797321

Wullings, B A; Van Beuningen, A R; Janse, J D; Akkermans, A D

1998-11-01

182

Specific Detection of Pasteurella multocida in Chickens with Fowl Cholera and in Pig Lung Tissues Using Fluorescent rRNA In Situ Hybridization  

PubMed Central

A Pasteurella multocida species-specific oligonucleotide probe, pmhyb449, targeting 16S rRNA was designed and evaluated by whole-cell hybridization against 22 selected reference strains in animal tissues. It differentiated P. multocida from other bacterial species of the families Pasteurellaceae and Enterobacteriaceae and also from divergent species of the order Cytophagales (except biovar 2 strains of Pasteurella avium and Pasteurella canis, which have high 16S rRNA similarity to P. multocida). The potential of the probe for specific identification and differentiation of P. multocida was further detected in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues P. multocida cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood vessels (wall and lumen). In pig lung, postmortem-injected P. multocida was detected in the alveoli (lumen and wall), and in both animals the bacterial cells were seen in the bronchi. The results showed that with the oligonucleotide probe pmhyb449, fluorescent in situ hybridization is a suitable and fast method for specific detection of P. multocida in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to P. multocida. PMID:11427580

Mbuthia, Paul Gichohi; Christensen, Henrik; Boye, Mette; Petersen, Kamille Majken Dumong; Bisgaard, Magne; Nyaga, Phillip Njeru; Olsen, John Elmerdahl

2001-01-01

183

Comparative Fluorescence in Situ Hybridization Mapping of a 431-kb Arabidopsis thaliana Bacterial Artificial Chromosome Contig Reveals the Role of Chromosomal Duplications in the Expansion of the Brassica rapa Genome  

Microsoft Academic Search

Comparative genome studies are important contributors to our understanding of genome evolution. Most comparative genome studies in plants have been based on genetic mapping of homologous DNA loci in different genomes. Large-scale comparative physical mapping has been hindered by the lack of efficient and affordable techniques. We report here the adaptation of fluorescence in situ hybridization (FISH) techniques for comparative

Scott A. Jackson; Zhukuan Cheng; Ming Li Wang; Howard M. Goodman; Jiming Jiang

2000-01-01

184

Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization? †  

PubMed Central

An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes. PMID:17293504

Smolina, Irina; Lee, Charles; Frank-Kamenetskii, Maxim

2007-01-01

185

Enumeration of Vibrio parahaemolyticus in the viable but nonculturable state using direct plate counts and recognition of individual gene fluorescence in situ hybridization.  

PubMed

Vibrio parahaemolyticus is a gram-negative, halophilic bacterium indigenous to marine and estuarine environments and it is capable of causing food and water-borne illness in humans. It can also cause disease in marine animals, including cultured species. Currently, culture-based techniques are used for quantification of V. parahaemolyticus in environmental samples; however, these can be misleading as they fail to detect V. parahaemolyticus in a viable but nonculturable (VBNC) state which leads to an underestimation of the population density. In this study, we used a novel fluorescence visualization technique, called recognition of individual gene fluorescence in situ hybridization (RING-FISH), which targets chromosomal DNA for enumeration. A polynucleotide probe labeled with Cyanine 3 (Cy3) was created corresponding to the ubiquitous V. parahaemolyticus gene that codes for thermolabile hemolysin (tlh). When coupled with the Kogure method to distinguish viable from dead cells, RING-FISH probes reliably enumerated total, viable V. parahaemolyticus. The probe was tested for sensitivity and specificity against a pure culture of tlh(+), tdh(-), trh(-)V. parahaemolyticus, pure cultures of Vibrio vulnificus, Vibrio harveyi, Vibrio alginolyticus and Vibrio fischeri, and a mixed environmental sample. This research will provide additional tools for a better understanding of the risk these environmental organisms pose to human health. PMID:21329738

Griffitt, Kimberly J; Noriea, Nicholas F; Johnson, Crystal N; Grimes, D Jay

2011-05-01

186

Numerical and Structural Genomic Aberrations Are Reliably Detectable in Tissue Microarrays of Formalin-Fixed Paraffin-Embedded Tumor Samples by Fluorescence In-Situ Hybridization  

PubMed Central

Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches. PMID:24733537

Horn, Heike; Bausinger, Julia; Staiger, Annette M.; Sohn, Maximilian; Schmelter, Christopher; Gruber, Kim; Kalla, Claudia; Ott, M. Michaela; Rosenwald, Andreas; Ott, German

2014-01-01

187

Mapping of low-frequency chimeric yeast artificial chromosome libraries from human chromosomes 16 and 21 by fluorescence in situ hybridization and quantitative image analysis  

SciTech Connect

Yeast artificial chromosome (YAC) clones from low-frequency chimeric libraries of human chromosomes 16 and 21 were mapped onto human diploid fibroblast metaphase chromosomes using fluorescence in situ hybridization (FISH) and digital imaging microscopy. YACs mapped onto chromosome 21 were selected to provide subregional location and ordering of known and unknown markers on the long arm of chromosome 21, particularly in the Down syndrome region (q22). YACs mapped onto chromosome 16 were selected to overlap regions spanning chromosome 16 cosmid maps. YAC clones were indirectly labeled with fluorescein, and the total DNA of the chromosome was counterstained with propidium iodide. A single image containing both the FISH signal and the whole chromosome was acquired for each chromosome of interest containing the fluorescent probe signal in a metaphase spread. From the digitized image, the fluorescence intensity profile through the long axis of the chromosome gave the total chromosome length and the probe position. The map position of the probe was expressed as the fractional length (FL) of the total chromosome relative to the end of the short arm (Flpter). From each clone hybridized, 20-40 chromosome images were analyzed. Thirty-eight YACs were mapped onto chromosome 16, and their FLs were distributed along the short and long arms. On chromosome 21, 47 YACs were mapped, including 12 containing known markers. To confirm the order of a dense population of YACs within the Down syndrome region, a two-color mapping strategy was used in which an anonymous YAC was located relative to one or two known markers on the metaphase chromosome. The chromosome FL maps have a 1- to 2-Mb resolution, and the FL measurement of each probe has a typical standard error of 0.5-1 Mb. 14 refs., 3 figs., 3 tabs.

Marrone, B.L.; Campbell, E.W.; Anzick, S.L.; Shera, K.; Campbell, M.; Yoshida, T.M.; McCormick, M.K.; Deaven, L. (Los Alamos National Lab., NM (United States))

1994-05-01

188

Simultaneous 16S and 18S rRNA fluorescence in situ hybridization (FISH) on LR White sections demonstrated in Vestimentifera (Siboglinidae) tubeworms.  

PubMed

Traditional morphological identification of invertebrate marine species is limited in early life history stages for many taxa. In this study, we demonstrate, by example of Vestimentiferan tubeworms (Siboglinidae, Polychaeta), that the simultaneous fluorescence in situ hybridization (FISH) of both eukaryotic host and bacterial symbiont cells is possible on a single semi-thin (1 ?m) section. This allows the identification of host specimens to species level as well as offering visualization of bacteria distributed within the host tissue. Previously published 18S rRNA host-specific oligonucleotide probes for Riftia pachyptila, Tevnia jerichonana and a newly designed Oasisia alvinae probe, as well as a 16S rRNA probe targeting symbionts found in all host species, were applied. A number of standard fixation and hybridization parameters were tested and optimized for the best possible signal intensity and cellular resolution. Ethanol conserved samples embedded in LR White low viscosity resin yielded the best results with regard to both signal intensity and resolution. We show that extended storage times of specimens does not affect the quality of signals attained by FISH and use our protocol to identify morphologically unidentifiable tubeworm individuals from a small data set, conforming to previous findings in succession studies of the Siboglinidae family. PMID:21507466

Schimak, Mario P; Toenshoff, Elena R; Bright, Monika

2012-02-01

189

Altered interphase fluorescence in situ hybridization profiles of chromosomes 4, 8q24, and 9q34 in pancreatic ductal adenocarcinoma are associated with a poorer patient outcome.  

PubMed

Most patients with pancreatic ductal adenocarcinoma (PDAC) die within 6 months of diagnosis. However, 20% to 25% patients undergoing total tumor resection remain alive and disease-free 5 years after diagnostic surgery. Few studies on tumor markers have predicted patient prognosis and/or survival. We evaluated the effect of tumor cytogenetic copy number changes detected by interphase fluorescence in situ hybridization on overall survival (OS) of 55 PDAC patients. The prognostic value of copy number changes showing an effect on OS was validated in an external cohort of 44 surgically resected PDAC patients by comparative genomic hybridization arrays, and the genes coded in altered chromosomes with prognostic value were identified by high-density single-nucleotide polymorphism arrays in 20 cases. Copy number changes of chromosomes 4 and 9q34 with gains of 8q24 were independently associated with shorter OS. On the basis of these three chromosomal alterations, a score is proposed that identifies patients with significantly different (P < 0.001) 5-year OS rates: 60% ± 20%, 16% ± 8%, and 0% ± 0%, respectively. Our results show an association between tumor cytogenetics and OS of PDAC patients and provide the basis for further prognostic stratification of patients undergoing complete tumor resection. Further studies to identify specific genes coded in these chromosomes and their functional consequences are necessary to understand the clinical effect of these changes. PMID:25157969

Gutiérrez, María L; Muñoz-Bellvis, Luis; Sarasquete, María E; Hernández-Mejía, David G; Abad, María Del Mar; Bengoechea, Oscar; Corchete, Luis; González-González, María; García-García, Jacinto; Gonzalez, Marcos; Mota, Ines; Orfao, Alberto; Sayagues, José M

2014-11-01

190

Whole-Mount In Situ Hybridization Using DIG-Labeled Probes in Planarian.  

PubMed

In recent years freshwater flatworms (planarian) have become a powerful model for studies of regeneration and stem cell biology. Whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH) are key and most commonly used techniques to determine and visualize gene expression patterns in planaria. Here, we present the established version of whole-mount in situ hybridization (WISH) and whole-mount fluorescence in situ hybridization (WFISH) protocol optimized over the last years by several labs from the rapidly growing planaria field and give an overview of recently introduced modifications which can be critical in the study of low abundant transcripts. PMID:25218375

Rybak-Wolf, Agnieszka; Solana, Jordi

2014-01-01

191

Simultaneous quantification of active carbon- and nitrogen-fixing communities and estimation of fixation rates using fluorescence in situ hybridization and flow cytometry.  

PubMed

Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and nifH (nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and (14)C/(15)N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean. PMID:25172848

McInnes, Allison S; Shepard, Alicia K; Raes, Eric J; Waite, Anya M; Quigg, Antonietta

2014-11-01

192

Fluorescence in situ hybridization and qPCR to detect Merkel cell polyomavirus physical status and load in Merkel cell carcinomas.  

PubMed

The Merkel cell polyomavirus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). Clonal integration and tumor-specific mutations in the large T antigen are strong arguments that MCPyV is a human tumor virus. However, the relationship between viral presence and cancer induction remains discussed controversially. Since almost all studies on virus prevalence are based on PCR techniques, we performed MCPyV fluorescence in situ hybridization (FISH) on MCC to gain information about the quality of the viral presence on the single cell level. MCPyV-FISH was performed on tissue microarrays containing 62 formalin-fixed and paraffin-embedded tissue samples including all tumor grades of 42 patients. The hybridization patterns were correlated to the qPCR data determined on corresponding whole tissue sections. Indeed, MCPyV-FISH and qPCR data were highly correlated, i.e. 83% for FISH-positive and 93% for FISH-negative cores. Accordingly, the mean of the qPCR values of all MCPyV-positive cores differed significantly from the mean of the negative cores (p = 0.0076). Importantly, two hybridization patterns were definable in the MCPyV-FISH: a punctate pattern (85%) indicating viral integration, which correlated with a moderate viral abundance and a combination of the punctate with a diffuse pattern (15%), suggesting a possible coexistence of integrated and episomal virus which was associated with very high viral load and VP1 expression. Thus, MCPyV-FISH adds important information on the single cell level within the histomorphological context and could therefore be an important tool to further elucidate MCPyV related carcinogenesis. PMID:24771111

Haugg, Anke M; Rennspiess, Dorit; Hausen, Axel Zur; Speel, Ernst-Jan M; Cathomas, Gieri; Becker, Jürgen C; Schrama, David

2014-12-15

193

A physical map of 15 loci on human chromosome 5q23-q33 by two-color fluorescence in situ hybridization  

SciTech Connect

The q23-q33 region of human chromosome 5 encodes a large number of growth factors, growth factor receptors, and hormone/neurotransmitter receptors. This is also the general region into which several disease genes have been mapped, including diastrophic dysplasia, Treacher Collins syndrome, hereditary startle disease, the myeloid disorders that are associated with the 5q-syndrome, autosomal-dominant forms of hereditary deafness, and limb girdle muscular dystrophy. The authors have developed a framework physical map of this region using cosmid clones isolated from the Los Alamos arrayed chromosome 5-specific library. Entry points into this library included 14 probes to genes within this interval and one anonymous polymorphic marker locus. A physical map has been constructed using fluorescence in situ hybridization of these cosmids on metaphase and interphase chromosomes, and this is in good agreement with the radiation hybrid map of the region. The derived order of loci across the region is cen-IL4-IL5-IRF1-IL3-IL9-EGR1-CD14-FGFA-GRL-D5S207-ADRB2-SPARC-RPS14-CSF1R-ADRA1, and the total distance spanned by these loci is approximately 15 Mb. The framework map, genomic clones, and contig expansion within 5q23-q33 should provide valuable resources for the eventual isolation of the clinically relevant loci that reside in this region. 31 refs., 3 figs., 2 tabs.

Saltman, D.L.; Dolganov, G.M. (Genelabs Inc. Redwood City, CA (United States)); Warrington, J.A.; Wasmuth, J.J. (Univ. of California, Irvine (United States)); Lovett, M. (Univ. of Texas Southwestern Medical Center, Dallas (United States))

1993-06-01

194

A physical map of 15 loci on human chromosome 5q23-q33 by two-color fluorescence in situ hybridization.  

PubMed

The q23-q33 region of human chromosome 5 encodes a large number of growth factors, growth factor receptors, and hormone/neurotransmitter receptors. This is also the general region into which several disease genes have been mapped, including diastrophic dysplasia, Treacher Collins syndrome, hereditary startle disease, the myeloid disorders that are associated with the 5q-syndrome, autosomal-dominant forms of hereditary deafness, and limb girdle muscular dystrophy. We have developed a framework physical map of this region using cosmid clones isolated from the Los Alamos arrayed chromosome 5-specific library. Entry points into this library included 14 probes to genes within this interval and one anonymous polymorphic marker locus. A physical map has been constructed using fluorescence in situ hybridization of these cosmids on metaphase and interphase chromosomes, and this is in good agreement with the radiation hybrid map of the region. The derived order of loci across the region is cen-IL4-IL5-IRF1-IL3-IL9-EGR1-CD1 4-FGFA-GRL-D5S207-ADRB2-SPARC-RPS14+ ++-CSF1R- ADRA1, and the total distance spanned by these loci is approximately 15 Mb. The framework map, genomic clones, and contig expansion within 5q23-q33 should provide valuable resources for the eventual isolation of the clinically relevant loci that reside in this region. PMID:8325647

Saltman, D L; Dolganov, G M; Warrington, J A; Wasmuth, J J; Lovett, M

1993-06-01

195

Genomic aberrations in salivary duct carcinoma arising in Warthin tumor of parotid gland: DNA microarray and HER2 fluorescence in situ hybridization.  

PubMed

Carcinoma arising from Warthin tumor is extremely rare. A 79-year-old man was admitted for a firm, well-defined, 5-cm left infra-auricular mass. Aspiration cytology showed many lymphohistiocytes and oncocytes in a proteinaceous background, compatible with Warthin tumor. A left superficial parotidectomy showed a solid mass around the cyst wall. The tumor cells of the solid area were arranged as infiltrative ducts with a few foci of malignant transformation. Virtual karyotyping disclosed a complex pattern of genetic aberrations with a focal amplification in 12q14-q21.2. This chromosomal region contains the MDM2 (murine double minute) gene, which regulates p53 inactivation. HER2 fluorescence in situ hybridization showed a focal amplification. Subsequently, the patient underwent total parotidectomy and ipsilateral neck dissection for a recurrence. To our knowledge, this is the first case of salivary duct carcinoma arising from Warthin tumor. The essential molecular pathway has not been reported, we presume an important role of MDM2 amplification- P53 inactivation. PMID:21877991

Kim, Hyun-Jung; Yoo, Young Sam; Park, Kyeongmee; Kwon, Ji-Eun; Kim, Jung Yeon; Monzon, Federico A

2011-09-01

196

Effects of Compost on Colonization of Roots of Plants Grown in Metalliferous Mine Tailings, as Examined by Fluorescence In Situ Hybridization?  

PubMed Central

The relationship between compost amendment, plant biomass produced, and bacterial root colonization as measured by fluorescence in situ hybridization was examined following plant growth in mine tailings. Mine tailings can remain devoid of vegetation for decades after deposition due to a combination of factors that include heavy metal toxicity, low pH, poor substrate structure and water-holding capacity, and a severely impacted heterotrophic microbial community. Research has shown that plant establishment, a desired remedial objective to reduce eolian and water erosion of such tailings, is enhanced by organic matter amendment and is correlated with significant increases in rhizosphere populations of neutrophilic heterotrophic bacteria. Results show that for the acidic metalliferous tailings tested in this study, compost amendment was associated with significantly increased bacterial colonization of roots and increased production of plant biomass. In contrast, for a Vinton control soil, increased compost had no effect on root colonization and resulted only in increased plant biomass at high levels of compost amendment. These data suggest that the positive association between compost amendment and root colonization is important in the stressed mine tailings environment where root colonization may enhance both microbial and plant survival and growth. PMID:19047384

Iverson, Sadie L.; Maier, Raina M.

2009-01-01

197

Radiation-induced chromosome aberrations in ataxia telangiectasia cells: high frequency of deletions and misrejoining detected by fluorescence in situ hybridization  

NASA Technical Reports Server (NTRS)

The mechanisms underlying the hyper-radiosensitivity of AT cells were investigated by analyzing chromosome aberrations in the G(2) and M phases of the cell cycle using a combination of chemically induced premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) with chromosome painting probes. Confluent cultures of normal fibroblast cells (AG1522) and fibroblast cells derived from an individual with AT (GM02052) were exposed to gamma rays and allowed to repair at 37 degrees C for 24 h. At doses that resulted in 10% survival, GM02052 cells were approximately five times more sensitive to gamma rays than AG1522 cells. For a given dose, GM02052 cells contained a much higher frequency of deletions and misrejoining than AG1522 cells. For both cell types, a good correlation was found between the percentage of aberrant cells and cell survival. The average number of color junctions, which represent the frequency of chromosome misrejoining, was also found to correlate well with survival. However, in a similar surviving population of GM02052 and AG1522 cells, induced by 1 Gy and 6 Gy, respectively, AG1522 cells contained four times more color junctions and half as many deletions as GM02052 cells. These results indicate that both repair deficiency and misrepair may be involved in the hyper-radiosensitivity of AT cells.

Kawata, Tetsuya; Ito, Hisao; George, Kerry; Wu, Honglu; Uno, Takashi; Isobe, Kouichi; Cucinotta, Francis A.

2003-01-01

198

Detection of integrated murine leukemia viruses in a mouse model of acute myeloid leukemia by fluorescence in situ hybridization combined with tyramide signal amplification.  

PubMed

This study was undertaken to develop a reliable method to enumerate and map somatically acquired, clonal, murine leukemia virus (MuLV) proviral insertions in acute myeloid leukemia (AML) cells from the BXH-2 mouse strain. This was achieved by using fluorescence in situ hybridization combined with tyramide signal amplification (FISH-TSA) and an 8.8 kilobase pair (kb) full-length ecotropic MuLV or 2.0 kb MuLV envelope (env) gene probe. Two-color FISH was utilized combining chromosome-specific probes for regions near the telomere and/or centromere and the MuLV probes. The technique reliably detected germline and somatically acquired, tumor-specific, MuLV proviruses in BXH-2 AML cell lines. It was possible to readily verify homozygous insertions at endogenous ecotropic MuLV loci, Emv1 (chromosome 5), Emv2 (chromosome 8) and a BXH-2 strain-specific locus (chromosome 11). This strategy also verified the presence of molecularly cloned proviral insertions within the mouse Nf1 gene and another locus on distal chromosome 11, as well as on chromosome 7 and chromosome 9 in BXH-2 AML cell line B117. The technique was also used to detect several new tumor-specific, proviral insertions in BXH-2 AML cell lines. PMID:10958940

Acar, H; Copeland, N G; Gilbert, D J; Jenkins, N A; Largaespada, D A

2000-08-01

199

Assessment of Her-2/neu status using immunocytochemistry and fluorescence in situ hybridization on fine-needle aspiration cytology smears: experience from a tertiary care centre in India.  

PubMed

Breast carcinoma shows amplification/overexpression of Her-2/neu in ?20-30% of cases. The determination of Her-2/neu expression accurately is vital in clinical practice as it has significant predictive value and eligibility for anti Her-2/neu therapy. Amplification and overexpression of Her-2/neu gene is traditionally identified by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) on tissue sections; only a few studies have evaluated feasibility of these techniques on cytological smears. One hundred cases of breast cancer with fine-needle aspiration cytology (FNAC) samples and corresponding surgically resected specimen were selected. Immunocytochemistry (ICC) and FISH for Her-2/neu was done on FNA smears, whereas IHC was performed on corresponding tissue sections. Diagnostic accuracy of ICC was 99% when compared with IHC. Comparison of FISH results with IHC showed 100% concordance. Unlike many centers in West, FNAC is still routinely performed in developing countries like India where vast majority of breast cancer cases present as palpable lumps. The high rates of accuracy of ICC and FISH for Her-2/neu detection can make FNAC a relevant first line of investigation as a cost effective model with a rapid turn-around time, providing complete information necessary for initial management of breast cancer patients. PMID:24376261

Durgapal, Prashant; Mathur, Sandeep R; Kalamuddin, Md; Datta Gupta, Siddhartha; Parshad, Rajinder; Julka, Pramod K; Panda, S K

2014-08-01

200

X chromosome evolution in the suni and eland antelope: detection of homologous regions by fluorescence in situ hybridization and G-banding.  

PubMed

Comparative cytogenetics and fluorescence in situ hybridization (FISH) were used to track structural rearrangements in the X chromosomes of two African antelope species, the eland (Taurotragus oryx; tribe Tragelaphini) and the suni (Neotragus moschatus; tribe Neotragini). Using two microdissected cattle chromosome painting probes (one specific for Xp-containing sequences corresponding to Xp24-->p12 of the cattle X, and one specific for Xq-containing sequences corresponding to Xq12-->qter), we show that intrachromosomal rearrangements distinguish the X chromosomes of these species. Furthermore, there is clear evidence that, superimposed on this background of intrachromosomal evolutionary change, the sequences contained in the cattle Xp painting probe appear to have moved as a complete unit during the repatterning of the bovid X. Although we are unable to infer the order of the rearranged chromosomal segments, given the large regions of homology detected by the chromosome paints, we nonetheless believe that this approach (combining conventional and molecular cytogenetic studies on the X chromosome) will prove useful for inferring phylogenetic relationships in the family Bovidae, particularly as more probes become available. PMID:9284920

Robinson, T J; Harrison, W R; Ponce de León, A; Elder, F F

1997-01-01

201

Human carbonyl reductase (CBR) localized to band 21q22. 1 by high-resolution fluorescence in situ hybridization displays gene dosage effects in trisomy 21 cells  

SciTech Connect

Human carbonyl reductase (CBR) belongs to a group of NADPH-dependent enzymes called aldo-keto reductases. The enzyme can function as an aldo-keto reductase or as a quinone reductase with potential for modulating quinone-mediated oxygen free radicals. The CBR gene was mapped by high-resolution fluorescence in situ hybridization to band 21q22.12, very close to the SOD1 locus at position 2lq22.11. CBR displayed gene dosage effects in trisomy 21 human lymphoblasts at the DNA and mRNA levels. Lymphoblasts with increasing chromosome 21 ploidy also showed increased aldo-keto reductase activity and increased quinone reductase activity. Both aldo-keto reductase activity and quinone reductase activity have been shown to be associated with carbonyl reductase. The location of CBR near SOD1 and the increased enzyme activity and potential for free radical modulation in trisomy 21 cells implicate CBR as a candidate for contributing to the pathology of certain diseases such as Down syndrome and Alzheimer disease. 28 refs., 1 fig., 1 tab.

