The importance of fuel dissociation and propargyl + allyl association for the formation of benzene in a fuel-rich 1-hexene flame.

Fuel decomposition and benzene formation processes in a premixed, laminar, low-pressure, fuel-rich flame of 1-hexene (C(6)H(12), CH(2)=CH-CH(2)-CH(2)-CH(2)-CH(3)) are investigated by comparing quantitative mole fraction profiles of flame species with kinetic modeling results. The premixed flame, which is stabilized on a flat-flame burner under a reduced pressure of 30 Torr (= 40 mbar), is analyzed by flame-sampling molecular-beam time-of-flight mass spectrometry which uses photoionization by tunable vacuum-ultraviolet synchrotron radiation. The temperature profile of the flame is measured by OH laser-induced fluorescence. The model calculations include the latest rate coefficients for 1-hexene decomposition (J. H. Kiefer et al., J. Phys. Chem. A, 2009, 113, 13570) and for the propargyl (C(3)H(3)) + allyl (a-C(3)H(5)) reaction (J. A. Miller et al., J. Phys. Chem. A, 2010, 114, 4881). The predicted mole fractions as a function of distance from the burner are acceptable and often even in very good agreement with the experimentally observed profiles, thus allowing an assessment of the importance of various fuel decomposition reactions and benzene formation routes. The results clearly indicate that in contrast to the normal reactions of fuel destruction by radical attack, 1-hexene is destroyed mainly by decomposition via unimolecular dissociation forming allyl (a-C(3)H(5)) and n-propyl (n-C(3)H(7)). Minor fuel-consumption pathways include H-abstraction reactions producing various isomeric C(6)H(11) radicals with subsequent ß-scissions into C(2), C(3), and C(4) intermediates. The reaction path analysis also highlights a significant contribution through the propargyl (C(3)H(3)) + allyl (a-C(3)H(5)) reaction to the formation of benzene. In this flame, benzene is dominantly formed through H-assisted isomerization of fulvene, which itself is almost exclusively produced by the C(3)H(3) + a-C(3)H(5) reaction.

Novel FISH probes designed to detect IGK-MYC and IGL-MYC rearrangements in B-cell lineage malignancy identify a new breakpoint cluster region designated BVR2.

Detection of translocations involving MYC at 8q24.1 in B-cell lineage malignancies (BCL) is important for diagnostic and prognostic purposes. However, routine detection of MYC translocations is often hampered by the wide variation in breakpoint location within the MYC region, particularly when a gene other than IGH, such as IGK or IGL, is involved. To address this issue, we developed and validated four fluorescence in situ hybridization (FISH) probes: two break apart probes to detect IGK and IGL translocations, and two dual-color, dual-fusion FISH (D-FISH) probes to detect IGK-MYC and IGL-MYC. MYC rearrangements (four IGK-MYC, 12 IGL-MYC and four unknown partner gene-MYC) were correctly identified in 20 of 20 archival BCL specimens known to have MYC rearrangements not involving IGH. Seven specimens, all of which lacked MYC rearrangements using a commercial IGH/MYC D-FISH probe, were found to have 8q24 breakpoints within a cluster region >350-645 kb 3' from MYC, provisionally designated as Burkitt variant rearrangement region 2 (BVR2). FISH is a useful ancillary tool in identifying MYC rearrangements. In light of the discovery of the distally located BVR2 breakpoint cluster region, it is important to use MYC FISH probes that cover a breakpoint region at least 1.0 Mb 3' of MYC.

Sirtuin 1 suppresses mitochondrial dysfunction of ischemic mouse livers in a mitofusin 2-dependent manner.

Ischemia/reperfusion (I/R) injury is a major cause of morbidity and mortality after liver surgery. The role of Sirtuin 1 (SIRT1) in hepatic I/R injury remains elusive. Using human and mouse livers, we investigated the effects of I/R on hepatocellular SIRT1. SIRT1 expression was significantly decreased after I/R. Genetic overexpression or pharmacological activation of SIRT1 markedly suppressed defective autophagy, onset of the mitochondrial permeability transition, and hepatocyte death after I/R, whereas SIRT1-null hepatocytes exhibited increased sensitivity to I/R injury. Biochemical approaches revealed that SIRT1 interacts with mitofusin-2 (MFN2). Furthermore, MFN2, but not MFN1, was deacetylated by SIRT1. Moreover, SIRT1 overexpression substantially increased autophagy in wild-type cells, but not in MFN2-deficient cells. Thus, our results demonstrate that the loss of SIRT1 causes a sequential chain of defective autophagy, mitochondrial dysfunction, and hepatocyte death after I/R.

Analysis of Hurthle cell neoplasms of the thyroid by interphase fluorescence in situ hybridization.

Recent studies have indicated that numerical chromosomal abnormalities including changes in p53 and cyclin D1 may be involved in Hurthle cell tumorigenesis. We analyzed a series of Hurthle cell neoplasms of the thyroid to evaluate the diagnostic and prognostic utility of numerical anomalies by DNA fluorescent probes for cyclin D1 and p53 gene loci and chromosomes 5, 7, 11, 12, 17, and 22. Interphase fluorescence in situ hybridization (FISH) analysis was performed on paraffin-embedded tissue sections from 10 Hurthle cell adenomas, 19 Hurthle cell carcinomas, and 7 normal thyroid tissues used as controls. Directly labeled fluorescent DNA probes for the centromere region of chromosomes 7, 11, 12, and 17 and locus-specific probes for chromosomes 5 and 22, cyclin D1, and p53 were utilized for dual-probe hybridizations. Sixty percent (6 of 10) Hurthle cell adenomas and 63% (12 of 19) Hurthle cell carcinomas showed chromosome gains. Twenty percent (2 of 10) Hurthle cell adenomas and 26% (5 of 19) Hurthle cell carcinomas showed chromosome losses. Normal thyroid tissues used as controls showed no chromosomal abnormalities. Among Hurthle cell tumors with chromosomal abnormalities, adenomas averaged 2.7 gains and 0.3 losses per case, and carcinomas averaged 3.3 gains and 0.6 losses per case. The two adenomas with chromosome losses each showed loss of one chromosome, whereas the five carcinomas with losses averaged 1.8 losses per case. Chromosome 22 was the most common loss identified, occurring in three of the 11 patients who died of disease. These results indicate that chromosomal imbalances as gains are common in both benign and malignant Hurthle cell neoplasms, but Hurthle cell carcinomas tend to have more chromosome losses than adenomas. Among Hurthle cell carcinomas in this study, chromosome losses were identified only from patients who died of disease. The loss of chromosome 22 may have prognostic value in Hurthle cell carcinoma of the thyroid.

Detection of t(2;5) in anaplastic large cell lymphoma: comparison of immunohistochemical studies, FISH, and RT-PCR in paraffin-embedded tissue.

Anaplastic large cell lymphoma (ALCL) is associated with the t(2;5)(p23;q35) translocation involving the anaplastic lymphoma kinase gene (ALK) and the nucleophosmin gene (NPM), which result in expression of a novel fusion protein, NPM-ALK (p80). Clinicopathologic studies have shown that ALK expression in ALCL is associated with improved 5-year survival rates when compared with ALCL lacking ALK expression. This study used paraffin-embedded tissue to compare interphase fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of t(2;5) with immunohistochemical analysis for the detection of ALK protein expression in 27 patients with CD30-positive ALCLs. ALK protein expression was detected with ALK1 antibody in 14 of the 27 patients. The neoplastic cells in 13 of these 14 lymphomas reacted with the p80NPM/ALK antibody. FISH, using a two-color ALK DNA probe, correlated 100% with the immunohistochemical results: a translocation involving the ALK gene was detected in all 14 lymphomas that reacted with anti-ALK1. RT-PCR, performed on 21 lymphomas, detected NPM-ALK mRNA in five of the lymphomas, all of which reacted with anti-ALK1 and showed ALK gene rearrangement by FISH. Lymphomas showing ALK1 reactivity occurred in a younger patient population (median age, 19.5 years) and were associated with improved 5-year survival rates (84%), as compared with lymphomas lacking ALK1 reactivity (median age, 68.0 years; 5-year survival rate, 35%; p = 0.008). We conclude that immunohistochemical studies, using antibody ALK1. and FISH for ALK gene rearrangement are equally effective for identifying patients with ALCL who have a favorable clinical outcome.

Detection of newborn aneuploidy by interphase fluorescence in situ hybridization.

OBJECTIVE: To test fluorescence in situ hybridization (FISH) probes for rapid detection of aneuploidy of chromosomes 13, 18, 21, X, and Y from newborn uncultured blood samples. MATERIAL AND METHODS: Directly labeled, multicolored, commercially available FISH probes for the five aforementioned chromosomes were validated, and their hybridization efficiencies were established. In a blinded study, eight trisomic samples were tested by this FISH method. RESULTS: The hybridization efficiency based on metaphase evaluation of each of the five probes was 100%, and no cross-hybridization occurred. The mean interphase hybridization efficiencies of the probes for chromosomes 13, 18, 21, X, and Y were 97.4 %, 89.4 %, 96.1%, 94.4 %, and 100 %, respectively. The eight abnormal samples were identified as trisomy 21 (in six), trisomy 13 (in one), and trisomy 18 (in one). CONCLUSION: The screening of aneuploidy of newborns for chromosomes 13, 18, 21, X, or Y by interphase FISH is rapid, reliable, and cost-effective. The test is especially suitable for medically urgent cases as a screen, followed by a standard chromosome analysis.

Application of multicolor fluorescent in situ hybridization for enhanced characterization of chromosomal abnormalities in congenital disorders.