Lemieux, N. (Universite de Montreal (Canada)); Malfoy, B. (Institut Curie Section de Biologie, Paris (France)); Forrest, G.L. (Beckman Research Institute at the City of Hope, Duarte, CA (United States))

1993-01-01

202

Synovial sarcoma arising in the vulva cytogenetically confirmed by SYT break-apart rearrangement fluorescence in situ hybridization: A case report and discussion of diagnostic methods  

PubMed Central

Synovial sarcoma (SS) is a soft tissue sarcoma of unknown histogenesis that rarely occurs in the female genital tract. We report a case of SS occurring in the right vulva of a young Japanese female. The tumor was composed of poorly differentiated rounded cell areas, surrounded by fibroblastic spindle-shaped cell areas. Immunohistochemically, the tumor cells were focally positive for cytokeratin, vimentin, CD99, Bcl-2 and neuron-specific enolase. The tumor was suspected, but was difficult to confirm as it was an SS based solely on light-microscopic and immunohistochemical findings. Although reverse transcription polymerase chain reaction (RT-PCR) failed to detect SS-specific SYT-SSX fusion gene transcripts using an RNA sample extracted from the formalin-fixed paraffin-embedded tumor tissue, SYT break-apart rearrangement fluorescence in situ hybridization (SYT bar-FISH) successfully confirmed our diagnosis of SS for the tumor. Thus, SYT bar-FISH may be more suitable for the purpose of the molecular diagnosis of SS than conventional RT-PCR when using archival formalin-fixed paraffin-embedded tissue specimens. PMID:23162630

KAWAUCHI, SHIGETO; IHARA, KOICHIRO; NISHIKAWA, KEI; SUGINO, NORIHIRO; TAKAHASHI, MUTSUO; SASAKI1, KOHSUKE

2012-01-01

203

Deletion of chromosome band 13q14 as detected by fluorescence in situ hybridization is a prognostic factor in patients with multiple myeloma who are receiving allogeneic dose-reduced stem cell transplantation  

Microsoft Academic Search

We investigated in a retrospective multi- center study the impact of chromosome arm 13q deletion (13q) as detected by fluorescence in situ hybridization (FISH) on outcome after dose-reduced al- lografting in patients with multiple my- eloma. In 68 of 140 patients, data on chromosome 13q status were available. Most patients included had advanced myeloma. At 2 years, patients with 13q

Nicolaus Kroger; Georgia Schilling; Hermann Einsele; Peter Liebisch; Avichai Shimoni; Arnon Nagler; Jose A. Perez-Simon; Jesus F. San Miguel; Michael Kiehl; Axel Fauser; Rainer Schwerdtfeger; Hannes Wandt; Herbert G. Sayer; Han Myint; Hans Klingemann; Tatjana Zabelina; Judith Dierlamm; Axel Hinke; Axel R. Zander

2004-01-01

204

Chromosomal study of lettuce and its allied species (Lactuca spp., Asteraceae) by means of karyotype analysis and fluorescence in situ hybridization.  

PubMed

In this study, in addition to the karyotype analysis, the chromosomal distributions of 5 S and 18 S rDNAs, and the Arabidopsis-type (T3AG3) telomeric sequences were detected by means of fluorescence in situ hybridization (FISH) to promote the information of chromosomal organization and evolution in the cultivated lettuce and its wild relatives, L. sativa, L. serriola, L. saligna and L. virosa. The karyotype analysis revealed the dissimilarity between L. virosa and the remaining species. In all four Lactuca species studied, one 5 S rDNA and two 18 S rDNA loci were detected. The simultaneous FISH of 5 S and 18 S rDNAs revealed that both rDNA loci of L. sativa, L. serriola and L. saligna were identical, however, that of L. virosa was different from the other species. These analyses indicate the closer relationships between L. sativa/L. serriola and L. saligna rather than L. virosa. Arabidopsis-type telomeric sequences were detected at both ends of their chromatids of all chromosomes not in the other regions. This observation suggests the lack of telomere-mediated chromosomal rearrangements among the Lactuca chromosomes. PMID:18215246

Matoba, Hideyuki; Mizutani, Takayuki; Nagano, Katsuya; Hoshi, Yoshikazu; Uchiyama, Hiroshi

2007-12-01

205

Clonal profiling of mixed lobular and ductal carcinoma revealed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.  

PubMed

A needle biopsy of a mass in the right breast of a 36-year-old woman revealed invasive ductal carcinoma (IDC), and approximately 20% of cancer cells showed unequivocal membranous staining with the HercepTest. After systemic therapy with trastuzumab and paclitaxel followed by FEC (fluorouracil + epirubicin + cyclophosphamide), a right mastectomy was performed. By histological and immunohistochemical examinations, the resected tumor consisted mainly of E-cadherin-negative invasive lobular carcinoma (ILC), and the rest was ERBB2-positive IDC; thus, the diagnosis was mixed ductal and lobular carcinoma. Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization (FISH) analyses revealed that ILC and IDC shared high-level amplification of CCND1 in homogeneously staining regions (HSR) and that IDC had an additional HSR-type amplicon of ERBB2. These findings strongly indicate that IDC and ILC had a common precursor cell with CCND1 amplification. Review of the biopsy specimen with FISH showed IDC with gene amplifications of CCND1 and ERBB2 as a minor component, IDC without amplification of CCND1 or ERBB2 as a major component, and a minute portion of ILC with CCND1 amplification. We speculate that chemotherapy and trastuzumab caused a marked reduction in IDC; however, ILC with CCND1 amplification was resistant to chemotherapy and grew. PMID:24888777

Tajiri, Ryosuke; Inokuchi, Masafumi; Sawada-Kitamura, Seiko; Kawashima, Hiroko; Nakamura, Ritsuko; Oyama, Takeru; Dobashi, Yoh; Ooi, Akishi

2014-05-01

206

Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization  

PubMed Central

Background There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders (?=?Laminaria japonica Aresch.), with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic chromosomes. A sex-specific marker is one of the best ways to make this determination. Methodology/Principal Findings To obtain clear images, chromosome preparation was improved using maceration enzymes and fluorochrome 4?, 6-diamidino-2-phenylindole (DAPI). The chromosome number of both male and female haploid gametophytes was 31, and there were 62 chromosomes in diploid sporophytes. Although the female chromosomes ranged from 0.77 µm to 2.61 µm in size and were larger than the corresponding ones in the males (from 0.57 µm to 2.16 µm), there was not a very large X chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH), this marker could be localized on one unique chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes. Conclusions/Significance Our data suggest that this marker was a female chromosome-specific DNA sequence. This is the first report of molecular marker localization on algal chromosomes. This research provides evidence for the benefit of using FISH for identifying molecular markers for sex identification, isolation of specific genes linked to this marker in the females, and sex determination of S. japonica gametophytes in the future. PMID:23166593

Gu, JunGang; Li, LiHua; Zhou, ZhiGang

2012-01-01

207

Fluorescence in situ hybridization as an adjunct tool in the diagnosis of primary and metastatic renal cell carcinoma in fine needle aspiration specimens.  

PubMed

We investigated the role of fluorescence in situ hybridization (FISH) in the diagnosis of primary renal neoplasms and lesions suspicious for metastatic renal cell carcinoma. ?Consecutive fine-needle aspiration biopsies (FNAB) of 39 renal masses and 41 metastatic tumours suspicious for renal cell origin were assessed with an immunohistochemical panel for CK7, RCC antigen, CD10, AMACR, PAX8, vimentin, and CD117. In addition, FISH was performed using probes for chromosomes 1p, 3p, 7, 17, X, and Y. ?A total of 31 of 39 primary renal masses and 33 of 41 metastatic tumors suspicious for renal origin demonstrated typical cytological and immunohistochemical (IHC) features of subtypes of renal neoplasms (40 clear cell renal cell carcinoma (RCC), 20 papillary RCC, and 4 renal oncocytomas). FISH analysis of 15 randomly selected cases each of primary and metastatic lesions revealed chromosomal abnormalities consistent with the diagnosis in 73% of these cases. Of 8 primary renal masses demonstrating atypical microscopic features and noncontributory IHC profiles, FISH was helpful in subtyping 5 (62%) of these lesions (2 clear cell RCC, 1 solid variant of oncocytic papillary RCC, 1 mixed clear cell and papillary RCC, and 1 chromophobe RCC with papillary architecture). Of 8 metastatic tumors clinically suspicious for renal cell origin and supportive, but nondiagnostic IHC, FISH revealed supportive chromosomal changes in 6 (75%) cases. ?In conclusion FISH analysis on FNAB material, even with limited tissue, may be contributory to the diagnosis and subtyping of RCC in diagnostically challenging biopsies. Diagn. Cytopathol. 2014;42:1013-1023. © 2014 Wiley Periodicals, Inc. PMID:24692327

Kos, Zuzana; Williams, Phillip A; Belanger, Eric C; Mai, Kien T

2014-12-01

208

Evaluation of a Fluorescence In Situ Hybridization Assay for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Lowenstein-Jensen and Mycobacteria Growth Indicator Tube Cultures Using Peptide Nucleic Acid Probes  

Microsoft Academic Search

A new fluorescence in situ hybridization assay based on peptide nucleic acid probes (MTB and NTM probes targeting tuberculous and nontuberculous species, respectively) for the identification of Mycobacterium tuber- culosis complex and differentiation between tuberculous and nontuberculous mycobacteria (NTM) was evalu- ated using Lowenstein-Jensen (LJ) solid cultures from 100 consecutive sputum samples and 50 acid-fast bacillus (AFB)-positive sputum samples as

POONPILAS HONGMANEE; HENRIK STENDER; OLE F. RASMUSSEN

209

Dual-color fluorescence in situ hybridization reveals an association of chromosome 8q22 but not 8p21 imbalance with high grade invasive breast carcinoma.  

PubMed

We previously reported molecular karyotype analysis of invasive breast tumour core needle biopsies by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) (Walker et al, Genes Chromosomes Cancer, 2008 May;47(5):405-17). That study identified frequently recurring gains and losses involving chromosome bands 8q22 and 8p21, respectively. Moreover, these data highlighted an association between 8q22 gain and typically aggressive grade 3 tumors. Here we validate and extend our previous investigations through FISH analysis of tumor touch imprints prepared from excised breast tumor specimens. Compared to post-surgical tumor excisions, core needle biopsies are known to be histologically less precise when predicting tumor grade. Therefore investigating these chromosomal aberrations in tumor samples that offer more reliable pathological assessment is likely to give a better overall indication of association. A series of 60 breast tumors were screened for genomic copy number changes at 8q22 and 8p21 by dual-color FISH. Results confirm previous findings that 8p loss (39%) and 8q gain (74%) occur frequently in invasive breast cancer. Both absolute quantification of 8q22 gain across the sample cohort, and a separate relative assessment by 8q22:8p21 copy number ratio, showed that the incidence of 8q22 gain significantly increased with grade (p = 0.004, absolute and p = 0.02, relative). In contrast, no association was found between 8p21 loss and tumor grade. These findings support the notion that 8q22 is a region of interest for invasive breast cancer pathogenesis, potentially harboring one or more genes that, when amplified, precipitate the molecular events that define high tumor grade. PMID:23936250

Walker, Logan C; McDonald, Margaret; Wells, J Elisabeth; Harris, Gavin C; Robinson, Bridget A; Morris, Christine M

2013-01-01

210

Dual-Color Fluorescence In Situ Hybridization Reveals an Association of Chromosome 8q22 but Not 8p21 Imbalance with High Grade Invasive Breast Carcinoma  

PubMed Central

We previously reported molecular karyotype analysis of invasive breast tumour core needle biopsies by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) (Walker et al, Genes Chromosomes Cancer, 2008 May;47(5):405-17). That study identified frequently recurring gains and losses involving chromosome bands 8q22 and 8p21, respectively. Moreover, these data highlighted an association between 8q22 gain and typically aggressive grade 3 tumors. Here we validate and extend our previous investigations through FISH analysis of tumor touch imprints prepared from excised breast tumor specimens. Compared to post-surgical tumor excisions, core needle biopsies are known to be histologically less precise when predicting tumor grade. Therefore investigating these chromosomal aberrations in tumor samples that offer more reliable pathological assessment is likely to give a better overall indication of association. A series of 60 breast tumors were screened for genomic copy number changes at 8q22 and 8p21 by dual-color FISH. Results confirm previous findings that 8p loss (39%) and 8q gain (74%) occur frequently in invasive breast cancer. Both absolute quantification of 8q22 gain across the sample cohort, and a separate relative assessment by 8q22:8p21 copy number ratio, showed that the incidence of 8q22 gain significantly increased with grade (p?=?0.004, absolute and p?=?0.02, relative). In contrast, no association was found between 8p21 loss and tumor grade. These findings support the notion that 8q22 is a region of interest for invasive breast cancer pathogenesis, potentially harboring one or more genes that, when amplified, precipitate the molecular events that define high tumor grade. PMID:23936250

Walker, Logan C.; McDonald, Margaret; Wells, J. Elisabeth; Harris, Gavin C.; Robinson, Bridget A.; Morris, Christine M.

2013-01-01

211

Epidermal Growth Factor Receptor Protein Expression and Gene Amplification in Normal, Hyperplastic, and Cancerous Glottic Tissue: Immunohistochemical and Fluorescent in Situ Hybridization Study on Tissue Microarrays  

PubMed Central

Aim To evaluate the importance of epidermal growth factor receptor (EGFR) protein overexpression and gene amplification in carcinogenesis of glottic cancer. Method In order to evaluate EGFR expression at protein and gene level, immunohistochemical (IHC) analysis and fluorescent in situ hybridization (FISH) were performed on tissue microarrays of laryngeal tissue (145 samples) – 38 samples of normal mucosa, 46 samples of hyperplastic lesions, and 61 samples of cancerous lesions. Results Membranous (mEGFR) and cytoplasmic (cEGFR) EGFR expression was significantly different between the analyzed groups. The differences were most striking in the suprabasal-transforming zone. IHC evaluation showed that high and low mEGFR staining contributed to the differentiation of dysplastic lesions, simple hyperplasia, and cancerous tissue, as well as between different degrees of atypia in hyperplastic lesions (P?in simple and abnormal hyperplastic lesions, but it was confirmed in 2/21 atypical hyperplasias, indicating that gene amplification can facilitate identification of malignant potential in hyperplastic lesions. In cancerous tissue, EGFR gene amplification was found in 8/50 samples. EGFR gene amplification was found in preinvasive cancer in one patient. In invasive carcinomas, gene amplification was not associated with stage or grade. Carcinomas with gene amplification showed significantly higher cEGFR expression (basal layer P?=?0.003; suprabasal layer P?=?0.002). Conclusions This study confirmed an increase in EGFR protein expression and gene amplification with the increase in biological aggressiveness of glottic lesions. A correlation between EGFR gene amplification and protein expression was established. Gene amplification proved to be an early event in glottic carcinogenesis, indicating its importance for glottic cancer prevention, early detection, and protocol selection. PMID:19673037

Braut, Tamara; Krstulja, Mira; Kujundzic, Milodar; Manestar, Dubravko; Hadzisejdic, Ita; Jonjic, Nives; Grahovac, Blazenka; Manestar, Darko

2009-01-01

212

Detection of Y chromosome sequences in a 45,X/46,XXq - patient by Southern blot analysis of PCR-amplified DNA and fluorescent in situ hybridization (FISH)  

SciTech Connect

In some cases of gonadal dysgenesis, cytogenetic analysis seems to be discordant with the phenotype of the patients. We have applied techniques such as Southern blot analysis and fluorescent in situ hybridization (FISH) to resolve the phenotype/genotype discrepancy in a patient with ambiguous genitalia in whom the peripheral blood karotype was 45,X. Gonadectomy at age 7 months showed the gonadal tissue to be prepubertal testis on the left side and a streak gonad on the right. The karyotype obtained from the left gonad was 45,X/46,XXq- and that from the right gonad was 45,X. Three different techniques, PCR amplification, FISH, and chromosome painting for X and Y chromosomes, confirmed the presence of Y chromosome sequences. Five different tissues were evaluated. The highest percentage of Y chromosome positive cells were detected in the left gonad, followed by the peripheral blood lymphocytes, skin fibroblasts, and buccal mucosa. No Y chromosomal material could be identified in the right gonad. Since the Xq- chromosome is present in the left gonad (testis), it is likely that the Xq- contains Y chromosomal material. Sophisticated analysis in this patient showed that she has at least 2 cell lines, one of which contains Y chromosomal material. These techniques elucidated the molecular basis of the genital ambiguity for this patient. When Y chromosome sequences are present in patients with Ullrich-Turner syndrome or gonadal dysgenesis, the risk for gonadal malignancy is significantly increased. Hence, molecular diagnostic methods to ascertain for the presence of Y chromosome sequences may expedite the evaluation of patients with the ambiguous genitalia. 21 refs., 4 figs., 2 tabs.

Kocova, M.; Siegel, S.F.; Wenger, S.L. [Univ. of Pittsburgh School of Medicine, Pittsburgh, PA (United States)

1995-02-13

213

FUS (16p11) gene rearrangement as detected by fluorescence in-situ hybridization in cutaneous low-grade fibromyxoid sarcoma: a potential diagnostic tool.  

PubMed

Low-grade fibromyxoid sarcoma (LGFMS) is a rare, typically deep-seated soft tissue neoplasm with deceptively bland cytology and metastatic potential. A t(7;16)(q34;p11) translocation, yielding a FUS/CREB3L2 fusion gene, has been identified in approximately 80%-90% of deep soft tissue LGFMS. Cutaneous fibromyxoid neoplasms occur not infrequently; dermatopathologists rarely consider LGFMS in the differential diagnosis, as this lesion is uncommon in the skin. We identified a group of superficial LGFMS and a spectrum of other cutaneous fibromyxoid neoplasms and performed fluorescence in situ hybridization (FISH) to assess the frequency of FUS rearrangement. FISH for the chromosomal rearrangement of FUS (16p11), using a dual-color, break-apart probe (Abbott Molecular/Vysis, Des Plaines, IL), was performed on formalin-fixed paraffin-embedded tissue sections from superficial LGFMS (n = 6), myxomas (n = 10), and myxofibrosarcoma/myxoid malignant fibrous histiocytomas (myxoid MFH) (n = 5). One hundred nonoverlapping tumor nuclei per case were evaluated for either fused (normal) or split (translocated) signals. Of the LGFMS, 4 of 6 (67%) showed a rearrangement of FUS (range: 72%-80% positive nuclei per 100 nuclei). The other neoplasms within the differential diagnosis were devoid of any rearrangement involving FUS (range: 0%-2% positive nuclei per 100 nuclei). Our observed frequency of FUS rearrangement in superficial LGFMS is consistent with those published in the literature for more deeply seated lesions. When applied to suspicious superficial myxoid or fibromyxoid neoplasms, the FUS FISH probe in formalin-fixed paraffin-embedded tissue can be a useful ancillary technique for diagnosis of this uncommon and deceptively bland tumor. PMID:21399449

Patel, Rajiv M; Downs-Kelly, Erinn; Dandekar, Monisha N; Fanburg-Smith, Julie C; Billings, Steven D; Tubbs, Raymond R; Goldblum, John R

2011-04-01

214

DIG In Situ Hybridization Protocol (with detergent)  

E-print Network

Cat. #1 093 274) at 4o C overnight. Dilute antibody 1:1000 in B2 GOAT. DAY THREE *Wash 3X30 minutes temperature in the dark for 10 minutes to 3 days, depending on abundance of transcript. B4 Solution: 45 µl NBTDIG In Situ Hybridization Protocol (with detergent) Leslie Vosshall 2 September 2000 DAY ONE

215

Large-scale polymorphism near the ends of several human chromosomes analyzed by using fluorescence in situ hybridization (FISH)  

SciTech Connect

We have discovered a large DNA segment that is polymorphically present at the ends of several human chromosomes. The segment, f7501, was originally derived form a human chromosome 19-specific cosmid library. FISH was used to determine the cosmid`s chromosomal distribution on 44 unrelated humans and several closely related primates. The human subjects represent a diversity of reproductively isolated ethnic populations. FISH analysis revealed that sequences highly homologous to the cosmid`s insert are present on both homologs at 3q, 15q,. and 19p in almost all individuals (88, 85, and 87 of 88 homologs, respectively). Other chromosomes sites were labeled much more rarely in the sampled individuals. For example, 56 of the 88 analyzed chromosomes 11 were labeled (18+/+, 6-/-, and 20+/- individuals). In contrast, 2q was labeled on only 1/88 sampled chromosomes. The termini of 2q, 5q, 6p, 6q, 7p, 8p, 9p, 9q, 11p, 12q, 16p, 19q, and 20q and an interstitial site at 2q13-14 were labeled in at least one individual of the set. EcoR1-fragments derived from the cosmid showed the same hybridization pattern as the entire cosmid, indicating that at least 40 kbp is shared by these chromosome ends. Ethnic differences in the allele frequency of these polymorphic variants was observed. For example, signals were observed on 8/10 and 7/10 of the chromosomes 7p and 16q, respectively, derived form Biakan Pygmies, but these sites were infrequently labeled in non-Pygmy human populations (2/68, respectively). This region has undergone significant changes in chromosome location during human evolution. Strong signal was seen on chimpanzee and gorilla chromosome 3, which is homologous to human chromosome 4, a chromosome unlabeled in any of the humans we have analyzed.

Trask, B.J.; Friedman, C. [Univ. of Washington, Seattle, WA (United States); Giorgi, D. [CNRS, Montpelier (France)] [and others

1994-09-01

216

Localization of ZNF164, ZNF146, GGTA1, SOX2, PRLR and EEF2 on homoeologous cattle, sheep and goat chromosomes by fluorescent in situ hybridization and comparison with the human gene map  

Microsoft Academic Search

The six following genes: zinc finger proteins 164 (ZNF164) and 146 (ZNF146), alpha-galactosyltransferase 1 (GGTA1), SRY-related HMG-box 2 (SOX2), prolactin receptor (PRLR) and elongation factor 2 (EEF2) have been localized by fluorescent in situ hybridization respectively on bovine and caprine chromosomes 17, 18, 11, 1, 20 and 7 and on sheep chromosomes 17, 14, 3, 1, 16, and 5. The

H. Hayes; C. Le Chalony; G. Goubin; D. Mercier; E. Payen; C. Bignon; K. Kohno

1996-01-01

217

Male infertility and copy number variants (CNVs) in the dog: a two-pronged approach using Computer Assisted Sperm Analysis (CASA) and Fluorescent In Situ Hybridization (FISH)  

PubMed Central

Background Infertility affects ~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors. Results We identified two genomic regions hybridized by BACs CH82-321J09 and CH82-509B23 showing duplication patterns in all samples except for an azoospermic dog. These two regions harbor two important genes for spermatogenesis: DNM2 and TEKT1. The genomic region encompassed by the BAC clone CH82-324I01 showed a single-copy pattern in all samples except for one dog, assessed with low-quality sperm, displaying a marked duplication pattern. This genomic region harbors SOX8, a key gene for testis development. Conclusion We present the first study involving functional and genetic analyses in male infertility. We set up an extremely reliable analysis on dog sperm cells with a highly consistent statistical significance, and we succeeded in conducting FISH experiments on sperm cells using BAC clones as probes. We found copy number differences in infertile compared with fertile dogs for genomic regions encompassing TEKT1, DNM2, and SOX8, suggesting those genes could have a role if deleted or duplicated with respect to the reference copy number in fertility biology. This method is of particular interest in the dog due to the recognized role of this species as an animal model for the study of human genetic diseases and could be useful for other species of economic interest and for endangered animal species. PMID:24373333

2013-01-01

218

Nucleic acid in-situ hybridization detection of infectious agents  

NASA Astrophysics Data System (ADS)

Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.

Thompson, Curtis T.

2000-04-01

219

Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography  

PubMed Central

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification. PMID:16332863

Ginige, Maneesha P.; Keller, Jurg; Blackall, Linda L.

2005-01-01

220

Characterization of Childhood Precursor T-Lymphoblastic Lymphoma by Immunophenotyping and Fluorescent In-Situ Hybridization: A Report from the Children's Oncology Group  

PubMed Central

Background T-lymphoblastic lymphoma (T-LBL) accounts for 25–30% of childhood non-Hodgkin’s lymphoma and is closely related to T-lymphoblastic leukemia (T-ALL). Recently, we demonstrated distinct differences in gene expression between childhood T-LBL and T-ALL, but molecular pathogenesis and relevant protein expression patterns in T-LBL remain poorly understood. Procedure Children with T-LBL with disseminated disease were registered and treated on COG protocol 5971. Paraffin-embedded tumor tissue was obtained at diagnosis for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) studies. We determined the pattern and intensity of staining for c-Myc, Skp2, Mib-1, p53, TCL-1, bcl-2, and bcl-6 proteins by IHC and c-Myc, p53, bcl-2, bcl-6, and TCR ?/? molecular alterations by FISH in 22 pediatric T-LBL cases. Results The majority of T-LBL samples expressed Mib-1 (59%) and c-Myc (77%) proteins in greater than 50% of the cells, but Skp2 (14%), p53 (14%), and bcl-2 (23%) expression was less common. FISH studies demonstrated 18% gains and 10% losses in c-Myc, 16% gains in p53, 12% gains and 6% losses in bcl-2, and 6% gains and 19% losses in bcl-6 with little direct correlation between the IHC and FISH studies. Conclusions Childhood T-LBL is a highly proliferative tumor associated with enhanced expression of c-Myc protein, but without detectable c-Myc molecular alterations. FISH studies did not identify consistent etiologies of molecular dysregulation, and future studies with other molecular approaches may be required to elucidate the molecular pathogenesis of childhood T-LBL. PMID:18618503

Smock, Kristi J.; Nelson, Marilu; Tripp, Sheryl R.; Sanger, Warren G.; Abromowitch, Minnie; Cairo, Mitchell S.; Perkins, Sherrie L.

2009-01-01

221

Diagnostic utility of NCOA2 fluorescence in situ hybridization and Stat6 immunohistochemistry staining for soft tissue angiofibroma and morphologically similar fibrovascular tumors.  

PubMed

Soft tissue angiofibroma (STA), a recently suggested new histologic entity, is a benign fibrovascular soft tissue tumor composed of bland spindle-shaped tumor cells with abundant collagenous to myxoid stroma and branching small vessels. The lesion has a characteristic AHRR-NCOA2 fusion gene derived from chromosomal translocation of t(5;8)(p15;q13). However, morphologically similar tumors containing abundant fibrovascular and myxoid stroma can complicate diagnosis. We designed an original DNA probe for detecting NCOA2 split signals on fluorescence in situ hybridization (FISH) and estimated its utility with 20 fibrovascular tumors: 4 each of STAs, solitary fibrous tumors (SFTs), and cellular angiofibromas and 3 each of low-grade myxofibrosarcomas, myxoid liposarcomas, and low-grade fibromyxoid sarcomas. We also performed FISH for 13q14 deletion and immunohistochemistry (IHC) staining for estrogen receptor, progesterone receptor, retinoblastoma protein, and MUC-4 expression. Furthermore, IHC for Stat6 was conducted in the 20 cases analyzed by FISH and in an additional 26 SFTs. We found moderate to strong nuclear Stat6 expression in all SFTs but no expression in the other tumors. Both estrogen receptor and progesterone receptor expressions were observed in STAs, SFTs, and cellular angiofibromas. Expression of retinoblastoma protein was found in less than 10% of cells in all tumor types except myxoid liposarcoma. The low-grade fibromyxoid sarcomas were strongly positive for MUC-4. All STAs showed NCOA2 split signals on FISH. All tumors, regardless of histologic type, had 13q14 deletion. The NCOA2 FISH technique is a practical method for confirming STA diagnosis. The combination of NCOA2 FISH and Stat6 IHC proved effective for the differential diagnosis of STA, even when using small biopsy specimens. PMID:24856853

Sugita, Shintaro; Aoyama, Tomoyuki; Kondo, Kei; Keira, Yoshiko; Ogino, Jiro; Nakanishi, Katsuya; Kaya, Mitsunori; Emori, Makoto; Tsukahara, Tomohide; Nakajima, Hisaya; Takagi, Masayuki; Hasegawa, Tadashi

2014-08-01

222

Detection of Escherichia coli O157 by peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) and comparison to a standard culture method.  

PubMed

Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 × 10(-2) to 1 × 10(2) CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day. PMID:23934486

Almeida, C; Sousa, J M; Rocha, R; Cerqueira, L; Fanning, S; Azevedo, N F; Vieira, M J

2013-10-01

223

Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method  

PubMed Central

Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 × 10?2 to 1 × 102 CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day. PMID:23934486

Almeida, C.; Sousa, J. M.; Rocha, R.; Cerqueira, L.; Fanning, S.; Azevedo, N. F.

2013-01-01

224

High-resolution mapping of the human 4q21 and the mouse 5E3 SCYB chemokine cluster by fiber-fluorescence in situ hybridization.  

PubMed

The CXC chemokine or small inducible cytokine B (SCYB) subfamily includes the T-cell chemoattractants MIG (CXCL9, SCYB9), IP-10 (CXCL10, SCYB10), and I-TAC (CXCL11, SCYB11). These three highly homologous chemokines lack the glutamic acid-leucine-arginine (ELR) motif and signal via the CXCR3 receptor. Previous work showed that the genes encoding these chemokines are localized in an individual mini-cluster on human Chromosome (Chr) 4 at position 4q21.2. Recently, we identified mouse Scyb11 and mapped this gene by fluorescence in situ hybridization (FISH) to mouse Chr 5E3, the orthologous locus to human 4q21 where the other two homologous mouse genes, Scyb9 and Scyb10, have also been localized. Since SCYB10 and SCYB11 are not represented in the recently published draft sequence of the human genome, we wanted to clarify exactly the order and distances of the three chemokine genes using two-color FISH on stretched DNA fiber preparations. Here, we report the simultaneous localization of all three genes and provide high-resolution visual maps of this chemokine cluster from both mouse and human. The three chemokine genes were found within a range of 32 kb on mouse and 29 kb on human DNA fiber targets. The precise physical distances were defined, and an almost identical arrangement of the human and mouse homologues was identified, indicating that this CXC chemokine mini-cluster has been completely conserved evolutionarily since the divergence of mouse and human. Our results refine previous maps of the three genes, support the hypothesis that they resulted from gene duplication that took place in a common ancestor of mouse and human, and provide complementary information on a region of the draft sequence of human Chr 4 that is not yet covered. PMID:11685476

Erdel, M; Theurl, M; Meyer, M; Duba, H C; Utermann, G; Werner-Felmayer, G

2001-09-01

225

Targeting the treponemal microbiome of digital dermatitis infections by high-resolution phylogenetic analyses and comparison with fluorescent in situ hybridization.  

PubMed

Modern pyrosequencing technology allows for a more comprehensive approach than traditional Sanger sequencing for elucidating the etiology of bovine digital dermatitis. We sought to describe the composition and diversity of treponemes in digital dermatitis lesions by using deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with species-level taxonomic identification. Treponema-specific 16S rRNA gene PCRs and pyrosequencing were performed on biopsy specimens originating from 10 different Catalan dairy herds (n = 36) with digital dermatitis, and this analysis yielded 75,297 sequences. We identified 20 different taxa, including a potentially novel phylotype that displayed 95% sequence identity to members of the Treponema denticola/Treponema pedis-like cluster. Species frequencies and abundances that were determined by pyrosequencing analysis were highly correlated with the results of fluorescent in situ hybridization using phylotype-specific oligonucleotide probes. In a limited number of animals from a single geographic region, we detected most of the Treponema phylotypes that were described in previous investigations of digital dermatitis. Additionally, we identified a number of phylotypes that mapped to oral treponemes of humans and dogs that had not been reported for digital dermatitis lesions. The results presented here support previous observations of a polytreponemal etiology of infections, with Treponema phagedenis-like, Treponema medium/Treponema vincentii-like, and T. denticola/T. pedis-like phylotypes being highly associated with disease. Using this new approach, it has become feasible to study large herds and their surrounding environments, which might provide a basis for a better understanding of the pathogenesis of this disease. PMID:23658264

Klitgaard, Kirstine; Foix Bretó, Antoni; Boye, Mette; Jensen, Tim K

2013-07-01

226

Targeting the Treponemal Microbiome of Digital Dermatitis Infections by High-Resolution Phylogenetic Analyses and Comparison with Fluorescent In Situ Hybridization  

PubMed Central

Modern pyrosequencing technology allows for a more comprehensive approach than traditional Sanger sequencing for elucidating the etiology of bovine digital dermatitis. We sought to describe the composition and diversity of treponemes in digital dermatitis lesions by using deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with species-level taxonomic identification. Treponema-specific 16S rRNA gene PCRs and pyrosequencing were performed on biopsy specimens originating from 10 different Catalan dairy herds (n = 36) with digital dermatitis, and this analysis yielded 75,297 sequences. We identified 20 different taxa, including a potentially novel phylotype that displayed 95% sequence identity to members of the Treponema denticola/Treponema pedis-like cluster. Species frequencies and abundances that were determined by pyrosequencing analysis were highly correlated with the results of fluorescent in situ hybridization using phylotype-specific oligonucleotide probes. In a limited number of animals from a single geographic region, we detected most of the Treponema phylotypes that were described in previous investigations of digital dermatitis. Additionally, we identified a number of phylotypes that mapped to oral treponemes of humans and dogs that had not been reported for digital dermatitis lesions. The results presented here support previous observations of a polytreponemal etiology of infections, with Treponema phagedenis-like, Treponema medium/Treponema vincentii-like, and T. denticola/T. pedis-like phylotypes being highly associated with disease. Using this new approach, it has become feasible to study large herds and their surrounding environments, which might provide a basis for a better understanding of the pathogenesis of this disease. PMID:23658264

Foix Breto, Antoni; Boye, Mette; Jensen, Tim K.

2013-01-01

227

Chromosomal differences between European and North American Atlantic salmon discovered by linkage mapping and supported by fluorescence in situ hybridization analysis  

PubMed Central

Background Geographical isolation has generated a distinct difference between Atlantic salmon of European and North American Atlantic origin. The European Atlantic salmon generally has 29 pairs of chromosomes and 74 chromosome arms whereas it has been reported that the North American Atlantic salmon has 27 chromosome pairs and an NF of 72. In order to predict the major chromosomal rearrangements causing these differences, we constructed a dense linkage map for Atlantic salmon of North American origin and compared it with the well-developed map for European Atlantic salmon. Results The presented male and female genetic maps for the North American subspecies of Atlantic salmon, contains 3,662 SNPs located on 27 linkage groups. The total lengths of the female and male linkage maps were 2,153?cM and 968?cM respectively, with males characteristically showing recombination only at the telomeres. We compared these maps with recently published SNP maps from European Atlantic salmon, and predicted three chromosomal reorganization events that we then tested using fluorescence in situ hybridization (FISH) analysis. The proposed rearrangements, which define the differences in the karyotypes of the North American Atlantic salmon relative to the European Atlantic salmon, include the translocation of the p arm of ssa01 to ssa23 and polymorphic fusions: ssa26 with ssa28, and ssa08 with ssa29. Conclusions This study identified major chromosomal differences between European and North American Atlantic salmon. However, while gross structural differences were significant, the order of genetic markers at the fine-resolution scale was remarkably conserved. This is a good indication that information from the International Cooperation to Sequence the Atlantic salmon Genome, which is sequencing a European Atlantic salmon, can be transferred to Atlantic salmon from North America. PMID:22928605

2012-01-01

228

Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH).  

PubMed

Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH. PMID:16545875

Kubota, Kengo; Ohashi, Akiyoshi; Imachi, Hiroyuki; Harada, Hideki

2006-09-01

229

Chromosomal instability in gastric mucosa-associated lymphoid tissue lymphomas: a fluorescent in situ hybridization study using a tissue microarray approach.  

PubMed

Extranodal marginal zone B-cell lymphomas (mucosa-associated lymphoid tissue [MALT] lymphomas) of the gastrointestinal tract have been known to have characteristic chromosomal aberrations including trisomies of chromosomes 3, 12, and 18. However, knowledge of the clinical significance of cytogenetic changes in MALT lymphomas is still limited. In the present study, the frequency of the numeric and structural aberrations of the chromosomes 1, 3, 12, 18 and X and of the MALT1 gene as well as their potential clinical significance were analyzed by using fluorescent in situ hybridization on a tissue microarray containing 257 tissue samples from 203 cases of surgically resected primary gastric lymphomas including 115 cases of MALT lymphomas, 88 cases of diffuse large B-cell lymphomas (DLBCLs, 75 with an associated MALT lymphoma, so-called ex-MALT DLBCL, and 13 de novo), and 54 controls cases of Helicobacter pylori-associated chronic gastritis. Clinical follow-up information was available in 137 cases. Trisomies 1, 3, 12, and 18 were detected in 3.3%, 44.4%, 12.3%, and 19.2% of MALT lymphomas and in 11.1%, 42.2%, 26.5%, and 22.0% of ex-MALT DLBCLs, respectively. In addition, we found gains of the X chromosome in 36.4% of MALT lymphomas, in 34.5% of ex-MALT DLBCLs, and in 36.4% of de novo DLBCLs. Structural and/or numeric abnormalities of the MALT1 gene were observed in 37.0% of MALT lymphomas and in 22.2% of ex-MALT DLBCLs. In de novo DLBCL, trisomies for chromosomes 3, 12, 18, and X were found in 42.9%, 10.0%, 11.1%, and 36.4%, respectively, whereas alterations of MALT1 (namely, translocations) were found in 20.0% of the cases. An unexpected high and previously unreported gain of chromosome X in gastric MALT lymphomas was found. This tumor appears, therefore, to be a genetically unstable neoplasia. Our results point out that t(11;18) and aneuploidy may be both involved in lymphomagenesis and that at least a subset of MALT lymphomas may progress toward high-grade neoplasia. PMID:18234275

Bernasconi, Barbara; Karamitopoulou-Diamantis, Eva; Karamitopolou-Diamantiis, Eva; Tornillo, Luigi; Lugli, Alessandro; Di Vizio, Dolores; Dirnhofer, Stephan; Wengmann, Stephan; Glatz-Krieger, Katharyna; Fend, Falko; Capella, Carlo; Insabato, Luigi; Terracciano, Luigi M

2008-04-01

230

In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes  

Microsoft Academic Search

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in do- mestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these tech- niques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In

SATOSHI OKABE; HISASHI SATOH; YOSHIMASA WATANABE

1999-01-01

231

Electron Paramagnetic Resonance and Fluorescence In Situ Hybridization-Based Investigations of Individual Doses for Persons Living at Metlino in the Upper Reaches of the Techa River  

SciTech Connect

Waterborne releases from the Mayak Production Association in Russia during 1949–1956 resulted in significant doses to persons living downstream; the most contaminated village was Metlino about 7 km downstream. Internal and external doses have been estimated for these residents using the Techa River Dosimetry System–2000; the primary purpose is to support epidemiological studies of the members of the Extended Techa River Cohort (ETRC). Efforts to validate the calculations of external and internal dose are considered essential. Two methods used for the validation of external dose are electron paramagnetic resonance (EPR) measurements of teeth and fluorescence in situ hybridization (FISH) measurements of chromosome translocations in circulating lymphocytes. For EPR, 36 measurements on 26 teeth from 16 donors from Metlino were made at the GSF (16 measurements) and the IMP (20 measurements); the correlation between measurements made at the two laboratories has been found to be 0.99. Background measurements were also made on 218 teeth (63 molars, 128 premolars, and 27 incisors). FISH measurements were made for 31 residents of Metlino at the GSF. These measurements were handicapped by the analysis of a limited number of cells; for several individuals no stable translocations were observed. FISH measurements were also made for 39 individuals believed to be unexposed. The majority of EPR-measurement results fell within the range of 70 to 2700 mGy (including background). The results of FISH-based measurements fell within the range of nondetectable to 2 Gy (background subtracted). The results of individual measurements using EPR and FISH methods were generally consistent with each other and with results of other assays, including thermoluminescent measurements of quartz extracted from bricks taken from old buildings. Results were also consistent with those estimated with the TRDS-2000. Thus, the limited sets of data currently available tend to validate the present calculations of external dose for members of the ETRC. However, more validation work is considered essential to the credibility of the dose calculations being made with the TRDS-2000.

Degteva, M. O.; Anspaugh, L. R.; Akleyev, A. V.; Jacob, Peter; Ivanov, Denis V.; Wieser, Albrecht; Vorobiova, M. I.; Shishkina, Elena A.; Shved, Valentina A.; Vozilova, Alexandra; Bayankin, Sergey N.; Napier, Bruce A.

2005-02-01

232

Assignment of the human dihydropyrimidine dehydrogenase gene (DPYD) to chromosome region 1p22 by fluorescence in situ hybridization  

SciTech Connect

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the three-step pathway of uracil and thymine catabolism leading to the formation of {beta}-alanine and {beta}-aminobutyric acid, respectively. Several studies have demonstrated the importance of DPD in cancer patients, particularly in those lacking or having only low levels of activity. Patients exhibiting severe toxicity when administered 5-fluorouracil were shown to have low DPD activity. Studies of affected families demonstrated that the deficiency was inherited in an autosomal recessive pattern. DPD deficiency is one of several inherited disorders of pyrimidine metabolism, clinically termed thymine-uracil-uria. 14 refs., 1 fig.

Takai, Setsuo [International Medical Center, Tokyo (Japan)] [International Medical Center, Tokyo (Japan); Fernandez-Salguero, Pedro; Kimura, Shioko [National Cancer Institute, Bethesda, MD (United States)] [and others] [National Cancer Institute, Bethesda, MD (United States); and others

1994-12-01

233

Miller-Dieker syndrome associated with duplication of 17p13.3 confirmed by fluorescence in situ hybridization (FISH)  

SciTech Connect

Miller-Dieker syndrome is characterized by profound mental retardation, craniofacial abnormalities, and lissencephaly (smooth brain). Microscopic or submicroscopic deletions of the 17p13.3 region have been reported in Miller-Dieker patients. We report a patient with this syndrome in whom a duplication of the 17p13.3 region was detected by FISH. The 9-year-old female proband was referred because of features of Miller-Dieker syndrome: microcephaly, profound psychomotor retardation, seizures, characteristic facies, and lissencephaly shown by MRI studies. High-resolution G-banding failed to demonstrate an abnormality in chromosome 17. However, FISH analysis with the DNA probe (Oncor No. 5101) specific for Miller-Dieker region of chromosome 17p13.3 demonstrated duplication of this segment instead of the classic deletion. We know of no other report of Miller-Dieker syndrome associated with duplication of 17p13.3. The family study revealed normal chromosomes in both parents by cytogenetic and FISH analysis. Our investigation suggests that duplications, as well as deletions, of the 17p13.3 region are associated with the Miller-Dieker syndrome. The presence of deletions or duplications of the same chromosomal region in patients with features of Miller-Dieker syndrome suggests that its pathogenesis may be due to gene dosage effects.

Li, S.; Tuck-Muller, C.M.; Martinez, J.E. [Univ. of South Alabama, Mobile, AL (United States)] [and others

1994-09-01

234

DNA/DNA in situ hybridization with enzyme linked probes  

SciTech Connect

A non-radioactive in situ nucleic acid hybridization method which requires no antibodies, haptens, avidin or biotin intermediateries is presented. Horseradish peroxidase (HRP) labeled nucleic acid probes are hybridized in situ for 2 hours or less, followed by brief washing of hybridized cells and the direct detection of in situ hybrids with diaminobenzidine (DAB). Application of this method to the detection of Human Papilloma Virus (HPV) in human cells is shown.

Grillo, S.; Mosher, M.; Charles, P.; Henry, S.; Taub, F.

1987-05-01

235

Inter-observer reproducibility of HER2 immunohistochemical assessment and concordance with fluorescent in situ hybridization (FISH): pathologist assessment compared to quantitative image analysis  

PubMed Central

Background In breast cancer patients, HER2 overexpression is routinely assessed by immunohistochemistry (IHC) and equivocal cases are subject to fluorescent in situ hybridization (FISH). Our study compares HER2 scoring by histopathologists with automated quantitation of staining, and determines the concordance of IHC scores with FISH results. Methods A tissue microarray was constructed from 1,212 invasive breast carcinoma cases with linked treatment and outcome information. IHC slides were semi-quantitatively scored by two independent pathologists on a range of 0 to 3+, and also analyzed with an Ariol automated system by two operators. 616 cases were scorable by both IHC and FISH. Results Using data from unequivocal positive (3+) or negative (0, 1+) results, both visual and automated scores were highly consistent: there was excellent concordance between two pathologists (kappa = 1.000, 95% CI: 1-1), between two machines (kappa = 1.000, 95% CI: 1-1), and between both visual and both machine scores (kappa = 0.898, 95% CI: 0.775–0.979). Two pathologists successfully distinguished negative, positive and equivocal cases (kappa = 0.929, 95% CI: 0.909–0.946), with excellent agreement with machine 1 scores (kappa = 0.835, 95% CI: 0.806–0.862; kappa = 0.837, 95% CI: 0.81–0.862), and good agreement with machine 2 scores (kappa = 0.698, 95% CI: 0.6723–0.723; kappa = 0.709, 95% CI: 0.684–0.732), whereas the two machines showed good agreement (kappa = 0.806, 95% CI: 0.785–0.826). When comparing categorized IHC scores and FISH results, the agreement was excellent for visual 1 (kappa = 0.814, 95% CI: 0.768–0.856), good for visual 2 (kappa = 0.763, 95% CI: 0.712–0.81) and machine 1 (kappa = 0.665, 95% CI: 0.609–0.718), and moderate for machine 2 (kappa = 0.535, 95% CI: 0.485–0.584). Conclusion A fully automated image analysis system run by an experienced operator can provide results consistent with visual HER2 scoring. Further development of such systems will likely improve the accuracy of detection and categorization of membranous staining, making this technique suitable for use in quality assurance programs and eventually in clinical practice. PMID:19476653

2009-01-01

236

In situ preparation and fluorescence quenching properties of polythiophene\\/ZnO nanocrystals hybrids through atom-transfer radical polymerization and hydrolysis  

Microsoft Academic Search

In this paper, a new approach for in situ preparing nanocomposites of conjugated polymers (CPs) and semiconductor nanocrystals was developed. Polythiophene grafted poly(zinc methacrylate) (PTh-g-PZMA) copolymer was synthesized by atom-transfer radical polymerization (ATRP) of zinc methacrylate (ZMA) initiated from the macroinitiator poly(2,5-(3-(bromoisopropyl-carbonyl-oxymethylene) thiophene)) (PTh-Br) with pendant initiator groups. Subsequently, the polythiophene grafted poly(methacrylate)\\/ZnO (PTh-g-PMA\\/ZnO) hybrid heterojunction nanocomposites were successfully prepared

Xiaoming Peng; Lin Zhang; Yiwang Chen; Fan Li; Weihua Zhou

2010-01-01

237

Novel, rapid DNA-based on-chip bacterial identification system combining dielectrophoresis and amplification-free fluorescent resonance energy transfer assisted in-situ hybridization (FRET-ISH)  

NASA Astrophysics Data System (ADS)

Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in-situ hybridization (FRET-ISH) species identification. Identification is achieved completely on chip in less than thirty minutes from receipt of sample compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.

Packard, Michelle M.; Shusteff, Maxim; Alocilja, Evangelyn

2011-10-01

238

FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RNA-TARGETED OLIGONUCLEOTIDE PROBE  

EPA Science Inventory

A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem, in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligo...

239

Detection of in-situ hybridization to human metaphase chromosomes by atomic force microscopy  

NASA Astrophysics Data System (ADS)

Detection of in situ hybridization to human metaphase chromosomes provides important information about gene mappings and about analysis of chromosomal disorders. We applied atomic force microscopy (AFM) to the detection of in situ hybridization to get better resolution as compared to light microscopy. Chromosomes were spread over a glass substrate and hybridized with DNA probes labeled with biotin or digoxigenin. The hybridized probes were reacted with streptavidin or anti-digoxigenin antibody, both of which were conjugated with 5-nm gold colloidal particles. We missed direct detection of the conjugated gold colloidal particles by micro-meter scale AFM scanning , but obtained clear topographic difference between the site of hybridization and the chromosome arm with the help of silver enhancement. We thus clearly detected the in situ hybridization using chromosome painting probes, alpha satellite probes, and locus specific gene probes by AFM. The in situ hybridization to DNA fiber was also detected by AFM. The detection of in situ hybridization by AFM has advantages over fluorescence in situ hybridization: no reduction of signal intensity under light irradiation. Application of AFM to the detection of in situ hybridization will be a useful method to analyze chromosomes.

Okamoto, Naoaki; Ishikawa, Mitsuru

2000-04-01

240

Detection of {open_quotes}cryptic{close_quotes}karyotypic rearrangements in closely related primate species by fluorescence in situ hybridization (FISH) using human subtelomeric DNA probes  

SciTech Connect

Specific human subtelomeric DNA probes were used to reveal cryptic chromosomal rearrangements that cannot be detected by conventional high resolution cytogenetic techniques, or by chromosomal in situ suppression hybridization using whole chromosome paint analysis. Two cosmids containing different subtelomeric DNA sequences were derived from human chromosome 19 and designated as 7501 and 16432. Cosmid 7501 was hybridized to chromosomes from humans, chimpanzee, gorilla and orangutan. In humans, 7501 consistently labeled chromosomes 3q, 15q, and 19p. Additional chromosomes were labeled in different individuals, indicating a polymorphic distribution of this sequence in the human genome. In contrast, 7501 consistently and strongly labeled only the q arm terminus of chromosome 3 in both chimp and gorilla. The identification of the chromosome was made by two-color FISH analysis using human chromosome 4-specific paint and homologous to human chromosome 4. None of the human subjects showed labeling of chromosome 4 with 7501. This finding suggests that in the course of human evolution, subsequent to the divergence of humans and African apes, a cryptic translocation occurred between the ancestral human chromosome 4 and one or more of the other human chromosomes that now contain this DNA segment. In orangutan, 7501 labeled a single acrocentric chromosome pair, a distinctly different chromosome than that labeled in chimp and gorilla. Comparison of chromosome sites labeled with cosmid 16432 showed the distribution of signals on chromosome 1q arm is the same for humans and chimp, but different in the gorilla. Humans and chimps show distinct labeling on sites 1q terminus and 1q41-42. In gorilla, there is instead a large cluster of intense signal near the terminus of 1q that clearly does not extend all the way to the terminus. A paracentric inversion or an unequal cross-over event may account for the observed difference between these species.

Youngblom, J.J. [California State University-Stanislaus, Turlock, CA (United States); Trask, B.J.; Friedman, C. [Univ. of Washington, Seattle, WA (United States)] [and others

1994-09-01

241

Molecular cytogenetic characteristics of the human hepatocellular carcinoma cell line HCCLM3 with high metastatic potential: comparative genomic hybridization and multiplex fluorescence in situ hybridization  

Microsoft Academic Search

The HCCLM3 cell line was established at the authors' institute from the lung metastatic lesions of BALB\\/c nude mice bearing human hepatocellular carcinoma (HCC) from the metastatic HCC cell line MHCC97-H. It has been shown to have a high potential for lung metastases and extensive metastases when the cells are inoculated subcutaneously or orthotopically in athymic nude mice. In the

Jiong Yang; Lun-Xiu Qin; Yan Li; Sheng-Long Ye; Yin-Kun Liu; Dong-Mei Gao; Jie Chen; Zhao-You Tang

2005-01-01

242

The oxytocin receptor gene (OXTR) localizes to human chromosome 3p25 by fluorescence in situ hybridization and PCR analysis of somatic cell hybrids  

SciTech Connect

The human oxytocin receptor regulates parturition and myometrial contractility, breast milk let-down, and reproductive behaviors in the mammalian central nervous system. Kimura et al. recently identified a human oxytocin receptor cDNA by means of expression cloning from a human myometrial cDNA library. To elucidate further the molecular mechanisms that regulate oxytocin receptor gene expression and to define the expected Mendelian inheritance of possible human disease states, we must determine the number of genes, their localization, and their organization and structure. We summarize below our data indicating that the human oxytocin receptor gene is localized to 3p25 and exists as a single copy in the haploid genome. 9 refs., 2 figs.

Simmons, C.F. Jr.; Clancy, T.E.; Quan, R. [Children`s Hospital, Boston, MA (United States)] [and others] [Children`s Hospital, Boston, MA (United States); and others

1995-04-10

243

Chromosomal mapping of 18S-28S and 5S rRNA genes by two-colour fluorescent in situ hybridization in six sturgeon species.  

PubMed

The number and distribution of the 18S-28S and 5S rRNA (rDNA) gene sequences were examined on mitotic chromosomes of six sturgeon species by two-colour in situ hybridization. Four of the six species, Huso huso, Acipenser stellatus, Acipenser sturio, and Acipenser ruthenus, with about 120 chromosomes, showed from six to eight 18S-28S rDNA signals, while 5S rDNA signals were on only one chromosome pair. The two species with 250-270 chromosomes, Acipenser baerii and Acipenser transmontanus, showed from 10 to 12 18S-28S sites and two chromosome pairs bearing 5S rDNA signals. In all examined species, the rather intense 5S rDNA signals apparently overlapped those of 18S-28S rDNA. These data support the diploid-tetraploid relationships between the two chromosome groups of sturgeons. The close association between the two rDNA families in species belonging to an ancestral fish order, such as Acipenseriformes, supports the hypothesis that the association represents a primitive condition. PMID:12834065

Fontana, Francesco; Lanfredi, Massimo; Congiu, Leonardo; Leis, Marilena; Chicca, Milvia; Rossi, Remigio

2003-06-01

244

Human chromosome 3: high-resolution fluorescence in situ hybridization mapping of 40 unique Not l linking clones homologous to genes and cDNAs  

Microsoft Academic Search

Forty newNotl linking clones representing sequence tagged sites (STSs) were mapped by fluorescencein situ hybridization (FISH) to different regions of human chromosome 3 (HSA3). Clone NL1-245, containing human aminoacylase 1, was localized to 3p21.2–p21.1. Our previous localization of the CLC-2 chloride channel protein gene was refined to 3q27. Clone NL2-316 most likely contains a translocon-associated protein -subunit gene and was

A. I. Protopopov; R. Z. Gizatullin; N. V. Vorobieva; M. V. Protopopova; C. Kiss; V. I. Kashuba; G. Klein; L. L. Kisselev; A. S. Graphodatsky; E. R. Zabarovsky

1996-01-01

245

Comparative fluorescence in situ hybridization mapping of a 431-kb Arabidopsis thaliana bacterial artificial chromosome contig reveals the role of chromosomal duplications in the expansion of the Brassica rapa genome.  

PubMed Central

Comparative genome studies are important contributors to our understanding of genome evolution. Most comparative genome studies in plants have been based on genetic mapping of homologous DNA loci in different genomes. Large-scale comparative physical mapping has been hindered by the lack of efficient and affordable techniques. We report here the adaptation of fluorescence in situ hybridization (FISH) techniques for comparative physical mapping between Arabidopsis thaliana and Brassica rapa. A set of six bacterial artificial chromosomes (BACs) representing a 431-kb contiguous region of chromosome 2 of A. thaliana was mapped on both chromosomes and DNA fibers of B. rapa. This DNA fragment has a single location in the A. thaliana genome, but hybridized to four to six B. rapa chromosomes, indicating multiple duplications in the B. rapa genome. The sizes of the fiber-FISH signals from the same BACs were not longer in B. rapa than those in A. thaliana, suggesting that this genomic region is duplicated but not expanded in the B. rapa genome. The comparative fiber-FISH mapping results support that chromosomal duplications, rather than regional expansion due to accumulation of repetitive sequences in the intergenic regions, played the major role in the evolution of the B. rapa genome. PMID:11014828

Jackson, S A; Cheng, Z; Wang, M L; Goodman, H M; Jiang, J

2000-01-01

246

Characterization of the temporal persistence of chromosomal abnormalities in the semen of Hodkin`s disease patients after treatment with NOVP chemotherapy using multi-chromosome fluorescence in situ hybridization  

SciTech Connect

Three-chromosome fluorescence in situ hybridization (FISH) was applied to sperm of men with Hodgkin`s disease to measure the persistence of chromosomally abnormal sperm within the time interval of 3 to 33 months after the end of treatment. NOVP chemotherapy includes the agents novantrone, oncovin, vinblastine, and prednisone, two of which are spindle poisons expected to induce aneuploidy. Semen samples were evaluated for the frequencies of fluorescence phenotypes representing hyperhaploidy, hypohaploidy, and genomic duplications using DNA probes specific for repetitive sequences on chromosomes X,Y, and 8. Using this procedure, NOVP was previously shown to induce chromosomally abnormal sperm in treated patients. In a longitudinal assessment of 11 semen samples from 2 men, frequencies of abnormal sperm appeared to return to pre-treatment levels at {approximately}6 months after the end of treatment and remained at these levels up to 33 months after the end of treatment. However, pre-treatment frequencies of chromosomally abnormal cells in Hodgkin`s patients were elevated above those found in normal healthy men. Additional patients are being evaluated to determine how long after therapy Hodgkin`s disease patients remain at increased risk for producing chromosomally abnormal sperm.

Cassel, M.J.; Robbins, W.A.; Wyrobek, A.J. [Lawrence Livermore National Laboratory, CA (United States); Meistrich, M.L. [Univ. of Texas, Houston, TX (United States)

1994-12-31

247

High-Resolution Mapping of Human Chromosome 11 by in Situ Hybridization with Cosmid Clones  

Microsoft Academic Search

Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by

Peter Lichter; Chieh-Ju Chang Tang; Katherine Call; Gary Hermanson; Glen A. Evans; David Housman; David C. Ward

1990-01-01

248

Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization  

Microsoft Academic Search

Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum ×

H. M. Thomas; W. G. Morgan; M. R. Meredith; M. W. Humphreys; J. M. Leggett

1994-01-01

249

Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation  

NASA Astrophysics Data System (ADS)

Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) represents a potential tool for the in-situ characterization of bioengineered tissues. In this study, we evaluate the application of TR-LIFS to non-intrusive monitoring of matrix composition during osteogenetic differentiation. Human adipose-derived stem cells, harvested from 3 patients, were induced in osteogenic media for 3, 5, and 7 weeks. Samples were subsequently collected and probed for time-resolved fluorescence emission with a pulsed nitrogen laser. Fluorescence parameters, derived from both spectral- and time-domain, were used for sample characterization. The samples were further analyzed using Western blot analysis and computer-based densitometry. A significant change in the fluorescence parameters was detected for samples beyond 3 weeks of osteogenic differentiation. The spectroscopic observations: 1) show increase of collagen I when contrasted against the time-resolved fluorescence spectra of commercially available collagens; and 2) are in agreement with Western blot analysis that demonstrated significant increase in collagen I content between 3- vs. 5-weeks and 3- vs. 7-weeks and no changes for collagens III, IV, and V. Our results suggest that TR-LIFS can be used as a non-invasive means for the detection of specific collagens in maturing connective tissues.

Marcu, Laura; Elbarbary, Amir; Zuk, Patricia; De Ugarte, Daniel A.; Benhaim, Prosper; Kurt, Hamza; Hedrick, Marc H.; Ashjian, Peter

2003-07-01

250

In situ Measurements of Phytoplankton Fluorescence Using Low Cost Electronics  

PubMed Central

Chlorophyll a fluorometry has long been used as a method to study phytoplankton in the ocean. In situ fluorometry is used frequently in oceanography to provide depth-resolved estimates of phytoplankton biomass. However, the high price of commercially manufactured in situ fluorometers has made them unavailable to some individuals and institutions. Presented here is an investigation into building an in situ fluorometer using low cost electronics. The goal was to construct an easily reproducible in situ fluorometer from simple and widely available electronic components. The simplicity and modest cost of the sensor makes it valuable to students and professionals alike. Open source sharing of architecture and software will allow students to reconstruct and customize the sensor on a small budget. Research applications that require numerous in situ fluorometers or expendable fluorometers can also benefit from this study. The sensor costs US$150.00 and can be constructed with little to no previous experience. The sensor uses a blue LED to excite chlorophyll a and measures fluorescence using a silicon photodiode. The sensor is controlled by an Arduino microcontroller that also serves as a data logger. PMID:23783738

Leeuw, Thomas; Boss, Emmanuel S.; Wright, Dana L.

2013-01-01

251

In situ measurements of phytoplankton fluorescence using low cost electronics.  

PubMed

Chlorophyll a fluorometry has long been used as a method to study phytoplankton in the ocean. In situ fluorometry is used frequently in oceanography to provide depth-resolved estimates of phytoplankton biomass. However, the high price of commercially manufactured in situ fluorometers has made them unavailable to some individuals and institutions. Presented here is an investigation into building an in situ fluorometer using low cost electronics. The goal was to construct an easily reproducible in situ fluorometer from simple and widely available electronic components. The simplicity and modest cost of the sensor makes it valuable to students and professionals alike. Open source sharing of architecture and software will allow students to reconstruct and customize the sensor on a small budget. Research applications that require numerous in situ fluorometers or expendable fluorometers can also benefit from this study. The sensor costs US$150.00 and can be constructed with little to no previous experience. The sensor uses a blue LED to excite chlorophyll a and measures fluorescence using a silicon photodiode. The sensor is controlled by an Arduino microcontroller that also serves as a data logger. PMID:23783738

Leeuw, Thomas; Boss, Emmanuel S; Wright, Dana L

2013-01-01

252

Fluorescence in situ hybridization analysis using PAX8- and PPARG-specific probes reveals the presence of PAX8-PPARG translocation and 3p25 aneusomy in follicular thyroid neoplasms.  

PubMed

At the present time, the differentiation between follicular thyroid carcinoma (FTC) and adenoma can be made only postoperatively and is based on the presence of capsular or vascular invasion. The ability to differentiate preoperatively between the malignant and benign forms of follicular thyroid tumors assumes greater importance in any clinical setting. The PAX8-PPARG translocation has been reported to occur in the majority of FTC. In this study, a group of 60 follicular thyroid neoplasms [18 FTC, 1 Hurthle cell carcinoma (HCC), 24 follicular thyroid adenomas (FTA), 5 Hurthle cell adenomas (HCA), and 12 follicular variants of papillary thyroid carcinomas (FV-PTC)] were analyzed to determine the prevalence of the PAX8-PPARG translocation by fluorescence in situ hybridization. The PAX8-PPARG translocation was detected in 2/18 FTC (11.1%). In addition, 2/18 (11.1%) FTC and 1/5 (20%) HCA showed 3p25 aneusomy only. The frequency of the translocation detected in the study was lower compared to the earlier studies conducted in Western countries. This might be attributed to the ethnic background and geographic location. Detection of either the PAX8-PPARG translocation or the 3p25 aneusomy in FTC indicates that these are independent genetic events. It is hereby concluded that 3p25 aneusomy or PAX8-PPARG translocation may play an important role in the molecular pathogenesis of follicular thyroid tumors. PMID:19963130

Chia, Wai Kit; Sharifah, Noor Akmal; Reena, Rahayu Md Zin; Zubaidah, Zakaria; Clarence-Ko, Ching Huat; Rohaizak, Muhammad; Naqiyah, Ibrahim; Srijit, Das; Hisham, Abdullah Nor; Asmiati, Arbi; Rafie, Md Kaslan

2010-01-01

253

Green Fluorescent Protein as a Visual Marker in Somatic Hybridization  

PubMed Central

Using a transgenic citrus plant expressing Green Fluorescent Protein (GFP) as a parent in somatic fusion experiments, we investigated the suitability of GFP as an in vivo marker to follow the processes of protoplast fusion, regeneration and selection of hybrid plants. A high level of GFP expression was detected in transgenic citrus protoplasts, hybrid callus, embryos and plants. It is demonstrated that GFP can be used for the continuous monitoring of the fusion process, localization of hybrid colonies and callus, and selection of somatic hybrid embryos and plants. PMID:12096810

OLIVARES?FUSTER, O.; PEÑA, L.; DURAN?VILA, N.; NAVARRO, L.

2002-01-01

254

A Novel Three-Colour Fluorescence in Situ Hybridization Approach for the Detection of t(7;12)(q36;p13) in Acute Myeloid Leukaemia Reveals New Cryptic Three Way Translocation t(7;12;16)  

PubMed Central

The t(7;12)(q36;p13) translocation is a recurrent chromosome abnormality that involves the ETV6 gene on chromosome 12 and has been identified in 20–30% of infant patients with acute myeloid leukaemia (AML). The detection of t(7;12) rearrangements relies on the use of fluorescence in situ hybridization (FISH) because this translocation is hardly visible by chromosome banding methods. Furthermore, a fusion transcript HLXB9-ETV6 is found in approximately 50% of t(7;12) cases, making the reverse transcription PCR approach not an ideal screening method. Considering the report of few cases of variant translocations harbouring a cryptic t(7;12) rearrangement, we believe that the actual incidence of this abnormality is higher than reported to date. The clinical outcome of t(7;12) patients is believed to be poor, therefore an early and accurate diagnosis is important in the clinical management and treatment. In this study, we have designed and tested a novel three-colour FISH approach that enabled us not only to confirm the presence of the t(7;12) in a number of patients studied previously, but also to identify a cryptic t(7;12) as part of a complex rearrangement. This new approach has proven to be an efficient and reliable method to be used in the diagnostic setting. PMID:24216708

Naiel, Abdulbasit; Vetter, Michael; Plekhanova, Olga; Fleischman, Elena; Sokova, Olga; Tsaur, Grigory; Harbott, Jochen; Tosi, Sabrina

2013-01-01

255

Fluorescence In Situ Hybridization Identifies Cryptic t(16;16)(p13;q22) Masked By del(16)(q22) in a Case of AML-M4 Eo  

PubMed Central

We report a patient presenting with acute myeloid leukemia (AML)-M4 Eo, in whom conventional cytogenetic analysis revealed a 46, XY, del(16)(q22) karyotype. Molecular analysis of the bone marrow cells using reverse transcriptase polymerase chain reaction (RT-PCR) identified a CBF?-MYH11, “type A” fusion transcript. However, despite a thorough reevaluation, a balanced chromosome 16 abnormality could not be definitively identified by cytogenetics. Since there exists a small possibility of obtaining a false-positive PCR result, fluorescence in situ hybridization (FISH) analysis using dual-color, break-apart probes for CBF? was performed to elucidate the mechanism of fusion gene formation and thus confirm the RT-PCR results. FISH analysis clearly revealed a cryptic t(16;16), which was probably masked by the del(16)(q22). FISH is the preferred diagnostic procedure to elucidate the CBF?-MYH11 fusion in this situation, and resolves the possibility of both false-positive and false-negative results with RT-PCR technique. Due to the improved prognosis of AML associated with the CBF?-MYH11 fusion compared to AML generally, we recommend the use of FISH for detection of inv(16)/t(16;16)/CBF?-MYH11 in patients with failed, complex, or apparently normal cytogenetics, and in whom the cell morphology indicates the strong possibility of this gene fusion. PMID:15269306

Merchant, Shakil H.; Haines, Skip; Hall, Bryan; Hozier, John; Viswanatha, David S.

2004-01-01

256

The Importance of 10q Status in an Outcomes-Based Comparison Between 1p/19q Fluorescence In Situ Hybridization and PCR-Based Microsatellite Loss of Heterozygosity Analysis of Oligodendrogliomas  

PubMed Central

1p/19q codeletion is a favorable prognostic marker of oligodendrogliomas. While fluorescence in situ hybridization (FISH) and microsatellite-based polymerase chain reaction (PCR) for loss of heterozygosity (LOH) are common methods to test for 1p/19q codeletion, it is unclear which test is better at prognostic stratification. This study analyzed outcomes of 111 oligodendrogliomas with both 1p/19q FISH and LOH done at the time of diagnosis. Overall concordance between the 2 assays was 81.1%. In grade III oligodendrogliomas, LOH was better than FISH at survival stratification (p < 0.0001 for LOH vs. p = 0.02 for FISH), although increasing the stringency of FISH interpretation criteria improved concordance and prognostic power. Oligodendrogliomas that were 1p/19q-codeleted by FISH but also had 10q LOH were negative for 1p/19q codeletion by PCR analysis in over 70% of cases, with very poor survival in the grade III subset. Thus, although PCR-based LOH is a better stratifier of 1p/19q status, FISH still has clinical and prognostic utility, especially if 10q data can be incorporated. PMID:22157622

Horbinski, Craig; Nikiforova, Marina N.; Hobbs, Jonathan; Cieply, Kathleen; Dacic, Sanja; Hamilton, Ronald L.

2011-01-01

257

Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris  

PubMed Central

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 106 cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 104 type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 106 type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 106 cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog. PMID:11571193

Dedysh, Svetlana N.; Derakshani, Manigee; Liesack, Werner

2001-01-01

258

Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization  

Microsoft Academic Search

An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluores- cently labeled decorator probes.

Irina Smolina; Charles Lee; Maxim Frank-Kamenetskii

2007-01-01

259

A new animal model for implant-related infected non-unions after intramedullary fixation of the tibia in rats with fluorescent in situ hybridization of bacteria in bone infection  

Microsoft Academic Search

There is no adequate animal model to mimic the difficult clinical situation of infected non-union of the tibia after intramedullary stabilization. The purpose was to establish an animal model of implant-related infected non-unions of the tibia in rats. Furthermore, it was evaluated if detection of bacteria by fluorescent in situ hybridisation (FISH) technique is possible in bone infection.17 rats were

Volker Alt; Katrin S. Lips; Christoph Henkenbehrens; Dominik Muhrer; Marcia Cavalcanti-Garcia; Ursula Sommer; Ulrich Thormann; Gabor Szalay; Christian Heiss; Theodoros Pavlidis; Eugen Domann; Reinhard Schnettler

2011-01-01

260

A Transgenomic Cytogenetic Sorghum (Sorghum propinquum) Bacterial Artificial Chromosome Fluorescence in Situ Hybridization Map of Maize (Zea mays L.) Pachytene Chromosome 9, Evidence for Regions of Genome Hyperexpansion  

PubMed Central

A cytogenetic FISH map of maize pachytene-stage chromosome 9 was produced with 32 maize marker-selected sorghum BACs as probes. The genetically mapped markers used are distributed along the linkage maps at an average spacing of 5 cM. Each locus was mapped by means of multicolor direct FISH with a fluorescently labeled probe mix containing a whole-chromosome paint, a single sorghum BAC clone, and the centromeric sequence, CentC. A maize-chromosome-addition line of oat was used for bright unambiguous identification of the maize 9 fiber within pachytene chromosome spreads. The locations of the sorghum BAC–FISH signals were determined, and each new cytogenetic locus was assigned a centiMcClintock position on the short (9S) or long (9L) arm. Nearly all of the markers appeared in the same order on linkage and cytogenetic maps but at different relative positions on the two. The CentC FISH signal was localized between cdo17 (at 9L.03) and tda66 (at 9S.03). Several regions of genome hyperexpansion on maize chromosome 9 were found by comparative analysis of relative marker spacing in maize and sorghum. This transgenomic cytogenetic FISH map creates anchors between various maps of maize and sorghum and creates additional tools and information for understanding the structure and evolution of the maize genome. PMID:17947405

Amarillo, F. Ina E.; Bass, Hank W.

2007-01-01

261

PEMERIKSAAN ABERASI KROMOSOM STABIL DENGAN TEHNIK FLUORESENCE IN SITU HYBRIDIZATION  

Microsoft Academic Search

MEASUREMENT OF STABLE CHROMOSOME ABERRATIONS BY FLUORESENCE IN SITU HYBRIDIZATION TECHNIQUE. Measurement of translocation as stable chromosome aberrations becomes a very important tool to detect cytogenetic damages in lymphocytes due to radiation exposure in prediction and assessment of immediate and late radiation effects. Translocation is also considered as optimum cytogeneric parameter for long-term retrospective biodosimetry. The aim of this study

Zubaidah Alatas; Pusat Teknologi

262

ALK Rearrangement in a Large Series of Consecutive Non-Small Cell Lung Cancers: Comparison Between a New Immunohistochemical Approach and Fluorescence In Situ Hybridization for the Screening of Patients Eligible for Crizotinib Treatment.  

PubMed

Context .- Echinoderm microtubule associated proteinlike 4-anaplastic lymphoma receptor tyrosine kinase (EML4-ALK) translocation has been described in a subset of patients with non-small cell lung cancer (NSCLC) and has been shown to have oncogenic activity. Fluorescence in situ hybridization (FISH) is used to detect ALK-positive NSCLC, but it is expensive, time-consuming, and difficult for routine application. Objective .- To evaluate the potential role of immunohistochemistry (IHC) as a screening tool to identify candidate cases for FISH analysis and for ALK inhibitor therapy in NSCLC. Design .- We performed FISH and IHC for ALK and mutational analysis for epidermal growth factor receptor (EGFR) and KRAS in 523 NSCLC specimens. We conducted IHC analysis with the monoclonal antibody D5F3 (Ventana Medical Systems, Tucson, Arizona) and a highly sensitive detection system. We also performed a MassARRAY-based analysis (Sequenom, San Diego, California) in a small subset of 11 samples to detect EML4-ALK rearrangement. Results .- Of the 523 NSCLC specimens, 20 (3.8%) were positive for ALK rearrangement by FISH analysis. EGFR and KRAS mutations were identified in 70 (13.4%) and 124 (23.7%) of the 523 tumor samples, respectively. ALK rearrangement and EGFR and KRAS mutations were mutually exclusive. Of 523 tumor samples analyzed, 18 (3.4%) were ALK(+) by IHC, 18 samples (3.4%) had concordant IHC and FISH results, and 2 ALK(+) cases (0.3%) by FISH failed to show ALK protein expression. In the 2 discrepant cases, we did not detect any mass peaks for the EML4-ALK variants by MassARRAY. Conclusions .- Our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis. PMID:24885803

Alì, Greta; Proietti, Agnese; Pelliccioni, Serena; Niccoli, Cristina; Lupi, Cristiana; Sensi, Elisa; Giannini, Riccardo; Borrelli, Nicla; Menghi, Maura; Chella, Antonio; Ribechini, Alessandro; Cappuzzo, Federico; Melfi, Franca; Lucchi, Marco; Mussi, Alfredo; Fontanini, Gabriella

2014-11-01

263

Dewaxing and Rehydrating Sections prior to In Situ Hybridization.  

PubMed

INTRODUCTIONThis protocol describes how to remove wax from embryo or tissue sections that have been affixed to glass slides. Traditionally, xylene has been used for this purpose, but less toxic solutions can also be employed. The embryo or tissue sections are then progressively rehydrated for compatibility with subsequent alcohol stains, aqueous stains, immunohistochemistry, or in situ hybridization. PMID:21356973

Nagy, Andras; Gertsenstein, Marina; Vintersten, Kristina; Behringer, Richard

2007-01-01

264

Cytogenetic Analysis Using Quantitative, High-Sensitivity, Fluorescence Hybridization  

Microsoft Academic Search

This report describes the use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations. Biotin-labeled DNA was hybridized to target chromosomes and subsequently rendered fluorescent by successive treatments with fluorescein-labeled avidin and biotinylated anti-avidin antibody. Human chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic

D. Pinkel; T. Straume; J. W. Gray

1986-01-01

265

In situ changes in native fluorescence from Bacillus subtilis during  

Microsoft Academic Search

Native fluorescence emission and excitation spectra were used to monitor changes in Bacillus subtilis (Bs) and Staphylococcus aureus (Sa) subjected to starvation conditions. Initially, the fluorescence spectra from the Bs and Sa was dominated by tryptophan emission. After the second day, a fluorescence band with an emission peak at 410 nm and an excitation peak at 345 nm appeared in

Alvin Katz; Alexandra Alimova; Glenn Minko; Howard E. Savage; Daniel V. Will; Mahendra Shah; Richard B. Rosen; Robert R. Alfano

2003-01-01

266

In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes  

PubMed Central

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH. PMID:10388720

Okabe, Satoshi; Satoh, Hisashi; Watanabe, Yoshimasa

1999-01-01

267

Rapid Discrimination of Haemophilus influenzae, H. parainfluenzae, and H. haemolyticus by Fluorescence In Situ Hybridization (FISH) and Two Matrix-Assisted Laser-Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Platforms  

PubMed Central

Background Due to considerable differences in pathogenicity, Haemophilus influenzae, H. parainfluenzae and H. haemolyticus have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal H. haemolyticus as H. influenzae. In a multi-center approach, we assessed the suitability of fluorescence in situ hybridization (FISH) and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) for the identification of H. influenzae, H. parainfluenzae and H. haemolyticus to species level. Methodology A strain collection of 84 Haemophilus spp. comprising 50 H. influenzae, 25 H. parainfluenzae, 7 H. haemolyticus, and 2 H. parahaemolyticus including 77 clinical isolates was analyzed by FISH with newly designed DNA probes, and two different MALDI-TOF-MS systems (Bruker, Shimadzu) with and without prior formic acid extraction. Principal Findings Among the 84 Haemophilus strains analyzed, FISH led to 71 correct results (85%), 13 uninterpretable results (15%), and no misidentifications. Shimadzu MALDI-TOF-MS resulted in 59 correct identifications (70%), 19 uninterpretable results (23%), and 6 misidentifications (7%), using colony material applied directly. Bruker MALDI-TOF-MS with prior formic acid extraction led to 74 correct results (88%), 4 uninterpretable results (5%) and 6 misidentifications (7%). The Bruker MALDI-TOF-MS misidentifications could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the system's database. In conclusion, no analyzed diagnostic procedure was free of errors. Diagnostic results have to be interpreted carefully and alternative tests should be applied in case of ambiguous test results on isolates from seriously ill patients. PMID:23646201

Frickmann, Hagen; Christner, Martin; Donat, Martina; Berger, Anja; Essig, Andreas; Podbielski, Andreas; Hagen, Ralf Matthias; Poppert, Sven

2013-01-01

268

Allelic Loss at SMAD4 in Polyps from Juvenile Polyposis Patients and Use of Fluorescence in Situ Hybridization to Demonstrate Clonal Origin of the Epithelium  

Microsoft Academic Search

Juvenile polyposis syndrome (JPS; Online Mendelian Inheritance in Man2 174900) is a rare Mendelian disorder in which individuals have typical hamartomatous polyps within the gastrointestinal tract. The stromal element of the polyps has classically been thought to be the proliferative component, although epithelial malignancies (largely gastro- intestinal cancers) occur more frequently than expected in JPS patients. Germ-line mutations in SMAD4

Kelly Woodford-Richens; Jill Williamson; Stephen Bevan; Joanne Young; Barbara Leggett; Ian Frayling; Yi Thway; Shirley Hodgson; Jin Cheon Kim; Takeo Iwama; Marco Novelli; Denise Sheer; Richard Poulsom; Nicholas Wright; Richard Houlston; Ian Tomlinson

269

The use of fluorescence in situ hybridization in the diagnosis of hidden mosaicism: apropos of three cases of sex chromosome anomalies.  

PubMed

FISH has been used as a complement to classical cytogenetics in the detection of mosaicism in sex chromosome anomalies. The aim of this study is to describe three cases in which the final diagnosis could only be achieved by FISH. Case 1 was an 8-year-old 46,XY girl with normal female genitalia referred to our service because of short stature. FISH analysis of lymphocytes with probes for the X and Y centromeres identified a 45,X/46,X,idic(Y) constitution, and established the diagnosis of Turner syndrome. Case 2 was a 21-month-old 46,XY boy with genital ambiguity (penile hypospadias, right testis, and left streak gonad). FISH analysis of lymphocytes and buccal smear identified a 45,X/46,XY karyotype, leading to diagnosis of mixed gonadal dysgenesis. Case 3 was a 47,XYY 19-year-old boy with delayed neuromotor development, learning disabilities, psychological problems, tall stature, small testes, elevated gonadotropins, and azoospermia. FISH analysis of lymphocytes and buccal smear identified a 47,XYY/48,XXYY constitution. Cases 1 and 2 illustrate the phenotypic variability of the 45,X/46,XY mosaicism, and the importance of detection of the 45,X cell line for proper management and follow-up. In case 3, abnormal gonadal function could be explained by the 48,XXYY cell line. The use of FISH in clinical practice is particularly relevant when classical cytogenetic analysis yields normal or uncertain results in patients with features of sex chromosome aneuploidy. PMID:23295296

Maciel-Guerra, Andréa Trevas; Paulo, Juliana De; Santos, Ana Paula; Guaragna-Filho, Guilherme; Andrade, Juliana Gabriel Ribeiro; Siviero-Miachon, Adriana Aparecida; Spinola-Castro, Angela Maria; Guerra-Júnior, Gil

2012-11-01

270

Analysis of HER2 status in breast carcinoma by fully automated HER2 fluorescence in situ hybridization (FISH): comparison of two immunohistochemical tests and manual FISH.  

PubMed

Easy and accurate HER2 testing is essential when considering the prognostic and predictive significance of HER2 in breast cancer. The use of a fully automated, quantitative FISH assay would be helpful to detect HER2 amplification in breast cancer tissue specimens with reduced inter-laboratory variability. We compared the concordance of HER2 status as assessed by an automated FISH staining system to manual FISH testing. Using 60 formalin-fixed paraffin-embedded breast carcinoma specimens, we assessed HER2 immunoexpression with two antibodies (DAKO HercepTest and CB11). In addition, HER2 status was evaluated with automated FISH using the Leica FISH System for BOND and a manual FISH using the Abbott PathVysion DNA Probe Kit. All but one specimen were successfully stained using both FISH methods. When the data were divided into two groups according to HER2/CEP17 ratio, positive and negative, the results from both the automated and manual FISH techniques were identical for all 59 evaluable specimens. The HER2 and CEP17 copy numbers and HER2/CEP17 ratio showed great agreement between both FISH methods. The automated FISH technique was interpretable with signal intensity similar to those of the manual FISH technique. In contrast with manual FISH, the automated FISH technique showed well-preserved architecture due to low membrane digestion. HER2 immunohistochemistry and FISH results showed substantial significant agreement (? = 1.0, p < 0.001). HER2 status can be reliably determined using a fully automated HER2 FISH system with high concordance to the well-established manual FISH method. Because of stable signal intensity and high staining quality, the automated FISH technique may be more appropriate than manual FISH for routine applications. PMID:24372629

Yoon, Nara; Do, In-Gu; Cho, Eun Yoon

2014-09-01

271

Quantitative Use of Fluorescent In Situ Hybridization To Examine Relationships between Mycolic AcidContaining Actinomycetes and Foaming in Activated Sludge Plants  

Microsoft Academic Search

The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acid- containing actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata,

RUSSELL J. DAVENPORT; THOMAS P. CURTIS; MICHAEL GOODFELLOW; FIONA M. STAINSBY; MARC BINGLEY

2000-01-01

272

Assignment of the human angiotensin II type 2 receptor gene (AGTR2) to chromosome Xq22-q23 by fluorescence in situ hybridization  

SciTech Connect

Angiotensin II (AII), the biologically active effector of the renin-angiotensin system, is a major regulator of blood pressure and electrolyte balance and a growth factor for diverse cell types. AII exerts its physiological effects by interacting with two pharmacologically distinct subtypes of receptors, designated AT{sub 1}, and AT{sub 2}. Most of the known responses to AII are mediated by the AT{sub 1} subtype, whereas the function of the AT{sub 2} receptor remains largely unknown. AT{sub 2} receptor expression is abundant in particular tissues such as adrenal medulla, specific brain regions, uterine myometrium, and ovarian granuloma cells. This specific localization in adult coupled to the demonstration that some actions of AII such as secretion of luteinizing hormone and prolactine, dilation of brain arterioles, or drinking response in rats can be inhibited in vitro by an AT{sub 2} receptor antagonist suggests that the AT{sub 2} subtype may play a role in neuronal and reproductive function. In addition, a growing amount of evidence indicates that the AT{sub 2} receptor may play a most important role in processes involving cellular growth and differentiation. It is abundantly and widely expressed in the mesenchymal tissues of the developing fetus and in the immature brain and is up-regulated in the heart and in vascular smooth muscle cells in the first days following birth. Moreover, AT{sub 2} receptor expression is enhanced in the adult in wound healing, in the neointima of injured vessels, and in pheochromocytoma. 12 refs., 1 fig.

Chassagne, C.; Meloche, S. [Hotel-Dieu de Montreal, Quebec (Canada)] [Hotel-Dieu de Montreal, Quebec (Canada); Beatty, B.G. [Hospital for Sick Children, Toronto, Ontario (Canada)] [Hospital for Sick Children, Toronto, Ontario (Canada)

1995-01-20

273

Cytogenetic Characterization and Fluorescence in situ Hybridization of (GATA)10 Repeats on Established Primary Cell Cultures from Indian Water Snake (Natrix piscator) and Indian Mugger (Crocodylus palustris) Embryos  

Microsoft Academic Search

Sex determination among reptiles has continued to draw the attention of geneticists and the mechanisms involved have been extensively studied and documented in the past 3 decades. The setting up of primary cell lines of reptilian tissues is an important tool in the present study which is a unique aspect not applied in earlier studies. Establishing the cell lines from

L. Rao; R. Turlapati; M. Patel; B. Panda; D. Tosh; S. Mangalipalli; A. Tiwari; V. P. Orunganti; D. Rose; A. Anand; M. K. Kulashekaran; S. R. Priya; R. K. Mishra; K. Majumdar; R. K. Aggarwal; L. Singh

2009-01-01

274

Fluorescence in situ hybridization of uncultured zoosporic fungi: Testing with clone-FISH and application to freshwater samples using CARD-FISH  

Microsoft Academic Search

Recently, molecular environmental surveys of the eukaryotic microbial community in lakes have revealed a high diversity of sequences belonging to uncultured zoosporic fungi commonly known as chytrids. These microorganisms have two different stages in their life cycle and are known as algal parasites (i.e. host-attached infective sporangia) and as food sources for zooplankton (i.e. free-living zooflagellate propagules) in aquatic systems.

Marlène Jobard; Serena Rasconi; Télesphore Sime-Ngando

2010-01-01

275

Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography  

Microsoft Academic Search

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). ( 13 C)acetate was used in SIP to label the DNA of the denitrifiers. The ( 13 C)DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13 C library

Maneesha P. Ginige; Jurg Keller; Linda L. Blackall

2005-01-01

276

Human 60-kDa SS-A/Ro ribonucleoprotein autoantigen gene (SSA2) localized to 1q31 by fluorescence in situ hybridization  

SciTech Connect

Human autoantibodies to intracellular antigens are often found in patients with systemic rheumatic diseases. Our laboratory and others have reported the cloning of the cDNAs for two protein autoantigens. The 60-kDa protein is a member of the RNA binding protein family containing an RNA binding motif and has been shown to be primarily responsible for direct interaction with hY-RNAs. The assignment of the 60-kDa autoantigen gene (SSA2) to 1q31 may allow further investigation of the genetic basis of human autoimmune diseases in which ss-A/Ro antibodies are observed. 14 refs., 1 fig.

Chan, E.K.L.; Tan, E.M. [Scripps Research Institute, La Jolla, CA (United States)] [Scripps Research Institute, La Jolla, CA (United States); Ward, D.C. [Yale Univ. School of Medicine, New Haven, CT (United States)] [and others] [Yale Univ. School of Medicine, New Haven, CT (United States); and others

1994-09-01

277

Multicenter Evaluation of a Candida albicans Peptide Nucleic Acid Fluorescent In Situ Hybridization Probe for Characterization of Yeast Isolates from Blood Cultures  

Microsoft Academic Search

Yeasts, long known to cause thrush and vulvovaginal infec- tions, have emerged as a significant cause of nosocomial infec- tions (18, 21). Although Candida albicans usually is the most frequently isolated organism from patients with fungemia, other species of Candida are also important pathogens in hos- pitalized patients (18, 21). Furthermore, some of the Candida species other than C. albicans

D. A. Wilson; M. J. Joyce; L. S. Hall; L. B. Reller; G. D. Roberts; G. S. Hall; B. D. Alexander; G. W. Procop

2005-01-01

278

Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry  

PubMed Central

Background Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples. Methods Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system. Results All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. Conclusions The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations. PMID:25248157

Conde, Esther; Suarez-Gauthier, Ana; Benito, Amparo; Garrido, Pilar; Garcia-Campelo, Rosario; Biscuola, Michele; Paz-Ares, Luis; Hardisson, David; de Castro, Javier; Camacho, M. Carmen; Rodriguez-Abreu, Delvys; Abdulkader, Ihab; Ramirez, Josep; Reguart, Noemi; Salido, Marta; Pijuan, Lara; Arriola, Edurne; Sanz, Julian; Folgueras, Victoria; Villanueva, Noemi; Gomez-Roman, Javier; Hidalgo, Manuel; Lopez-Rios, Fernando

2014-01-01

279

Differential diagnosis of cyclin D2+ mantle cell lymphoma based on fluorescence in situ hybridization and quantitative real-time-PCR  

PubMed Central

Mantle cell lymphoma is characterized by the t(11;14) chromosomal translocation, resulting in the overexpression of cyclin D1 (CycD1). Recently, cases of mantle cell lymphoma negative for cycD1 but positive for cycD2 or cycD3 were identified by gene expression profiling and confirmed by immunohistochemistry. We analyzed 4 cases of cycD2+ mantle cell lymphoma with a translocation involving the CCND2 locus, and its differential diagnosis from 35 mature B-cell non-Hodgkin’s lymphomas based on immunohistochemistry, quantitative RT-PCR and FISH analysis. Bona fide cycD2+ mantle cell lymphoma carried translocations involving the CCND2 gene, and IGH and IGK loci were identified as partners. As a result of this translocation, cycD2 mRNA was highly over-expressed when compared with normal lymphoid tissue and other B-cell non-Hodgkin’s lymphomas, including chronic lymphocytic leukemia, making this technique ideally suited to identify cycD2+mantle cell lymphoma. In contrast, positive immunostaining for cycD2 was found in most B-cell non-Hodgkin’s lymphomas, and therefore, it is not specific for a diagnosis of cycD2+mantle cell lymphoma. PMID:19608671

Quintanilla-Martinez, Leticia; Slotta-Huspenina, Julia; Koch, Ina; Klier, Margit; Hsi, Eric D.; de Leval, Laurence; Klapper, Wolfram; Gesk, Stefan; Siebert, Reiner; Fend, Falko

2009-01-01

280

Localization of the horse (Equus caballus) ?-globin gene complex to chromosome 13 by luorescence in situ hybridization  

Microsoft Academic Search

The ?-globin gene complex in Equus caballus has been mapped by fluorescence in situ hybridization to the telomeric region of the long arm of chromosome 13. This is the first equine gene to be mapped to this chromosome.Copyright © 1993 S. Karger AG, Basel

E. A. Oakenfull; V. J. Buckle; J. B. Clegg

1993-01-01

281

Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community  

PubMed Central

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO3?-N mg of mixed-liquor volatile suspended solids (MLVSS)?1 h?1 to a steady-state value of 0.06 mg of NO3?-N mg of MLVSS?1 h?1 over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [13C]methanol to biomark the DNA of the denitrifiers. The extracted [13C]DNA and [12C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [13C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [12C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [14C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges. PMID:14711691

Ginige, Maneesha P.; Hugenholtz, Philip; Daims, Holger; Wagner, Michael; Keller, Jurg; Blackall, Linda L.

2004-01-01

282

Membrane connectivity estimated by digital image analysis of HER2 immunohistochemistry is concordant with visual scoring and fluorescence in situ hybridization results: algorithm evaluation on breast cancer tissue microarrays  

PubMed Central

Introduction The human epidermal growth factor receptor 2 (HER2) is an established biomarker for management of patients with breast cancer. While conventional testing of HER2 protein expression is based on semi-quantitative visual scoring of the immunohistochemistry (IHC) result, efforts to reduce inter-observer variation and to produce continuous estimates of the IHC data are potentiated by digital image analysis technologies. Methods HER2 IHC was performed on the tissue microarrays (TMAs) of 195 patients with an early ductal carcinoma of the breast. Digital images of the IHC slides were obtained by Aperio ScanScope GL Slide Scanner. Membrane connectivity algorithm (HER2-CONNECT™, Visiopharm) was used for digital image analysis (DA). A pathologist evaluated the images on the screen twice (visual evaluations: VE1 and VE2). HER2 fluorescence in situ hybridization (FISH) was performed on the corresponding sections of the TMAs. The agreement between the IHC HER2 scores, obtained by VE1, VE2, and DA was tested for individual TMA spots and patient's maximum TMA spot values (VE1max, VE2max, DAmax). The latter were compared with the FISH data. Correlation of the continuous variable of the membrane connectivity estimate with the FISH data was tested. Results The pathologist intra-observer agreement (VE1 and VE2) on HER2 IHC score was almost perfect: kappa 0.91 (by spot) and 0.88 (by patient). The agreement between visual evaluation and digital image analysis was almost perfect at the spot level (kappa 0.86 and 0.87, with VE1 and VE2 respectively) and at the patient level (kappa 0.80 and 0.86, with VE1max and VE2max, respectively). The DA was more accurate than VE in detection of FISH-positive patients by recruiting 3 or 2 additional FISH-positive patients to the IHC score 2+ category from the IHC 0/1+ category by VE1max or VE2max, respectively. The DA continuous variable of the membrane connectivity correlated with the FISH data (HER2 and CEP17 copy numbers, and HER2/CEP17 ratio). Conclusion HER2 IHC digital image analysis based on membrane connectivity estimate was in almost perfect agreement with the visual evaluation of the pathologist and more accurate in detection of HER2 FISH-positive patients. Most immediate benefit of integrating the DA algorithm into the routine pathology HER2 testing may be obtained by alerting/reassuring pathologists of potentially misinterpreted IHC 0/1+ versus 2+ cases. PMID:21943197

2011-01-01

283

In situ hybridization analysis indicates that 4AL-5AL-7BS translocation preceded subspecies differentiation of Triticum turgidum.  

PubMed

The important cyclic translocation 4AL-5AL-7BS is an evolutionary signature of polyploidy in wheat. This study aimed to determine its distribution within the subspecies of Triticum turgidum L., using genomic in situ hybridization and fluorescence in situ hybridization. As it exists in all eight subspecies, this translocation appeared before the differentiation of the subspecies of T. turgidum. This translocation probably first appeared in T. turgidum subsp. dicoccoides and was then transmitted into the other subspecies. Its existence in all of the analyzed subspecies suggests that this translocation may confer an adaptive advantage during the course of evolution. PMID:23789999

Hao, Ming; Luo, Jiangtao; Zhang, Lianquan; Yuan, Zhongwei; Zheng, Youliang; Zhang, Huaigang; Liu, Dengcai

2013-05-01

284

Hybrid-type temperature sensor for in situ measurement  

SciTech Connect

A hybrid-type surface temperature sensor combines the contact and noncontact methods, which allows us to overcome the shortcomings of both methods. The hybrid-type surface thermometer is composed mainly of two components: a metal film sheet that makes contact with an object and a radiometer that is used to detect the radiance of the rear surface of the metal film, which is actually a modified radiation thermometer. Temperature measurement using the hybrid-type thermometer with a several tens micrometer thick Hastelloy sheet, a highly heat and corrosion resistant alloy, is possible with a systematic error of -0.5 K and random errors of {+-}0.5 K, in the temperature range from 900 to 1000 K. This thermometer provides a useful means for calibration of in situ temperature measurement in various processes, especially in the silicon semiconductor industry. This article introduces the basic idea of the hybrid-type surface sensor, presents experimental results and discussions, and finally describes some applications.

Iuchi, Tohru; Hiraka, Kensuke [Sensor Photonics Research Center, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585 (Japan)

2006-11-15

285

1Nonradioactive In Situ Hybridization in Atherosclerotic Tissue.  

PubMed

In situ hybridization (ISH) is a powerful and important technique that allows the detection and microscopic localization of nucleic acids within the specific cell, tissue, or chromosome of interest. In addition, it offers increased sensitivity over traditional filter hybridization, since low-copy mRNA molecules in individual cells can be detected. At the time the ISH technique was developed by Pardue and Gall (1), there were restrictions in it since radioisotopes were the only labels for nucleic acids available and autoradiographic film was the only detection system. Current molecular biological cloning techniques have now enabled most researchers to prepare almost any specific probe of choice and, more importantly, modern nonradioactive labels with colorimetric detection have removed all the limitations and restrictions of radioactive labels. The principal advantages of nonradioactive hybridization compared with isotopic hybridization are increased speed, greater resolution, lower costs, and reduced radioactive exposure. Furthermore, it allows the opportunity for combining different labels in one ISH experiment. The procedures behind ISH localization of DNA or RNA are very similar and may be summarized in five areas: (1) sample and glass slide preparation, including fixation, mounting, and ISH pretreatment, (2) probe preparation/labeling, (3) hybridization, (4) probe removal/washing, and (5) detection. Nonradioactive probe labeling itself can be divided into two methods, i.e., direct and indirect. This chapter describes the preparation of atherosclerotic tissue for ISH, indirect labeling of probes with digoxigenin (DIG), and the detection protocols suitable for this type of tissue. The DIG labeling method was developed by Kessler (2) and is based on the steroid digoxigenin, which is isolated from Digitalis purpurea and D. lanata. The DIG molecule is linked to the C-5 position of uridine (UTP, dUTP, or ddUTP) via a spacer arm. The DIG-labeled nucleotides can be incorporated easily into nucleic acid probes by DNA polymerases such as DNA polymerase I,Taq DNA polymerase, T7 DNA polymerase, RNA polymerases, and terminal transferase. These various enzymes therefore allow DIG labeling by random priming, nick translation, PCR, 3'-end labeling/tailing, and in vitro transcription. Following hybridization, DIG-probes may be detected with high-affinity specific anti-DIG antibodies (3). These antibodies are conjugated with alkaline phosphatase, peroxidase, fluoroscein, rhodamine, AMCA (amino-methylcoumarin-acetic acid), or colloidal gold (for electron microscopy) enabling a very versatile detection system. This system can be made even more versatile and sensitive by using unconjugated anti-DIG followed by conjugated secondary antibodies. A detection sensitivity of about 0.1 pg (as determined by Southern blot) can be achieved with combinations of anti-DIG-alkaline phospatase and NBT or BCIP. In this chapter, I describe a protocol that we developed for nonradioactive in situ hybridization of atherosclerotic tissue for the detection of interleukin 8, tissue factor, and tissue factor pathway inhibitor in both frozen and paraffin-embedded tissue (4-7). PMID:21340943

Apostolopoulos, J

2001-01-01

286

In situ hybridization of plant meiotic and mitotic chromosomes: differences in signal detection.  

PubMed

The technique of in situ hybridization to both meiotic and mitotic chromosomes of Rumex acetosa is described. Differences in the efficiency of signal detection were observed between the two types of material. The implications of these results for in situ hybridization to other plant species are explored. PMID:1300148

Clark, M S; Parker, J S

1992-09-01

287

Genome discrimination by in situ hybridization in Icelandic species of Elymus and Elytrigia (Poaceae: Triticeae).  

PubMed

The genome constitution of Icelandic Elymus caninus, E. alaskanus, and Elytrigia repens was examined by fluorescence in situ hybridization using genomic DNA and selected cloned sequences as probes. Genomic in situ hybridization (GISH) of Hordeum brachyantherum ssp. californicum (diploid, H genome) probe confirmed the presence of an H genome in the two tetraploid Elymus species and identified its presence in the hexaploid Elytrigia repens. The H chromosomes were painted uniformly except for some chromosomes of Elytrigia repens which showed extended unlabelled pericentromeric and subterminal regions. A mixture of genomic DNA from H. marinum ssp. marinum (diploid, Xa genome) and H. murinum ssp. leporinum (tetraploid, Xu genome) did not hybridize to chromosomes of the Elymus species or Elytrigia repens, confirming that these genomes were different from the H genome. The St genomic probe from Pseudoroegneria spicata (diploid) did not discriminate between the genomes of the Elymus species, whereas it produced dispersed and spotty hybridization signals most likely on the two St genomes of Elytrigia repens. Chromosomes of the two genera Elymus and Elytrigia showed different patterns of hybridization with clones pTa71 and pAes41, while clones pTa1 and pSc119.2 hybridized only to Elytrigia chromosomes. Based on FISH with these genomic and cloned probes, the two Elymus species are genomically similar, but they are evidently different from Elytrigia repens. Therefore the genomes of Icelandic Elymus caninus and E. alaskanus remain as StH, whereas the genomes of Elytrigia repens are proposed as XXH. PMID:11341738

Orgaard, M; Anamthawat-Jónsson, K

2001-04-01

288

Quantitative analysis of chromosome in situ hybridization signal in paraffin-embedded tissue sections  

SciTech Connect

Interphase cytogenetic analysis using chromosome-specific probes is increasingly being used to detect chromosomal aberrations on paraffin-embedded tissue sections. However, quantitative analysis of the hybridization signal is confounded by the nuclear slicing that occurs during sectioning. To determine the sensitivity and accuracy of chromosome in situ hybridization for detecting numerical chromosomal aberrations on paraffin-embedded sections, in situ hybridization was performed on sections derived from mixtures of cell populations with know frequencies of numerical chromosomal aberrations and the Chromosome Index (CI) was calculated (i.e., total number of signal spots/number of nuclei counted) as a quantitative measure of chromosome copy number. The presence of 25% or more monosomic or tetrasomic cells in a given population was easily detected as a change in CI (P < 0.05). Lower degrees of polysomy could be detected as a small percentage of nuclear fragments with >2 signal spots. The CI was not significantly influenced by a change in section thickness from 4 to 8 {mu}M, by an increase in cell size from 478 to 986 {mu}M{sup 3}, or by the choice of detection method (fluorescence vs. conventional bright-field microscopy). Comparative analysis of touch preparations and tissue sections from the corresponding breast tumors showed that CI accurately reflects the average copy number of chromosomes in intact nuclei and may actually be superior to in situ hybridization on whole nuclei for the detection of numerical chromosomal changes in defined histologic areas. This method is thus a sensitive and accurate means of studying genetic changes in premalignant and malignant tissue, and of assessing the genetic changes associated with specific phenotypes. 27 refs., 8 figs., 3 tabs.

Dhingra, K.; Emami, K.; Hortobagyi, G.N. [Univ. of Texas M.D. Anderson Cancer Center, Houston, TX (United States)] [and others

1994-06-01

289

Resolving Genetic Functions within Microbial Populations: In Situ Analyses Using rRNA and mRNA Stable Isotope Probing Coupled with Single-Cell Raman-Fluorescence In Situ Hybridization  

Microsoft Academic Search

Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope (13C)-linked analyses to determine microbial identity and

Wei E. Huang; Andrew Ferguson; Andrew C. Singer; Kathryn Lawson; Ian P. Thompson; Robert M. Kalin; Michael J. Larkin; Mark J. Bailey; Andrew S. Whiteley

2009-01-01

290

Two-color in situ hybridization of whole-mount mouse embryos.  

PubMed

RNA in situ hybridization is a powerful technique used to identify the spatial localization of a specific RNA in a tissue section or whole tissue. In this protocol, we describe a reliable method for two-color in situ hybridization that can be used to accurately assess the expression of multiple genes with contrasting or overlapping expression patterns in whole mouse embryos. PMID:24318811

Biris, Kristin K; Yamaguchi, Terry P

2014-01-01

291

The Use of Whole-Mount "in Situ" Hybridization to Illustrate Gene Expression Regulation  

ERIC Educational Resources Information Center

"In situ" hybridization is a widely used technique for studying gene expression. Here, we describe two experiments addressed to postgraduate genetics students in which the effect of transcription factors on gene expression is analyzed in "Drosophila embryos of different genotypes by whole-mount in situ hybridization. In one of the…

Llamusí, Beatriz; Muñoz-Soriano, Verónica; Paricio, Nuria; Artero, Rubén

2014-01-01

292

Human sex determination by in situ hybridization using non-radioactive probes  

E-print Network

Human sex determination by in situ hybridization using non-radioactive probes M Benkhalifa1 N Animals; Toulouse-Auzeville, 10-13 July 1990) in situ hybridization / non-radioactive / sex determination and Human Genetics, Munich, Germany (Proceedings of the 9th European Colloquium on Cytogenetics of Domestic

Paris-Sud XI, Université de

293

Improved hybrid solar cells via in situ UV-polymerization.  

SciTech Connect

One approach for making inexpensive inorganic-organic hybrid photovoltaic (PV) cells is to fill highly ordered TiO{sub 2} nanotube (NT) arrays with solid organic hole conductors such as conjugated polymers. Here, a new in situ UV polymerization method for growing polythiophene (UV-PT) inside TiO{sub 2} NTs is presented and compared to the conventional approach of infiltrating NTs with pre-synthesized polymer. A nanotubular TiO{sub 2} substrate is immersed in a 2,5-diiodothiophene (DIT) monomer precursor solution and then irradiated with UV light. The selective UV photodissociation of the C-I bond produces monomer radicals with intact {pi}-ring structure that further produce longer oligothiophene/PT molecules. Complete photoluminescence quenching upon UV irradiation suggests coupling between radicals created from DIT and at the TiO{sub 2} surface via a charge transfer complex. Coupling with the TiO{sub 2} surface improves UV-PT crystallinity and {pi}-{pi} stacking; flat photocurrent values show that charge recombination during hole transport through the polymer is negligible. A non-ideal, backside-illuminated setup under illumination of 620-nm light yields a photocurrent density of {approx} 5 {micro}A cm{sup -2} - surprisingly much stronger than with comparable devices fabricated with polymer synthesized ex situ. Since in this backside architecture setup we illuminate the cell through the Ag top electrode, there is a possibility for Ag plasmon-enhanced solar energy conversion. By using this simple in situ UV polymerization method that couples the conjugated polymer to the TiO{sub 2} surface, the absorption of sunlight can be improved and the charge carrier mobility of the photoactive layer can be enhanced.

Tepavcevic, S.; Darling, S. B.; Dimitrijevic, N. M.; Rajh, T.; Sibener, S. J.; Univ. of Chicago

2009-08-03

294

Digoxigenin-Labeled In Situ Hybridization for the Detection of Streptococcus suis DNA in Polyserositis and a Comparison with Biotinylated In Situ Hybridization  

PubMed Central

ABSTRACT The objective of this study was to develop digoxigenin-labeled in situ hybridization (ISH) for the detection of Streptococcus suis in naturally infected pigs with polyserositis and to compare it with biotinylated ISH. Digoxigenin-labeled hybridization signals for S. suis were observed in cells that had infiltrated the fibrous polyserositis and microcolonies in the blood vessels. Mock hybridization showed no hybridization signals for endogenous digoxigenin. Biotinylated hybridization signals for S. suis were observed in cells that had infiltrated the fibrous polyserositis. However, similar hybridization signals were also observed in the fibrous inflammatory area using mock hybridization for endogenous biotin. The present study demonstrated that digoxigenin-labeled ISH is a valuable diagnostic tool for specific detection of S. suis in polyserositic tissues without nonspecific reactions compared with biotinylated ISH. PMID:23985415

KANG, Ikjae; SEO, Hwi Won; PARK, Changhoon; OH, Yeonsu; LEE, Jeehoon; YOU, Ok Heui; KIM, Sung-Hoon; GOTTSCHALK, Marcelo; CHAE, Chanhee

2013-01-01

295

[Physical localization of ribosomal genes and chromosome DAPI banding by in situ hybridization in Medicago sativa L].  

PubMed

Physical localization of ribosomal genes in diploid and tetraploid alfalfa (Medicago. sativa) was studied using fluorescent in situ hybridization (FISH). It was revealed that 45s gene was only located at nucleolus organizer region (NOR) with a single locus in both diploid and tetraploid alfalfa, while 5s gene had 2-3 loci on chromosomes. Using the genomic DNA from M. coerulea and M. falcata as probe to hybridize with tetraploid species in alfalfa, both diploid species were successfully hybridized with tetraploid chromosomes, only showing the difference in hybridization signals in different numbers of chromosomes. Chromosomes of alfalfa exhibited DAPI banding by FISH analysis. In general, the patterns of distribution of DAPI banding were consistent with C-banding for M. coerulea. The possible origination of tetraploid alfalfa was discussed based on DAPI banding patterns and FISH analysis for ribosomal genes . PMID:16520314

Chen, Jian-Min; Hong, Yi-Huan; Wang, You-Ping; Bowley, Steve; Wan, Jian-Min

2006-02-01

296

New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling  

Microsoft Academic Search

Multicolour in situ hybridisation (MFISH) is increasingly applied to karyotyping and detection of chromosomal abnormalities. So far 27 colour analyses have been described using fluorescently labelled chromosome painting probes in a so-called combinatorial approach. In this paper a new strategy is presented to use efficiently the currently available number of spectrally separated fluorophores in order to increase the multiplicity of

HJ Tanke; J Wiegant; RPM van Gijlswijk; V Bezrookove; H Pattenier; RJ Heetebrij; EG Talman; AK Raap; J Vrolijk

1999-01-01

297

Detection of Parvovirus B19 DNA in Bone Marrow Cells by Chemiluminescence In Situ Hybridization  

Microsoft Academic Search

A chemiluminescence in situ hybridization method was developed for the search of B19 parvovirus DNA in bone marrow cells, employing digoxigenin-labeled B19 DNA probes, immunoenzymatically detected with a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. The light emitted from the in situ- hybridized probe was analyzed and measured by a high-performance luminograph connected to an optical microscope and to a

MONICA MUSIANI; ALDO RODA; MARIALUISA ZERBINI; GIOVANNA GENTILOMI; PATRIZIA PASINI; GIORGIO GALLINELLA; ANDSIMONA VENTUROLI

298

Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability  

PubMed Central

Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability. PMID:24712299

2014-01-01

299

Genomic Origin and Organization of the Allopolyploid Primula egaliksensis Investigated by in situ Hybridization  

PubMed Central

Background and Aims Earlier studies have suggested that the tetraploid Primula egaliksensis (2n = 40) originated from hybridization between the diploids P. mistassinica (2n = 18) and P. nutans (2n = 22), which were hypothesized to be the maternal and paternal parent, respectively. The present paper is aimed at verifying the hybrid nature of P. egaliksensis using cytogenetic tools, and to investigate the extent to which the parental genomes have undergone genomic reorganization. Methods Genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with ribosomal DNA (rDNA) probes, together with sequencing of the internal transcribed spacer (ITS) region of the rDNA, were used to identify the origin of P. egaliksensis and to explore its genomic organization, particularly at rDNA loci. Key Results GISH showed that P. egaliksensis inherited all chromosomes from P. mistassinica and P. nutans and did not reveal major intergenomic rearrangements between the parental genomes (e.g. interchromosomal translocations). However, karyological comparisons and FISH experiments suggested small-scale rearrangements, particularly at rDNA sites. Primula egaliksensis lacked the ITS-bearing heterochromatic knobs characteristic of the maternal parent P. mistassinica and maintained only the rDNA loci of P. nutans. These results corroborated sequence data indicating that most ITS sequences of P. egaliksensis were of the paternal repeat type. Conclusions The lack of major rearrangements may be a consequence of the considerable genetic divergence between the putative parents, while the rapid elimination of the ITS repeats from the maternal progenitor may be explained by the subterminal location of ITS loci or a potential role of nucleolar dominance in chromosome stabilization. These small-scale rearrangements may be indicative of genome diploidization, but further investigations are needed to confirm this assumption. PMID:18308718

Guggisberg, Alessia; Baroux, Celia; Grossniklaus, Ueli; Conti, Elena

2008-01-01

300

Investigation of polymer electrolyte membrane chemical degradation and degradation mitigation using in situ fluorescence spectroscopy  

PubMed Central

A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO2 nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO2 to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO2 into the catalyzed membrane provided an eightfold reduction in ROS generation rate. PMID:22219367

Prabhakaran, Venkateshkumar; Arges, Christopher G.; Ramani, Vijay

2012-01-01

301

Chlorophyll bleaching by UV-irradiation in vitro and in situ: Absorption and fluorescence studies  

NASA Astrophysics Data System (ADS)

Chlorophyll bleaching by UV-irradiation has been studied by absorbance and fluorescence spectroscopy in extracts containing mixtures of photosynthetic pigments, in acetone and n-hexane solutions, and in aqueous thylakoid suspensions. Chlorophyll undergoes destruction (bleaching) accompanied by fluorescent transient formation obeying first-order kinetics. The bleaching is governed by UV-photon energy input, as well as by different chlorophyll molecular organizations in solvents of different polarities ( in vitro), and in thylakoids ( in situ). UV-C-induced bleaching of chlorophylls in thylakoids is probably caused by different mechanisms compared to UV-A- and UV-B-induced bleaching.

Zvezdanovi?, Jelena; Cveti?, Tijana; Veljovi?-Jovanovi?, Sonja; Markovi?, Dejan

2009-01-01

302

Elucidating Structure and Function In Vivo With Hybrid Fluorescence and Magnetic Resonance Imaging  

Microsoft Academic Search

While the mathematics, physics, and technology behind magnetic resonance (MR) and fluorescence image formation are distinctively different, the two modalities have significant complementary features to impart strong preclinical and clinical application synergies. Traditionally, hybrid MR and fluorescence imaging implied the use of a system where optical and MR signals can be concurrently acquired. In this case, the common geometry allows

Mark Niedre; Vasilis Ntziachristos

2008-01-01

303

Surface layer variability in the Ross Sea, Antarctica as assessed by in situ fluorescence measurements  

NASA Astrophysics Data System (ADS)

Phytoplankton fluorescence, temperature and salinity were measured from December through February using in situ instruments deployed at two locations in the southern Ross Sea, Antarctica during the austral summers of three consecutive years (2003-2004, 2004-2005, and 2005-2006) to assess the short-term, seasonal and interannual variations in phytoplankton biomass and oceanographic conditions. The seasonal climatologies of physical forcing variables were also determined from satellite measurements, and the data from the two sites compared to the 2000-2009 mean. In situ fluorometers were deployed at three depths at 77°S, 172.7°E and 77.5°S, 180°. Significant differences between the two sites were consistently observed, confirming the anticipated high level of spatial and temporal heterogeneity. Chlorophyll fluorescence was maximal in late December, and generally decreased rapidly to modest levels in January and February. However, during 1 year (2003-2004) a secondary bloom was found, with summer maxima being similar to those observed during spring. Fluorescence displayed a strong diel cycle, with strong quenching during periods of maximum irradiance. The magnitude of this reduction was large (the minimum average fluorescence was 25% of the daily mean) and decreased with depth. Fluorescence varied interannually, with the absolute levels and temporal patterns being different among years. The two sites had different temperature/salinity properties as measured at 24 m, and both variables changed with time. During 2004-2005 we were able to continuously measure the photosynthetic quantum efficiency of PSII ( F v/ F m) at 11 m, which revealed a minimum in December, and an increase in January, whereas the absolute fluorescence ( F o) decreased simultaneously. We suggest that this reflected a mixing event, whereby available irradiance increased, allowing a short period of growth in a more favorable optical environment. While substantial variations from the mean physical forcing were observed, the linkage of these physical variations with fluorescence was not always clear. Short-term (over 24-h) changes in fluorescence occurred, and were likely related to advective events. Wind events altered fluorescence in the surface layer, and these redistributed phytoplankton in the surface. The variability in chlorophyll fluorescence and physical forcing over a variety of scales in the Ross Sea provides insights into temporal-spatial coupling of phytoplankton.

Smith, Walker O.; Asper, Vernon; Tozzi, Sasha; Liu, Xiao; Stammerjohn, Sharon E.

2011-01-01

304

In Situ Hybridization for the Precise Localization of Transcripts in Plants  

PubMed Central

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14. PMID:22143276

Javelle, Marie; Marco, Cristina F.; Timmermans, Marja

2011-01-01

305

In situ hybridization analysis of immunoglobulin heavy chain variable gene expression with family specific oligonucleotide probes  

Microsoft Academic Search

We have developed an improved in situ hybridization (ISH) technique for the analysis of human immunoglobulin heavy chain variable (VH) gene family expression in suspensions of human B lymphocytes. Oligonucleotide probes specific for framework region (FR) consensus germline sequences for each of the seven human VH gene families were designed and hybridization conditions were developed to accommodate the greatest degree

Charles H. Rundle; Harry W. Schroeder; William J. Koopman

1998-01-01

306

Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization  

Microsoft Academic Search

We have examined the cytological localization of rRNA synthesis, transport, and processing events within the mammalian cell\\u000a nucleolus by double-label fluorescent in situ hybridization analysis using probes for small selected segments of pre-rRNA,\\u000a which have known half-lives. In particular, a probe for an extremely short-lived 5? region that is not found separate of the\\u000a pre-rRNA identifies nascent transcripts within the

Inara B. Lazdins; Michael Delannoy; Barbara Sollner-Webb

1997-01-01

307

Progress in molecular diagnosis of Charcot-Marie-Tooth-disease type 1 (CMT 1, HMSN I) and hereditary neuropathy with liability to pressure palsies (HNPP) by fluorescence in situ hybridization (FISH)-detection of a potential genetic mosaicism  

SciTech Connect

We tested 20 CMT 1 patients characterized according to the criteria of the European CMT consortium by Southern hybridization of MspI restricted genomic DNA with probes pVAW409R1, pVAW412Hec and pEW401HE. In 11 of the 20 CMT 1 cases (55%), we observed a duplication in 17q11.2; one patient had a dinucleotide insertion in exon 6 of the PO-gene (5%). One HNPP case had a typical 17p11.2 deletion. Analysis of CA-repeats was performed with primers RM11GT and Mfd41; SSCP-analysis of the PO, PMP22 and Cx32-genes is in progress. FISH was carried out with probe pVAW409R1. 125 interphase nuclei were analyzed for each proband by counting the signals per nucleus. Normal cells show a characteristic distribution of signals: 1 signal in 5.9% of nuclei, 2 in 86.3% and 3 in 7.8%. A duplication is indicated by a shift to 3 signals in more than approximately 60% and 2 in less than 25% of the nuclei. In contrast, the 17p11.2 deletion of the HNPP patient shifts to 82.4% of nuclei with a single hybridization signal versus 14.4% with 2 signals. We detected one case with significantly abnormal distribution of interphase nuclei hybridization signals compared to cultures of normal cells and to those with 17p11.2 duplication or deletion: 3.2% nuclei revealed 1 signal, 48.0% two signals and 48.8% 3 signals, indicating a pathogenic but moderate dosis increase compared to the throughout duplicated cases. FISH with probe pVAW409R1 is a versatile tool to detect the HNPP deletion both in interphase nuclei and in metaphase chromosomes. In CMT 1 disease interphase nuclei are required for FISH analysis due to the small duplication of 1.5 Mbp. In contrast to Southern techniques, FISH is able to detect genetic mosaicism.

Bathke, K.; Liehr. T.; Ekici, A. [Institute for Human Genetics, Erlange (Germany)] [and others

1994-09-01

308

Rapid and sensitive in situ hybridization method for detecting and identifying lactic acid bacteria in wine  

Microsoft Academic Search

A rapid and sensitivein situhybridization method, using total genomic DNA probes, can specifically detect and identify the lactic acid bacteria in wine. The total genomic DNA probes were labelled with digoxygenin (DIG) by random priming and hybridized with the genomic DNA of the bacteria to be identified. The hybrids were detected with fluorescent anti-DIG Fab-fragments or with enzyme-conjugated anti-DIG Fab-fragments.

D Sohier; A Lonvaud-Funel

1998-01-01

309

A PDMS-Based Cylindrical Hybrid Lens for Enhanced Fluorescence Detection in Microfluidic Systems  

PubMed Central

Microfluidic systems based on fluorescence detection have been developed and applied for many biological and chemical applications. Because of the tiny amount of sample in the system; the induced fluorescence can be weak. Therefore, most microfluidic systems deploy multiple optical components or sophisticated equipment to enhance the efficiency of fluorescence detection. However, these strategies encounter common issues of complex manufacturing processes and high costs. In this study; a miniature, cylindrical and hybrid lens made of polydimethylsiloxane (PDMS) to improve the fluorescence detection in microfluidic systems is proposed. The hybrid lens integrates a laser focusing lens and a fluorescence collecting lens to achieve dual functions and simplify optical setup. Moreover, PDMS has advantages of low-cost and straightforward fabrication compared with conventional optical components. The performance of the proposed lens is first examined with two fluorescent dyes and the results show that the lens provides satisfactory enhancement for fluorescence detection of Rhodamine 6G and Nile Red. The overall increments in collected fluorescence signal and detection sensitivity are more than 220% of those without lens, and the detection limits of Rhodamine 6G and Nile red are lowered to 0.01 ?g/mL and 0.05 ?g/mL, respectively. The hybrid lens is further applied to the detection of Nile red-labeled Chlorella vulgaris cells and it increases both signal intensity and detection sensitivity by more than 520%. The proposed hybrid lens also dramatically reduces the variation in detected signal caused by the deviation in incident angle of excitation light. PMID:24531300

Lin, Bor-Shyh; Yang, Yu-Ching; Ho, Chong-Yi; Yang, Han-Yu; Wang, Hsiang-Yu

2014-01-01

310

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors  

Microsoft Academic Search

Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were

A. H. N. Hopman; F. C. S. Ramaekers; A. K. Raap; J. L. M. Beck; P. Devilee; M. Ploeg; G. P. Vooijs

1988-01-01

311

FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RIBOSOMAL RNA-TARGETED OLIGONUCLEOTIDE PROBE  

EPA Science Inventory

A fluroescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligonu...

312

Development of an automatic machine for in situ hybridization and immunohistochemistry.  

PubMed

An instrument for the automation of in situ hybridization and immunohistochemistry has been developed. This machine is capable of analyzing 20 microscope glass slides via all of the steps required for colorimetric in situ hybridization or immunohistochemistry. The slides are placed specimen-side down on a specialized Teflon slide-holder set in the reaction chamber of the machine. The system uses a unique type of capillary action between the slide and the holder. The holder has two small holes and is designed to apply, incubate and sequentially add and remove reagents from the slide surface. The system performs the complete processes of in situ hybridization and immunohistochemistry from dewaxing to colorization. Some applications were carried out using this instrument. Cultured cells infected with cytomegalovirus, adenovirus, or herpes simplex virus were hybridized with homologous biotinylated probes, and showed strong purple signals with alkaline phosphatase in the presence of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Automatic in situ hybridization using other colorimetric detection systems (e.g., peroxidase-labeled probes/diaminobenzidine/H2O2) was also examined in cells infected with Chlamydia trachomatis and in paraffin-embedded hepatic tissue sections from patients with hepatitis. For conventional immunohistochemical staining, formalin-fixed and paraffin-embedded tissues were used. Glial fibrillary acidic protein and gamma-immunoglobulins were detected automatically in human brain white matter and tonsillar tissues, respectively, as peroxidase-based reddish signals. The intensity of staining was equal to that achieved by manual methods. PMID:1776690

Takahashi, T; Ishiguro, K

1991-08-01

313

Detection of Staphylococcus aureus and Staphylococcus epidermidis in Clinical Samples by 16S rRNA-Directed In Situ Hybridization  

PubMed Central

Staphylococcus epidermidis and Staphylococcus aureus are the most common causes of medical device-associated infections, including septicemic loosenings of orthopedic implants. Frequently, the microbiological diagnosis of these infections remains ambiguous, since at least some staphylococci have the capacity to reduce their growth rate considerably. These strains exhibit a small-colony phenotype, and often they are not detectable by conventional microbiological techniques. Moreover, clinical isolates of S. aureus and S. epidermidis adhere to polymer and metal surfaces by the generation of thick, multilayered biofilms consisting of bacteria and extracellular polysaccharides. This study reports improved detection and identification of S. aureus and S. epidermidis by an in situ hybridization method with fluorescence-labeled oligonucleotide probes specific for staphylococcal 16S rRNA. The technique has proven to be suitable for the in situ detection of staphylococci, which is illustrated by the identification of S. epidermidis in a connective tissue sample obtained from a patient with septicemic loosening of a hip arthroplasty. We also show that this technique allows the detection of intracellularly persisting bacteria, including small-colony variants of S. aureus, and the differentiation of S. epidermidis from other clinically relevant staphylococci even when they are embedded in biofilms. These results suggest that the 16S rRNA in situ hybridization technique could represent a powerful diagnostic tool for the detection and differentiation of many other fastidious microorganisms. PMID:10405419

Krimmer, Vanessa; Merkert, Hilde; von Eiff, Christof; Frosch, Matthias; Eulert, Jochen; Lohr, Jochen F.; Hacker, Jorg; Ziebuhr, Wilma

1999-01-01

314

Coexistence of t(15;17) and t(15;16;17) detected by fluorescence in situ hybridization in a patient with acute promyelocytic leukemia: A case report and literature review  

PubMed Central

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), which results in the fusion of the promyelocytic leukemia (PML) gene at 15q22 with the retinoic acid ?-receptor (RARA) gene at 17q21. The current study presents the case of a 54-year-old female with APL carrying the atypical PML/RARA fusion signal due to a novel complex variant translocation t(15;16;17)(q22;q24;q21), as well as the classical PML/RARA fusion signal. Subsequent array comparative genomic hybridization revealed somatic, cryptic deletions on 3p25.3, 8q23.1 and 12p13.2-p13.1, and a duplication on 8q11.2; however, no genetic material loss or gain was observed in the breakpoint regions of chromosomes 15, 16 or 17. To the best of our knowledge, this is the first report of the coexistence of two abnormal clones, one classical and one variant, presenting simultaneously in addition to cryptic chromosome segmental imbalances in an adult APL patient. PMID:25120648

ZHANG, RUI; KIM, YOUNG-MI; WANG, XIANFU; LI, YAN; PANG, HUI; LEE, JI-YUN; LI, SHIBO

2014-01-01

315

Identification of a tumor marker chromosome by flow sorting, DNA amplification in vitro, and in situ hybridization of the amplified product  

SciTech Connect

A method combining flow sorting and molecular cytogenetic techniques was used to identify an unknown marker chromosome in the bladder tumor cell line J82. The marker chromosome was isolated by dual parameter sorting after staining with Hoechst 33258 and chromomycin [Lambda]3. DNA amplification of 300 isolated chromosomes by polymerase chain reaction using the Alu-primer Bk33 and the Lines-primer LH5 was carried out. The DNA was labelled using Bio-11-dUTP and applied to human lymphocyte metaphase cells in a suppressive in situ hybridization procedure. Fluorescence was visible over chromosome 20 and over the distal one-half of 6p. Together the fluorescent regions accounted for only 60% of the marker length, indicating a possible duplication of chromosome 20 material. This was subsequently confirmed by applying bicolor in-situ hybridization using chromosome 6- and 20-specific DNA libraries to metaphase cells of the J82 culture.

Boschman, G.; Rens, W.; Slater, R.; Aten, J. (Univ. of Amsterdam (Netherlands)); Buys, C.; Veen, A. van der; Osinga, J. (State Univ. of Groningen (Netherlands))

1993-01-01

316

Amplification of chromosomal DNA in situ  

DOEpatents

Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

Christian, Allen T. (Tracy, CA); Coleman, Matthew A. (Livermore, CA); Tucker, James D. (Livermore, CA)

2002-01-01

317

An efficient in situ hybridization protocol for multiple tissue sections and probes on miniaturized slides.  

PubMed

To facilitate detection of gene activity in tissue sections we combined common protocols of in situ hybridization on tissue sections (TSISH) with the technique of whole-mount in situ hybridization (WMISH). Miniature glass slides for mounting tissue sections were cut from regular microscope slides and handled for in situ hybridization in laboratory-made 2-ml containers (baskets) similar to those originally used for WMISH on Drosophila embryos. A salient point of the method is the use of airtight reaction vessels placed in a dry thermostat for critical hybridization steps as this facilitates reproducible and stringent hybridization conditions which are difficult to achieve on tissue sections otherwise. The practicability of the method is illustrated on consecutive serial frozen sections of murine neonatal cerebellum hybridized for math1 and neuroD, two developmentally regulated genes with distinct expression patterns. For both genes excellent spatial resolution and a highly dynamic range of signal intensity was obtained. The approach enables simple processing of multiple probes, allows the efficient and economic use of small tissue samples and is amenable to automation. PMID:12203097

Weisheit, Gunnar; Mertz, Diana; Schilling, Karl; Viebahn, Christoph

2002-09-01

318

[Identification of the triploid hybrid chromosomes of Elymus canadensis L. x Hordeum brivisubulatum Link. by genomic in situ hybridization].  

PubMed

AThe intergeneric F1 hybrid between Elymus canadensis L. and Hordeum brivisubulatum Link. is a triploid (2n = 3x = 21 ), in which 7 chromosomes,that is equal to the base number of parent's chromosomes, were lost. In order to indentify the chromosome constitution of the triploid F1 hybrid of E. canadensis L. x H. brivisubulatum Link., the genomic in situ hybridization of F1 root tip cell chromosome DNA that H. brivisubulatum H1 H1 H2H2 genome DNA labeled with Biotin-16-dUTP was conducted using E. canadensis SSH(c)H(c) genome as blocking DNA. The result showed that the chromosomes of triploid F1 hybrid consisted of 7 chromosomes from E. canadensis S genome and 14 chromosomes from H. brivisubulatum H1 H2 genome while the 7 chromosomes of Hc genome of E. canadensis were lost. PMID:15473327

Yu, Zhuo; Yun, Jin-Feng; Ma, You-Zhi; Xin, Zhi-Yong

2004-07-01

319

In-situ detection of viral nucleic acids using fluorescent probes  

NASA Astrophysics Data System (ADS)

The objective of this work was to develop and improve technologies in cytometry and molecular biology for the specific in situ detection of viral nucleic acids. The major application for this system was the detection and measurement of individual cells stained specifically for the Human Immunodeficiency Virus (HIV) in patients with Acquired Immune Deficiency Syndrome (AIDS). Staining procedures used nucleic acid either directly or indirect labeled with enzymes or fluorescent probes. A cytometry system was used to acquire digitized images of labeled cells and determine their individual staining density or intensity. Efforts are underway to improve the sensitivity of these assays using time-resolved methods.

Donovan, Richard M.

1990-07-01

320

Femtosecond optical tweezers for in-situ control of two-photon fluorescence.  

PubMed

We perform a comparison of optical tweezing using continuous wave (cw) and femtosecond lasers. Measurement of the relative Q-values in the femtosecond and cw regimes shows that femtosecond optical tweezers are just as effective as cw optical tweezers. We also demonstrate simultaneous optical tweezing and in-situ control of two-photon fluorescence (at 400nm) from dye-doped polymer microspheres. By switching the 800 nm tweezing laser source between femtosecond and cw regimes, we turned the fluorescent signal from the tweezed particle on and off while maintaining an equivalent tweezing action. Femtosecond lasers can thus be used for optical tweezing and simultaneously utilized to induce nonlinear multi-photon processes such as two-photon excitation or even photoporation. PMID:19483818

Agate, B; Brown, C; Sibbett, W; Dholakia, K

2004-06-28

321

Detection of HIV1 DNA and Messenger RNA in Individual Cells by PCR-Driven in Situ Hybridization and Flow Cytometry  

Microsoft Academic Search

Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence

Bruce K. Patterson; Michele Till; Patricia Otto; Charles Goolsby; Manohar R. Furtado; Lincoln J. McBride; Steven M. Wolinsky

1993-01-01

322

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.  

PubMed Central

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells. PMID:11423432

Tsuji, A; Sato, Y; Hirano, M; Suga, T; Koshimoto, H; Taguchi, T; Ohsuka, S

2001-01-01

323

Classifying Fruit Fly Early Embryonic Developmental Stage Based on Embryo In situ Hybridization Images  

Microsoft Academic Search

In this paper, we present a supervised classification system for sorting Drosophila embryonic in situ hybridization (ISH) images according to their developmental stages. The proposed system first segments the embryo from an image and registers it for subsequent texture feature extraction. In order to extract the most distinguishing features for classifying developmental stages, we identify several areas of interest in

Hua Zhong; Wei-bang Chen; Chengcui Zhang

2009-01-01

324

Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization.  

PubMed Central

Acidophilic pituitary adenomas commonly produce growth hormone (GH) or prolactin (PRL), according to studies employing immunohistochemical and ultrastructural methods. To examine this question, in situ hybridization with oligonucleotide probes was done on routinely processed tissues received in the pathology laboratory to analyze for the presence of GH and PRL messenger RNA (mRNA) in 4 normal pituitaries, 10 prolactinomas, and 16 GH-secreting adenomas. Most acidophilic cells in normal pituitaries expressed either GH or PRL hormone and the respective mRNAs, but GH mRNA and PRL hormone were also detected in some of the same cells. Patients with a clinical diagnosis of prolactinoma had cells with only PRL mRNA in their tumors, while most (14 of 16) patients with a clinical diagnosis of acromegaly or gigantism had both GH and PRL mRNAs in their tumors. The GH adenomas varied in these studies. In situ hybridization was helpful in characterizing the adenoma from a patient with acromegaly who had immunoreactive PRL, but no immunoreactive GH in the resected tumor; in situ hybridization analysis revealed mRNAs for both GH and PRL in the same tumor cells. Our findings indicate that pituitary adenomas from patients with acromegaly commonly express PRL mRNA. It is concluded that in situ hybridization provides new information about the clinical biology and the histopathologic classification of pituitary adenomas. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:2466405

Lloyd, R. V.; Cano, M.; Chandler, W. F.; Barkan, A. L.; Horvath, E.; Kovacs, K.

1989-01-01

325

Nested polymerase chain reaction and in situ hybridization for detection of nucleopolyhedrosis  

Microsoft Academic Search

A nested polymerase chain reaction (PCR) and in situ hybridization were developed for detection of baculoviruses in insects or other arthropods with nucleopolyhedrosis. The nested PCR was based on the sequences of polyhedrin genes from baculoviruses. Two sets of primers were designed, primers set, 35\\/36, was for the first step of amplification and yielded a product of around 680 bp,

Chung-Hsiung Wang; Hsi-Nan Yang; Hwei-Chung Liu; Guang-Hsung Kou; Chu-Fang Lo

2000-01-01

326

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries  

Microsoft Academic Search

Summary A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive

P. Lichter; T. Cremer; J. Borden; L. Manuelidis; D. C. Ward

1988-01-01

327

Transformation and segregation of GFP fluorescence and glyphosate resistance in horseweed (Conyza canadensis) hybrids.  

PubMed

The goal of this research was to generate a breeding population of horseweed segregating for glyphosate resistance. In order to generate a marker to select between hybrids of glyphosate resistant (GR) and glyphosate susceptible (GS) horseweed, a GR horseweed accession from western Tennessee was transformed with a green fluorescent protein (GFP) transgene. The GFP marker allowed for the simple and accurate determination of GR hybrid plants by visual observation. GR plants were shown to be transgenic via the green fluorescence under UV light, and resistant to glyphosate when sprayed with the field-use-rate 0.84 kg acid equivalent ha(-1) of glyphosate (i.e. Roundup) herbicide. An in vitro screen for glyphosate resistance in seedlings was developed, and a 5 microM glyphosate concentration was found to reduce dry weight in GS seedlings but not in GR seedlings. The GR plants containing GFP were then hand-crossed with GS plants from eastern Tennessee under greenhouse conditions, with GS plants acting as the pollen acceptor. Resulting seed was collected and germinated for GFP fluorescence screening. Seedlings that exhibited the transgenic GFP phenotype were selected as F(1) hybrids between GR and GS horseweed. Thirty GSxGR hybrids were produced on the basis of a green-fluorescent GFP phenotype of GR plants. GSxGFP/GR F(1) hybrids produced F(2) seeds, and F(2) plants were shown to segregate for GFP fluorescence and glyphosate resistance independently. Both traits segregated at a Mendelian 3:1 ratio, indicating a single gene is responsible for each phenotype. PMID:17024451

Halfhill, Matthew D; Good, Laura L; Basu, Chhandak; Burris, Jason; Main, Christopher L; Mueller, Thomas C; Stewart, C Neal

2007-03-01

328

Use of monodispersed, fluorescently labeled bacteria to estimate in situ protozoan bacterivory.  

PubMed

We have developed a procedure for preparing monodispersed, fluorescently labeled bacteria (FLB), which may be used to measure virtually instantaneous rates of protozoan bacterivory in natural waters. FLB can be prepared both from natural bacterioplankton assemblages and from clonal isolates and can be stored in frozen suspension or freeze-dried without apparent loss of fluorescence intensity. They are not toxic to protozoa and can be metabolized to support bacterivorous protozoan growth rates equal to those on the same strain of unstained, viable bacteria. In experiments comparing uptake of FLB with uptake of fluorescent latex microspheres by protozoan assemblages in a salt marsh tidal creek, we found that both pelagic oligotrichous ciliates and phagotrophic flagellates ingested FLB with a frequency 4- to 10-fold greater than they ingested the microspheres. Consequently, it appears that the use of latex microspheres leads to underestimation of protozoan bacterivory and that the FLB technique is superior for estimating instantaneous rates of in situ protozoan grazing on bacterioplankton. PMID:16347355

Sherr, B F; Sherr, E B; Fallon, R D

1987-05-01

329

In situ synthesis of photocatalytically active hybrids consisting of bacterial nanocellulose and anatase nanoparticles.  

PubMed

Bacterial nanocellulose (BNC) is an extraordinary biopolymer with a wide range of potential technical applications. The high specific surface area and the interconnected pore system of the nanofibrillar BNC network suggest applications as a carrier of catalysts. The present paper describes an in situ modification route for the preparation of a hybrid material consisting of BNC and photocatalytically active anatase (TiO(2)) nanoparticles (NPs). The influence of different NP concentrations on the BNC biosynthesis and the resulting supramolecular structure of the hybrids was investigated. It was found that the number of colony forming units (CFUs) and the consumption of glucose during biosynthesis remained unaffected compared to unmodified BNC. During the formation of the BNC network, the NPs were incorporated in the whole volume of the accruing hybrid. Their distribution within the hybrid material is affected by the anisotropic structure of BNC. The photocatalytic activity (PCA) of the BNC-TiO(2) hybrids was determined by methanol conversion (MC) under UV irradiation. These tests demonstrated that the NPs retained their PCA after incorporation into the BNC carrier structure. The PCA of the hybrid material depends on the amount of incorporated NPs. No alteration of the photocatalyst's efficiency was found during repeated PCA tests. In conclusion, the in situ integration of photocatalytically active NPs into BNC represents an attractive possibility to extend its fields of application to porous filtering media for drinking water purification and air cleaning. PMID:22925063

Wesarg, Falko; Schlott, Franziska; Grabow, Janet; Kurland, Heinz-Dieter; Heßler, Nadine; Kralisch, Dana; Müller, Frank A

2012-09-18

330

Development of a combined portable x-ray fluorescence and Raman spectrometer for in situ analysis  

NASA Astrophysics Data System (ADS)

In this work, we have built a portable X-ray fluorescence (XRF) spectrometer in a planar configuration coupled to a Raman head and a digital optical microscope, for in situ analysis. Several geometries for the XRF apparatus and digital microscope are possible in order to overcome spatial constraints and provide better measurement conditions. With this combined spectrometer, we are now able to perform XRF and Raman measurements in the same point without the need for sample collection, which can be crucial when dealing with cultural heritage objects, as well as forensic analysis. We show the capabilities of the spectrometer by measuring several standard reference materials, as well as other samples usually encountered in cultural heritage, geological, as well as biomedical studies.

Guerra, M.; Longelin, S.; Pessanha, S.; Manso, M.; Carvalho, M. L.

2014-06-01

331

Development of a combined portable x-ray fluorescence and Raman spectrometer for in situ analysis.  

PubMed

In this work, we have built a portable X-ray fluorescence (XRF) spectrometer in a planar configuration coupled to a Raman head and a digital optical microscope, for in situ analysis. Several geometries for the XRF apparatus and digital microscope are possible in order to overcome spatial constraints and provide better measurement conditions. With this combined spectrometer, we are now able to perform XRF and Raman measurements in the same point without the need for sample collection, which can be crucial when dealing with cultural heritage objects, as well as forensic analysis. We show the capabilities of the spectrometer by measuring several standard reference materials, as well as other samples usually encountered in cultural heritage, geological, as well as biomedical studies. PMID:24985805

Guerra, M; Longelin, S; Pessanha, S; Manso, M; Carvalho, M L

2014-06-01

332

In situ fluorescence probing of the chemical changes during sol-gel thin film formation  

SciTech Connect

Pyranine (8-hydroxy-1,3,6-trisulfonated pyrene) was used as an in situ fluorescence probe to monitor the chemical evolution during sol-gel thin film deposition of silica by the dip-coating process. The sensitivity of pyranine luminescence to protonation/deprotonation effects was used to quantify changes in the water/alcohol ratio in real time as the substrate was withdrawn from the sol reservoir. The spatially resolved spectral results showed that preferential evaporation of alcohol occurred, and that the solvent composition in the vicinity of the drying line reached values in excess of 80 vol% water. Correlation of the luminescence results with the interference pattern of the depositing film allowed the solvent composition to be mapped as a function of film thickness.

Nishida, Fumito; McKiernan, J.M.; Dunn, B.; Zink, J.I. [Univ. of California, Los Angeles, CA (United States); Brinker, C.J. [Sandia National Labs., Albuquerque, NM (United States); Hurd, A.J. [Univ. of New Mexico, Albuquerque, NM (United States)

1995-06-01

333

Changes in rRNA Levels during Stress Invalidates Results from mRNA Blotting: Fluorescence In Situ rRNA Hybridization Permits Renormalization for Estimation of Cellular mRNA Levels  

Microsoft Academic Search

Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth

MARTIN C. HANSEN; ALLAN K. NIELSEN; SØREN MOLIN; KARIN HAMMER; MOGENS KILSTRUP

2001-01-01

334

Transformation and segregation of GFP fluorescence and glyphosate resistance in horseweed ( Conyza canadensis ) hybrids  

Microsoft Academic Search

The goal of this research was to generate a breeding population of horseweed segregating for glyphosate resistance. In order\\u000a to generate a marker to select between hybrids of glyphosate resistant (GR) and glyphosate susceptible (GS) horseweed, a GR\\u000a horseweed accession from western Tennessee was transformed with a green fluorescent protein (GFP) transgene. The GFP marker\\u000a allowed for the simple and

Matthew D. Halfhill; Laura L. Good; Chhandak Basu; Jason Burris; Christopher L. Main; Thomas C. Mueller; C. Neal Stewart Jr

2007-01-01

335

Novel, in-situ Raman and fluorescence measurement techniques: Imaging using optical waveguides  

NASA Astrophysics Data System (ADS)

The following dissertation describes the development of methods for performing standoff and in- situ Raman and fluorescence spectroscopy for chemical imaging and non-imaging analytical applications. The use of Raman spectroscopy for the in- situ identification of crack cocaine and cocaine.HCl using a fiberoptic Raman probe and a portable Raman spectrograph has been demonstrated. We show that the Raman spectra of both forms of cocaine are easily distinguishable from common cutting agents and impurities such as benzocaine and lidocaine. We have also demonstrated the use of Raman spectroscopy for in-situ identification of drugs separated by thin layer chromatography. We have investigated the use of small, transportable, Raman systems for standoff Raman spectroscopy (e.g. <20 m). For this work, acousto-optical (AOTF) and liquid crystal tunable filters (LCTF) are being used both with, and in place of dispersive spectrographs and fixed filtering devices. In addition, we improved the flexibility of the system by the use of a modified holographic fiber-optic probe for light and image collection. A comparison of tunable filter technologies for standoff Raman imaging is discussed along with the merits of image transfer devices using small diameter image guides. A standoff Raman imaging system has been developed that utilizes a unique polymer collection mirror. The techniques used to produce these mirrors make it easy to design low f/# polymer mirrors. The performance of a low f/# polymer mirror system for standoff Raman chemical imaging has been demonstrated and evaluated. We have also demonstrated remote Raman hyperspectral imaging using a dimension-reduction, 2-dimensional (2-D) to 1-dimensional (1-D), fiber optic array. In these studies, a modified holographic fiber-optic probe was combined with the dimension-reduction fiber array for remote Raman imaging. The utility of this setup for standoff Raman imaging is demonstrated by monitoring the polymerization of dibromostyrene. To further demonstrate the utility of in- situ spectral imaging, we have shown that small diameter (350 ?m) image guides can be used for in-situ measurements of analyte transport in thin membranes. This has been applied to the measurement of H2O diffusion in Nafion™ membranes using the luminescent compound, [Ru(phen)2dppz] 2+, which is a H2O indicator.

Carter, Jerry Chance

336

Nuclear Structure Studied by Fluorescence Hybridization: Visualization of Individual Gene Transcription and RNA Splicing: A Thesis  

Microsoft Academic Search

The overall objective of this study has been to address some of the longstanding questions concerning functional organization of the interphase nucleus. This was achieved by using recently developed high-resolution fluorescence in situ hybridization techniques for a precise localization of specific DNA and RNA sequences in conjunction with immunocytochemistry and biochemical fractionation. This study is based on the philosophy that

Yigong P. Xing

1993-01-01

337

Electrospun in-situ hybrid polyurethane\\/nano-TiO 2 as wound dressings  

Microsoft Academic Search

By combining the organic-inorganic hybridization, wet phase inversion, and electrospinning, novel electrospun polyurethane\\u000a (PU) membranes with in-situ generated nano-TiO2 were prepared, which satisfied the requirements of an ideal wound dressing. The morphology of the PU-TiO2 mats and the cross sectional morphologies of the membranes were characterized by a scanning electron microscopy (SEM). The\\u000a average diameter of the individual fibers obtained

Lidan Yan; Shaoxiong Si; Yi Chen; Tun Yuan; Haojun Fan; Yongyi Yao; Qiyi Zhang

2011-01-01

338

Using in situ hybridization and PFGE Southern hybridization to detect translocation breakpoints in a BOR/TRPS patient cell line  

SciTech Connect

Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder characterized by ear malformations, cervical fistulae, hearing loss and renal abnormalities. We have integrated the Genethon YAC contig maps with additional markers in the chromosome 8q region genetically linked by a unique patient cell line. This cell line is from a patient who has both the branchio-oto-renal syndrome and tricho-rhino-phalangeal syndrome (TRPS). High resolution cytogenetics demonstrated a direct insertion of materials from 8q13.3q21.13 to 8q24.11. TRPS has been previously linked to deletions involving 8q24.11-q24.13. The rearrangement in this patient suggests that TRPS results from loss of gene function due to insertion at the 8q24.11 breakpoint and the possible location for the BOR gene is at either of the two breakpoints of 8q13.3 and 8q21.13. We have constructed cosmid contigs in 8q24.11. In situ hybridization with cosmids mapped to these locations as probes has helped to narrow down the breakpoints. Combinations of cosmids on either side or overlapping the 8q24.11 breakpoint show split signals on one chromosome 8q arm due to insertion of the materials from the proximal region. Cosmids mapped to the TRPS deletion region have been used to hybridize to pulsed field gel genomic blots of DNA from the patient cell line and detected rearranged genomic fragments. Both in situ hybridization and genomic PFGE Southern blot will be used to precisely locate the breakpoints.

Gu, J.Z.; Sapru, M.; Smith, D. [Univ. of Houston, TX (United States)] [and others

1994-09-01

339

Analysis of MET mRNA Expression in Gastric Cancers Using RNA In Situ Hybridization Assay: Its Clinical Implication and Comparison with Immunohistochemistry and Silver In Situ Hybridization  

PubMed Central

We investigated MET mRNA expression status using RNA in situ hybridization (ISH) technique in primary and metastatic lesions of 535 surgically resected gastric carcinoma (GC) cases. We compared the results with those of immunohistochemistry and silver in situ hybridization, and examined the association with clinicopathologic characteristics and prognosis. Among 535 primary GCs, 391 (73.1%) were scored 0, 87 (16.3%) were scored 1, 38 (7.1%) were scored 2, 12 (2.2%) were scored 3 and 7 (1.3%) were scored 4 by RNA ISH. High MET mRNA expression (score ?3) was associated with lymph node metastasis (P?=?.014), distant metastasis (P?=?.001), and higher TNM stage (P<.001). MET mRNA expression was correlated with protein expression (r?=?0.398; P<.001) and gene copy number (r?=?0.345; P<.001). The patients showing high-MET mRNA in primary or metastatic lesions had shorter overall survival than those showing low-MET mRNA (primary tumors, P?=?.002; metastatic lymph nodes, P<.001). The patients showing positive conversion of MET mRNA status in metastatic lymph node had shorter overall survival than those with no conversion (P?=?.011). Multivariate analysis demonstrated that high MET mRNA expression in metastatic lymph node was an independent prognostic factor for overall survival (P?=?.007). Therefore, this study suggests that MET mRNA expression assessed by RNA ISH could be useful as a potential marker to identify MET oncogene-addicted GC. PMID:25364819

Choi, Jiwoon; Lee, Hee Eun; Kim, Min A.; Jang, Bo Gun; Lee, Hye Seung; Kim, Woo Ho

2014-01-01

340

Site Specific Synthesis and in-situ Immobilization of Fluorescent Silver Nanoclusters on DNA Nanoscaffolds Using Tollens Reaction  

SciTech Connect

DNA strands with specific sequences and covalently attached sugar moieties were used for the site-specific incorporation of the sugar units on a DNA origami scaffold. This approach enabled the subsequent site-specific synthesis and in situ immobilization of fluorescent Ag clusters at predefined positions on the DNA nanoscaffold by treatment with the Tollens reagent.

Pal, Suchetan; Varghese, R.; Yan, Hao; Liu, Yan

2011-01-01

341

Tracking the composition and dominant components of the microbial community via polymerase chain reaction-denaturing gradient gel electrophoresis and fluorescence in situ hybridization during vermiconversion for liquid-state excess sludge stabilization.  

PubMed

To quantitatively explore the microbial community modified by earthworms, a vermifilter (VF, with earthworms) and a conventional biofilter (BF, without earthworms) were continuously operated to stabilize excess sludge. The results demonstrated a positive role imposed by earthworms on compositions and dominant components of microbial community in the VF. For one thing, the phyla Actinobacteria and Acidobacteria were only detected in the VF, which might explain for the higher Shannon index of bacteria in the VF (H = 2.58) than that in the BF (H = 1.99). For another, the total proportion of dominant bacteria in the VF increased by 23% compared to the BF. Moreover, quantification analysis explicitly noted that the dominant bacteria in VF were ?-proteobacteria (27 ± 2%) and ?-proteobacteria (24 ± 1%) while that in BF was Bacteroidetes (21 ± 1%). In conclusion, stimulated by earthworms, a unique microbial community developed in the VF, thus improving the stabilization of excess sludge. PMID:24971951

Xu, Ting; Xing, Meiyan; Yang, Jian; Lv, Baoyi; Duan, Ting; Nie, Jing

2014-09-01

342

In situ Study of DNA Attachment and Hybridization at Silicon Surfaces by Infrared Absorption Spectroscopy  

NASA Astrophysics Data System (ADS)

In this study, we have investigated in situ the process of DNA immobilization and the subsequent hybridization on Si surfaces by using infrared (IR) absorption spectroscopy in the geometry of multiple internal reflection (MIR-IRAS). We use 3-aminopropyl-triethoxysilane (APTES) to functionalize Si surfaces with the amine group for DNA attachment. MIR-IRAS data, together with ab initio calculations, demonstrate that the amine-terminated surfaces are covalently coupled to thiol-modified oligonucleotides using a crosslinker of sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC). Hybridization experiments reveal that MIR-IRAS enables us to detect DNA hybridization through IR spectral changes in the frequency region around 1685 cm-1 where the vibrational modes of the bases appear. The present results show that MIR-IRAS is a promising tool for the label-free detection of DNA hybridization as well as the in situ (in vitro) characterization of the conformation changes of DNA molecules immobilized on Si surfaces.

Ishibashi, Ken-ichi; Tanaka, Kohki; Hirano-Iwata, Ayumi; Miyamoto, Ko-ichiro; Kimura, Yasuo; Niwano, Michio

2008-04-01

343

Imaging Solar-Induced Chlorophyll Fluorescence with Hyperspectral Sensor in Situ  

NASA Astrophysics Data System (ADS)

Chlorophyll fluorescence is related to photosynthesis and it has become common practice to use chlorophyll fluorescence in plant ecophysiology studies in recent years. Active fluorescence method, which needs to generate laser to induce chlorophyll fluorescence, seems unsafe and is impossible to use on a satellite platform over 400 kilometers away in space. Therefore, solar-induced chlorophyll fluorescence (SIF) is an ideal substitution and a potential way to study vegetation condition. Imaging SIF can be a complement to the traditional SIF study with non-imaging spectrometer in the field experiments. With the fluorescence images, soil and leaves in different conditions can be distinguished easily and appropriate pixels can be selected in calculating SIF. The goal of this study is to use an imaging hyperspectral sensor companioned with a multi-angle observe platform to acquire vegetation apparent reflectance in order to image vegetation SIF in situ. The hyperspectral sensor has a 3.3 nm spectral resolution and less than 1 nm spectral sampling interval. SIF is extracted using Fraunhofer Line Depth (FLD) principle. Three FLD methods, namely the original FLD method (sFLD), the modified FLD (3FLD) and the improved FLD (iFLD) are used in both two major Fraunhofer bands, 687 nm and 760 nm. The results of this study show that, although radiance and reflectance of leaf varies, the peak of reflectance in 760 nm is obvious and stable. Around 687 nm, a peak of reflectance also occurs, but it is not obvious and the position varies. Furthermore, all of the three FLD methods get clear SIF images of wheat in 760 nm, while images calculated around 687 nm are comparatively vague. It seems that the depth and width of Fraunhofer line in 687 nm are so small that a sensor with higher spectral resolution should be used. In addition, using sFLD in 760 nm can get the clearest SIF images among all the methods. This is perhaps because of the overestimated SIF of sFLD method and, as a consequence, the contrast caused by SIF is amplified. Because more light was absorbed by upper leaves under light, SIF of upper leaves is bigger than fluorescence of lower leaves. Several new leaves and old leaves under light were selected and analyzed with statistical methods. The result shows that standard deviations in 760 nm of the three FLD methods are less than those in 687 nm and the results in 760 nm are more reasonable. Additionally, different compositions of the bands selected for the FLDs can also affect the results. When selecting the bands located out of the Fraunhofer line (?out), two principles should be considered. On one hand, the band selected should be kept in a relative long distance to the closest Fraunhofer line to prevent its effect. On the other hand, the presumptions of FLD methods would be correct only if the selected ?out is close enough to the band existed within the Fraunhofer line. In this study, for each FLD methods, the same composition of bands was selected for all the pixels and the differences among pixels were ignored. However, the shapes of reflectance spectrum of different pixels are actually different in details. It is impossible to guarantee that appropriate band composition was applied to each pixel. This can also affect the final SIF images. The way to select the optimal band composition should be studied in the future.

Wang, R.; Liu, Z.

2012-12-01

344

In situ and in vitro comparison of laser fluorescence with visual inspection in detecting occlusal caries lesions  

Microsoft Academic Search

The aim of this study was to compare the in situ and in vitro performances of a laser fluorescence (LF) device (DIAGNOdent\\u000a 2095) with visual inspection for the detection of occlusal caries in permanent teeth. Sixty-four sites were selected, and\\u000a visual inspection and LF assessments were carried out, in vitro, three times by two independent examiners, with a 1-week interval

Andréia Bolzan de Paula; Juliana Álvares Duarte Bonini Campos; Michele Baffi Diniz; Josimeri Hebling; Jonas Almeida Rodrigues

2011-01-01

345

In situ hybridization using a biotinylated DNA probe on formalin-fixed liver biopsies with hepatitis B virus infections: in situ hybridization superior to immunochemistry.  

PubMed

In situ hybridization (ISH) using a biotinylated hepatitis B virus (HBV) DNA probe was compared with immunohistochemistry (IHC) using antibody to HBsAg in liver biopsies of 98 patients with HBV-related hepatitis. The positive reaction for ISH and/or IHC was demonstrated in 83 cases from 98 cases studied. In 60 cases, the findings of ISH and IHC were concordant, and in 20 cases, ISH showed evidence of HBV infection by detecting HBV DNA, but IHC did not. The study here confirms the advantages of ISH detecting HBV-DNA over IHC detecting HBsAg. Since ISH using biotinylated probe is simple and rapid, it may be used as a routine diagnostic procedure. PMID:2194215

Choi, Y J

1990-05-01

346

[Application of in situ micro energy dispersive X-ray fluorescence analysis in mineralogy].  

PubMed

Thirteen rock samples were collected for studying the variation of element content in the mineral during the alteration process from Xinjiang, China. The IED-6000 in situ micro energy dispersive X-ray fluorescence developed by CDUT was applied to get chemical and physical data from minerals. The non-destructive spectrometer is based on a low-power Mo-anode X-ray tube and a Si-PIN peltier cooled X-ray detector. The unique design of the tube's probe allows very close coupling of polycapillary and makes the use of micro-area measurement feasible and efficient. The spectrometer can be integrated into any microscope for analysis. The long axis diameter of beam spot is about 110 microm. According to micro-EDXRF measurement, the tetrahedrite was corrected to pyrite, improving the efficiency and accuracy of the mineral identification. The feldspar of mineralized rock sample is rich in Cu and Zn which can be used as prospecting indicator elements. Element content of Cr, Mn and Co shows negative correlation with the degree of mineralization. PMID:24555398

Yang, Hai; Ge, Liang-Quan; Gu, Yi; Zhang, Qing-Xian; Xiong, Sheng-Qing

2013-11-01

347

Nerve growth factor mRNA in brain: localization by in situ hybridization  

SciTech Connect

Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons.

Rennert, P.D.; Heinrich, G.

1986-07-31

348

Silver nanoparticle-enhanced fluorescence in microtransponder-based immuno- and DNA hybridization assays  

PubMed Central

The aim of this study is to improve assay sensitivity in common solid-phase bioassay configurations as the result of using silver nanoparticles. The solid phase was provided by numerically indexed, silicon-based electronic chips, microtransponders (p-Chips) that have previously been used in multiplexed assays. Assay configurations investigated included an ELISA-type immunoassay and a DNA hybridization assay. The surface of p-Chips was derivatized with the silver island film (SIF) and a polymer, and then characterized with AFM and SEM. Silver nanoparticle sizes were in the range of 100 to 200 nm. Four fluorophores were tested for fluorescence enhancement; namely, green fluorescent protein, phycoerythrin, Cy3 and Alexa Fluor 555. We consistently observed significant fluorescence enhancement and sensitivity improvement in the p-Chip-based assays: the sensitivity in the cytokine IL-6 immunoassay was 4.3 pg/ml, which represented a 25-fold increase over the method not involving a SIF; and 50 pM in the hybridization assay, a 38-fold increase. The greatest enhancement was obtained for p-Chip surfaces derivatized first with the polymer and then coated with SIF. In conclusion, we show that the SIF-p-Chip-based platform is a highly sensitive method to quantify low-abundance biomolecules in nucleic acid-based assays and immunoassays. PMID:20798932

Li, Ji; Wang, Zhuying; Gryczynski, Ignacy

2010-01-01

349

A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback  

Microsoft Academic Search

We have developed a non-radioactive in situ hybridization technique for the localization of RNA in whole mount Drosophila embryos. After fixation, whole embryos are hybridized in situ with a DNA probe which has been labeled with digoxygenin. The hybridization products are detected by using a phosphatase-coupled antibody against digoxygenin. In parallel experiments, embryos can be treated with an antibody directed

Diethard Tautz; Christine Pfeifle

1989-01-01

350

Chromosome painting in plants: in situ hybridization with a DNA probe from a specific microdissected chromosome arm of common wheat.  

PubMed Central

We report here on the successful painting of a specific plant chromosome within its own genome. Isochromosomes for the long arm of chromosome 5 of the wheat B genome (5BL) were microdissected from first meiotic metaphase spreads of a monoisosomic 5BL line of the common wheat Triticum aestivum cv. Chinese Spring. The dissected isochromosomes were amplified by degenerate oligonucleotide-primed PCR in a single tube reaction. The amplified DNA was used as a complex probe mixture for fluorescent in situ hybridization on first meiotic metaphase spreads of lines carrying 5BL as a distinctive marker. Hybridization signals were observed, specifically, along the entire 5BL. In some of the cells, labeling was also detected in two bivalents, presumably those of the 5B "homoeologues" (partial homologues) found in common wheat (5A and 5D). The probe also revealed discrete domains in tapetal nuclei at interphase, further supporting the probe's high specificity. These data suggest that chromosome and homoeologous group-specific sequences are more abundant in 5BL than genome-specific sequences. Chromosome-painting probes, such as the one described here for 5BL, can facilitate the study of chromosome evolution in polyploid wheat. Images PMID:7991581

Vega, M; Abbo, S; Feldman, M; Levy, A A

1994-01-01

351

Assignment of the human RT6 gene to 11q13 by PCR screening of somatic cell hybrids and in situ hybridization  

SciTech Connect

RT6 is a T cell membrane protein that has attracted interest because a defect in RT6 expression is associated with susceptibility to autoimmune type I diabetes in DP-BB rats and NOD mice. Using PCR screening of human/rodent somatic cell hybrids and fluorescence in situ hybridization, the authors have determined that the gene for the human RT6 homologue is located at 11q13, centromeric to the gene for tyrosinase (TYR, albino locus) and telomeric to that for fibroblast growth factor 4 (FGF4). The data suggest that the human RT6 gene constitutes a new linkage group with TYR and the gene for olfactory marker protein (OMP) on 11q, which has a counterpart in mouse chromosome 7. Thus, in the human, the RT6 locus is dissociated from the hemoglobin [beta] chain locus (HBB) and its neighboring conserved linkage group at 11q15, in contrast to the mouse, in which RT6 shows a tighter linkage to Hbb than to Tyr. The results support the conclusion that there has been considerable intrachromosomal reshuffling of linked genes since the divergence of primates and rodents. 9 refs., 1 fig.

Koch-Nolte, F.; Haag, F.; Kuehl, M.; Thiele, H.G.; Singh, S. (Univ. Hospital, Hamburg (Germany)); Van Heyningen, V. (Medical Research Council, Edinburgh (United Kingdom)); Hoovers, J. (Univ. of Amsterdam (Netherlands)); Grzeschik, K.H. (Univ. of Marburg (Germany))

1993-11-01

352

Spatial gene expression quantification: a tool for analysis of in situ hybridizations in sea anemone Nematostella vectensis  

PubMed Central

Background Spatial gene expression quantification is required for modeling gene regulation in developing organisms. The fruit fly Drosophila melanogaster is the model system most widely applied for spatial gene expression analysis due to its unique embryonic properties: the shape does not change significantly during its early cleavage cycles and most genes are differentially expressed along a straight axis. This system of development is quite exceptional in the animal kingdom. In the sea anemone Nematostella vectensis the embryo changes its shape during early development; there are cell divisions and cell movement, like in most other metazoans. Nematostella is an attractive case study for spatial gene expression since its transparent body wall makes it accessible to various imaging techniques. Findings Our new quantification method produces standardized gene expression profiles from raw or annotated Nematostella in situ hybridizations by measuring the expression intensity along its cell layer. The procedure is based on digital morphologies derived from high-resolution fluorescence pictures. Additionally, complete descriptions of nonsymmetric expression patterns have been constructed by transforming the gene expression images into a three-dimensional representation. Conclusions We created a standard format for gene expression data, which enables quantitative analysis of in situ hybridizations from embryos with various shapes in different developmental stages. The obtained expression profiles are suitable as input for optimization of gene regulatory network models, and for correlation analysis of genes from dissimilar Nematostella morphologies. This approach is potentially applicable to many other metazoan model organisms and may also be suitable for processing data from three-dimensional imaging techniques. PMID:23039089

2012-01-01

353

Fluorescent labeling of Acanthamoeba assessed in situ from corneal sectioned microscopy  

PubMed Central

Acanthamoeba keratitis is a serious pathogenic corneal disease, with challenging diagnosis. Standard diagnostic methods include corneal biopsy (involving cell culture) and in vivo reflection corneal microscopy (in which the visualization of the pathogen is challenged by the presence of multiple reflectance corneal structures). We present a new imaging method based on fluorescence sectioned microscopy for visualization of Acanthamoeba. A fluorescent marker (MT-11-BDP), composed by a fluorescent group (BODIPY) inserted in miltefosine (a therapeutic agent against Acanthamoeba), was developed. A custom-developed fluorescent structured illumination sectioned corneal microscope (excitation wavelength: 488 nm; axial/lateral resolution: 2.6 ?m/0.4-0.6 ?m) was used to image intact enucleated rabbit eyes, injected with a solution of stained Acanthamoeba in the stroma. Fluorescent sectioned microscopic images of intact enucleated rabbit eyes revealed stained Acanthamoeba trophozoites within the stroma, easily identified by the contrasted fluorescent emission, size and shape. Control experiments show that the fluorescent maker is not internalized by corneal cells, making the developed marker specific to the pathogen. Fluorescent sectioned microscopy shows potential for specific diagnosis of Acanthamoeba keratitis. Corneal confocal microscopy, provided with a fluorescent channel, could be largely improved in specificity and sensitivity in combination with specific fluorescent marking. PMID:23082290

Marcos, Susana; Requejo-Isidro, Jose; Merayo-Lloves, Jesus; Acuña, A. Ulises; Hornillos, Valentin; Carrillo, Eugenia; Pérez-Merino, Pablo; del Olmo-Aguado, Susana; del Aguila, Carmen; Amat-Guerri, Francisco; Rivas, Luis

2012-01-01

354

In situ IO Measurements in the Marine Boundary Layer Using Laser-Induced Fluorescence Spectroscopy  

NASA Astrophysics Data System (ADS)

The iodine monoxide (IO) radical plays an important role in the chemistry of the marine boundary layer (MBL). It is formed by the reaction of ozone with iodine atoms generated by the photolysis of I2 and photo-labile iodocarbons and at night from the reaction of NO3 with I2 and O3. IO is implicated in ozone destruction, DMS oxidation and new particle formation. IO has been measured previously using LP-DOAS, with absorption paths of several kilometres, and MAX-DOAS both of which are associated with significant spatial averaging over the halogen source regions. An in situ laser-induced fluorescence (LIF) technique has been developed to detect {IO} radicals in the MBL (Whalley et. al. (2007), J. Atmos. Chem. 58: 19 - 39) and has been employed in two separate instruments. In both field instruments, an all solid-state pulsed laser operating at 445 nm is used to excite IO in the A 2 ?3/2 \\ (?'=2) \\ ? \\ X 2 ?3/2 \\ (?"=0) electronic transition, with off-resonant fluorescence detected at 520.3 nm in the (2,5) band. The sensitivity of each instrument is determined by the generation of known concentrations of IO (between 5 and 100 pptv) from the photolysis of N2O at 185 nm followed by the subsequent reaction of oxygen atoms with CF3I. The first instrument was deployed in Roscoff, North-western France during August/September 2006 as part of the RHaMBLe (Reactive Halogens in the Marine Boundary Layer) campaign. The instrument limit of detection was 0.4 pptv for a 5 min integration time, with an uncertainty of 23%. Located on a small jetty, the instrument measured significant levels of IO on 11 days, with up to 29 pptv observed (10 second average). IO displayed a clear diurnal profile with a maximum at low tide, and lower concentrations observed on some nights. This is compared with LP-DOAS (University of Leeds) measurements also made at the site. The second instrument was deployed twice in 2007. The instrumental limit of detection was also found to be 0.4 pptv for a 5 minute integration time, with an overall uncertainty of 23%. The first deployment was aboard the RSS Discovery to measure open ocean IO over the Mauritanian upwelling during May/June 2007. The second deployment was to Mace Head, Western Ireland during August 2007. At Mace Head, IO was observed on all 9 measurement days, with a maximum of 28 pptv observed (1 min average) coinciding with low (spring) tide.

Heard, D. E.; Bale, C. S.; Bloss, W. J.; Commane, R.; Furneaux, K. L.; Ingham, T.; Whalley, L. K.

2007-12-01

355

Calretinin distribution in the octopus brain: an immunohistochemical and in situ hybridization histochemical analysis.  

PubMed

The distribution of calretinin containing neurons examined by in situ hybridization mapping was compared with that obtained by immunocytochemistry in the brain of octopus. Results revealed a close correspondence between the two types of investigations. Western blot analysis disclosed a 29 kDa protein immunostained with anti-calretinin antibody. Calretinin containing neurons were localized mainly in the cortex of octopus lobes, including the vertical, frontal, basal, buccal, palliovisceral, pedal and branchial, with variations of staining intensity and density of immunoreactive cells. The amacrine cells surrounding calretinin containing neuronal bodies of the cortex were also labeled unlike the glial cells. The close correspondence of blotting analysis, immunocytochemistry and in situ hybridization indicates with no doubt that calretinin, like other calcium-binding proteins previously studied, is also present in the nervous system of cephalopods. Furthermore, although recent findings localize calretinin also in endocrine glands, the presence of this calcium-binding protein in the brain of octopus indicates that calretinin appeared early in the phylogeny as a neuronal protein already in invertebrates. PMID:17188660

Altobelli, Giovanna G; Cimini, Vincenzo

2007-02-01

356

Photosynthetic activity, photoprotection and photoinhibition in intertidal microphytobenthos as studied in situ using variable chlorophyll fluorescence  

NASA Astrophysics Data System (ADS)

The photosynthetic activity of microphytobenthos biofilms was studied in situ on an intertidal mudflat of the Ria de Aveiro, Portugal. Time series of physical variables characterizing the microenvironment at the sediment photic zone (incident solar irradiance, temperature, salinity), photophysiological parameters and productive biomass of undisturbed microalgal assemblages were measured during daytime low-tide periods along one spring-neap tidal cycle, with the objective of (1) characterizing the short-term variability in photosynthetic activity in situ, (2) relating it with the changing environmental conditions and (3) with the operation of physiologically (xanthophyll cycle) and behaviorally (vertical migration) based photoprotective processes, and (4) assessing the occurrence of photoinhibition. Pulse Amplitude Modulated (PAM) fluorometry was applied to measure photosynthetic activity (the effective and maximum quantum yield of photosystem II, ? F/ Fm' and Fv/ Fm; the photosynthesis index EFY; rapid light-response curves (RLC)), the photoprotective operation of the xanthophyll cycle and photoinhibition (non-photochemical quenching, NPQ; quantum efficiency of open RCs, Fv'/ Fm'), and vertical migration (productive biomass, Fo). The photosynthetic activity was found to be strongly affected by the cumulative light dose received during the morning low-tide periods. The fluorescence indices ? F/ Fm', EFY, Fv'/ Fm' and RLC parameters were more depressed under high irradiances when clear sky was present during the morning low tide than when foggy conditions reduced the light dose received during a comparable period. Productive biomass exhibited maximum values in the first hours of the morning, followed by a steep decrease when irradiance reached moderate levels, due to the downward migration of the microalgae. This photophobic migratory response appeared to display a photoprotective role, allowing ? F/ Fm' to remain near optimum values until irradiance reached values as high as 750 ?mol m -2 s -1. The response to high light also included the formation of NPQ, expected to represent mainly the operation of the xanthophyll cycle, which attained high values, above 5.9 for 1500 ?mol m -2 s -1. Despite the photoprotection provided by energy-dissipation processes and photophobic behaviour, the light response of most photophysiological parameters showed a clear counter-clockwise hysteresis pattern, indicating the occurrence of photoinhibition. Hysteresis was due to the incomplete recovery of photosynthetic activity during the afternoon low tide, and its magnitude was dependent on the morning light doses.

Serôdio, João; Vieira, Sónia; Cruz, Sónia

2008-06-01

357

Dual Labeling of Pseudomonas putida with Fluorescent Proteins for In Situ Monitoring of Conjugal Transfer of the TOL Plasmid  

PubMed Central

We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor P. putida cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies. PMID:12902279

Nancharaiah, Y. Venkata; Wattiau, Pierre; Wuertz, Stefan; Bathe, Stephan; Mohan, S. Venkata; Wilderer, Peter A.; Hausner, Martina

2003-01-01

358

High-magnification vascular imaging to reject false-positive sites in situ during Hexvix® fluorescence cystoscopy  

NASA Astrophysics Data System (ADS)

Fluorescence imaging for detection of non-muscle-invasive bladder cancer is based on the selective production and accumulation of fluorescing porphyrins-mainly, protoporphyrin IX-in cancerous tissues after the instillation of Hexvix®. Although the sensitivity of this procedure is very good, its specificity is somewhat limited due to fluorescence false-positive sites. Consequently, magnification cystoscopy has been investigated in order to discriminate false from true fluorescence positive findings. Both white-light and fluorescence modes are possible with the magnification cystoscope, allowing observation of the bladder wall with magnification ranging between 30× for standard observation and 650×. The optical zooming setup allows adjusting the magnification continuously in situ. In the high-magnification (HM) regime, the smallest diameter of the field of view is 600 microns and the resolution is 2.5 microns when in contact with the bladder wall. With this cystoscope, we characterized the superficial vascularization of the fluorescing sites in order to discriminate cancerous from noncancerous tissues. This procedure allowed us to establish a classification based on observed vascular patterns. Seventy-two patients subject to Hexvix® fluorescence cystoscopy were included in the study. Comparison of HM cystoscopy classification with histopathology results confirmed 32/33 (97%) cancerous biopsies and rejected 17/20 (85%) noncancerous lesions.

Lovisa, Blaise; Jichlinski, Patrice; Weber, Bernd-Claus; Aymon, Daniela; van den Bergh, Hubert; Wagnières, Georges

2010-09-01

359

Fluorescence-Based Bacterial Overlay Method for Simultaneous In Situ Quantification of Surface-Attached Bacteria?  

PubMed Central

For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, Streptococcus mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications. PMID:17308176

Muller, Rainer; Groger, Gerhard; Hiller, Karl-Anton; Schmalz, Gottfried; Ruhl, Stefan

2007-01-01

360

Making a Hybrid Microfluidic Platform Compatible for In Situ Imaging by Vacuum-Based Techniques  

SciTech Connect

A self-contained microfluidic-based device was designed and fabricated for in situ imaging of aqueous surfaces using vacuum techniques. The device is a hybrid between a microfluidic PDMS block and external accessories, all portable on a small platform (10 cm-8 cm). The key feature is that a small aperture with a diameter of 2-3 micrometers is opened to the vacuum, which serves as a detection window for in situ imaging of aqueous surfaces. Vacuum compatibility and temperature drop due to water vaporization are the two most important challenges in this invention. Theoretical calculations and fabrication strategies are presented from multiple design aspects. In addition, results from the time-of-flight secondary ion mass spectrometry (ToF-SIMS) of aqueous surfaces are presented.

Yang, Li; Yu, Xiao-Ying; Zhu, Zihua; Thevuthasan, Suntharampillai; Cowin, James P.

2011-10-26

361

In-situ silica incorporated carboxymethyl tamarind: development and application of a novel hybrid nanocomposite.  

PubMed

A novel hybrid nanocomposite has been prepared using in situ incorporation of nano-sized filler (silica) onto carboxymethyl tamarind kernel polysaccharide (CMT). Various characterizations were employed to confirm that silica nano particles have been incorporated onto the polymer matrix. Rheological characteristics reveal stronger CMT-Si interaction at 0.5 and 1 wt% level. Beyond 1 wt% Si concentration, the interaction is less and so there is little drop in shear viscosity. Flocculation efficiency increases with incorporation of nano filler, maximum efficacy being observed at 1 wt% silica concentration. All the nanocomposites exhibited better flocculation characteristics in comparison to pure CMT. PMID:21946078

Pal, Sagar; Gorain, M K; Giri, A; Bandyopadhyay, A; Panda, A B

2011-12-01

362

Karyotype Analysis of Four Vicia Species using In Situ Hybridization with Repetitive Sequences  

PubMed Central

Mitotic chromosomes of four Vicia species (V. sativa, V. grandiflora, V. pannonica and V. narbonensis) were subjected to in situ hybridization with probes derived from conserved plant repetitive DNA sequences (18S–25S and 5S rDNA, telomeres) and genus?specific satellite repeats (VicTR?A and VicTR?B). Numbers and positions of hybridization signals provided cytogenetic landmarks suitable for unambiguous identification of all chromosomes, and establishment of the karyotypes. The VicTR?A and ?B sequences, in particular, produced highly informative banding patterns that alone were sufficient for discrimination of all chromosomes. However, these patterns were not conserved among species and thus could not be employed for identification of homologous chromosomes. This fact, together with observed variations in positions and numbers of rDNA loci, suggests considerable divergence between karyotypes of the species studied. PMID:12770847

NAVRÁTILOVÁ, ALICE; NEUMANN, PAVEL; MACAS, JI?Í

2003-01-01

363

A New In Situ Method of Determining Relative Abundances and Charge States of Implanted Transition Metals in Individual Grains Using Synchrotron X-Ray Fluorescence  

SciTech Connect

We report on a new in situ method of determining relative abundances and charge states of implanted transition metals in individual grains using synchrotron X-ray fluorescence. In order to determine in situ the relative abundances and charge states of the transition metals in implanted solar wind in individual lunar plagioclase grains, we have developed a new microbeam x-ray fluorescence method using the synchrotron x-ray microprobe at the Advanced Photon Source (GSECARS sector 13) at Argonne National Laboratory.

Kitts, K.; Sutton, S.; Newville, M. (NIU); (UofC)

2007-03-06

364

Orientation of loci within the human major histocompatibility complex by chromosomal in situ hybridization.  

PubMed Central

We have determined the localization and orientation of two genetic probes within the human major histocompatibility complex by chromosomal in situ hybridization. Our data indicate that a cloned genomic probe cross-hybridizing to HLA-A, -B, and -C heavy chain loci is homologous to sequences located on chromosome 6 at band p21.3 while a subclone of the genomic HLA-DR alpha-chain gene corresponding to the nonpolymorphic p34 protein is homologous to sequences in band 6p21.1. Our data suggest that this technique may permit the estimation of map distances between linked gene loci, assuming a uniform frequency of map units in the human genome. The relative positions of these genes was confirmed in a mother and son carrying a chromosome rearrangement involving 6p and 14p in which the sequences hybridizing to a DR alpha-chain genomic clone were found at the distal end of the 6p--chromosome [der(6)] while the sequences hybridizing to the HLA-A, -B, -C alpha-chain probe were found in the 14p+ chromosome [der(14)]. Images PMID:6585830

Morton, C C; Kirsch, I R; Nance, W E; Evans, G A; Korman, A J; Strominger, J L

1984-01-01

365

Turn-on fluorescent dopamine sensing based on in situ formation of visible light emitting polydopamine nanoparticles.  

PubMed

Dopamine is the principle biomarker for diseases such as schizophrenia, Huntington's, and Parkinson's, and the need is urgent for rapid and sensitive detection methods for diagnosis and monitoring of such diseases. In this Article, we report a turn-on fluorescent method for rapid dopamine sensing which is based on monitoring the intrinsic fluorescence of in situ synthesized polydopamine nanoparticles. The assay uses only a common base and an acid, NaOH and HCl to initiate and stop the polymerization reaction, respectively, which makes the assay extremely simple and low cost. First, we studied the in situ optical properties of polydopamine nanoparticles, for the first time, which formed under different alkaline conditions in order to determine optimum experimental parameters. Then, under optimized conditions we demonstrated high sensitivity (40 nM) and excellent selectivity of the assay. With its good analytical figures of merit, the described method is very promising for detection of dopamine related diseases. PMID:24803112

Yildirim, Adem; Bayindir, Mehmet

2014-06-01

366

Identification of biological molecules in situ at high resolution via the fluorescence excited by a scanning electron beam.  

PubMed Central

Proteins, nucleic acids, and fluorescein-conjugated antibody are shown to be identifidable in situ via the fluorescence excited by the focused electron beam of a canning electron microscope. A molecular species is identified by its characteristic fluorescence spectrum and by a characteristic alteration of the spectrum with time under the electron beam. Primary protein fluorescence is relatively rapidly destroyed by the beam, but protein photoproduct fluorescence is more rugged and will in some cases permit detection of small numbers of protein molecules. Nucleic acid fluorescence is extremely long-lived and will permit detection of small numbers of nucleic acid residues. The theoretical resolution limit for localization of a particular molecular species -- about 20 A--is determined by the known maximum distance for molecular excitation by fast electrons. Drect extapolation from an observed resolution of 900 A in the localization of nucleic acid using a low-efficiency detector leads to an experimental resolution limit of less than 60 A. Fluorescence is strongly quenched by residual water in the specimen. Similar quenching is produced by some macromolecular associations and so may serve to localize such associations. Images PMID:768980

Hough, P V; McKinney, W R; Ledbeter, M C; Pollack, R E; Moos, H W

1976-01-01

367