OBJECTIVE: To determine the efficacy of multicolor fluorescent in situ hybridization (M-FISH), which paints each chromosome in a unique color, for identification of congenital derivative and marker chromosomes. MATERIAL, METHODS AND CASES: Commercially available M-FISH probes were used to label each chromosome in a specific fluorescent color. Six representative cases involving derivative chromosomes, markers, and subtle anomalies were analyzed by M-FISH. RESULTS: Three familial, rather subtle derivative chromosomes were identified by M-FISH with relative ease. A small ring that was unidentifiable by banded-chromosome analysis was identified by M-FISH. A case of a subtle telomeric anomaly could not be resolved without the use of telomeric-specific probes. The M-FISH results were confirmed by individual chromosome-specific painting probes. CONCLUSION: M-FISH was helpful for identifying a wide range of congenital chromosomal anomalies. However, for subtle chromosomal abnormalities, use of locus-specific probes may be necessary.

Prenatal detection of aneuploidy by directly labeled multicolored probes and interphase fluorescence in situ hybridization.

OBJECTIVE: To detect aneuploidy of chromosomes 13, 18, 21, X, and Y with use of new, directly labeled, multicolored, commercially available DNA probes from interphase cells of amniotic fluid (AF). MATERIAL AND METHODS: The hybridization sites of the five probes were validated by metaphase analysis. The fluorescence in situ hybridization (FISH) normal range was determined from a series of normal AF specimens and tested on a series of normal and abnormal specimens. RESULTS: The hybridization efficiencies of the five probes were 100%. The mean AF interphase disomic signal patterns for chromosomes 13, 18, 21, XX, and XY were 95.9%, 89.1%, 94.3%, 94.7%, and 98.7%, respectively. Of a total of 508 cases analyzed, 211 were aneuploid. All cases were correctly identified and no false results occurred (in comparison with karyotypic analysis), although maternal cell contamination was relatively common. CONCLUSION: Clinical screening for aneuploidy of chromosomes 13, 18, 21, X, and Y from interphase AF cells is possible with use of these probes and FISH. Cases of maternal cell contamination and mosaicism necessitate cautious interpretation. The FISH procedure is recommended for screening of common aneuploidies, followed by a complete chromosome analysis to detect anomalies.

Tandem translocation of chromosomes 22 and 15 with two preserved satellite stalk regions and deletion 22q13.3-qter.

We describe here a case of a tandem 22/15 translocation with deletion of the 22q13.3-qter region and retention of the NOR of chromosome 15. A 2(1/2)-year-old Korean girl was referred for chromosome analysis after a clinical evaluation for developmental delay. Physical examination revealed hypotonia, developmental delay, delay of gross motor milestones and speech delay. No dysmorphic features of face, hands or feet were evident in the patient. G-banded peripheral blood lymphocyte chromosomes showed a tandem translocation between chromosomes 22 and 15, with the satellite stalks of chromosome 15 apparently being retained. All-telomere FISH analysis using a TTAGGG repeat probe showed absent signals at the junction of the translocation. Sequential G-banding and FISH analysis using a beta satellite probe showed positive signals close to the junction of the translocation, an indication that the short arms of the chromosome 15 involved in the translocation are retained. FISH with a probe for arylsulfatase, mapped to 22q13.3 region, was negative on the translocation chromosome. Therefore, the 22q13.3 region is deleted.

Method for sequential staining of GTL-banded metaphases with fluorescent-labeled chromosome-specific paint probes.

We describe a method for use of fluorescent-labeled whole chromosome-specific paint probes on GTL-banded metaphases to utilize the combined potential of these techniques for defining chromosome abnormalities. The efficacy of this method was tested on 6 cases involving different chromosome abnormalities and various tissues, including blood, amniotic fluid, skin fibroblasts, and bone marrow.

Guidelines for buccal smear collection in breast-fed infants.

Buccal smear analysis is a noninvasive, fast, and relatively inexpensive diagnostic method. It is used commonly where rapid gender identification is necessary or, more recently, for detection of aneusomy, microdeletion syndromes, and a variety of polymerase chain reaction-based molecular genetic tests. Previously we have shown that maternal cells can contaminate buccal smears taken from breast-fed infants, resulting in difficulty with test interpretation. The aim of this study was to determine optimal timing and technique for buccal smear collection in breast-fed infants in order to avoid diagnostic errors. We analyzed prospectively 50 breast-fed male infants for presence of cells with XX signal pattern from buccal mucosa scrapings at different times after breast feeding. The efficiency of mucosal cleaning on elimination of maternal cells was evaluated by comparing the proportion of XX cells before and after wiping of buccal mucosa with a cotton swab. Maternal cells were present in 23 of 48 (47.9%) samples collected within 5 min of feeding. The proportion of XX signal pattern was significantly (P = 0.001) reduced in samples collected at 30 min (8/48, P = 0.001) and > or =60 min (2/29, P = 0.0002) after feeding. Mucosal cleaning prior to smear collection significantly decreased the number of XX positive samples from 23 of 48 to 10 of 48 (P = 0.002). Buccal smears should not be obtained in nursing neonates until at least 60 min after breast feeding. In addition, prior to sample collection, buccal mucosa should be cleaned thoroughly with a cotton swab applicator. The same guidelines are applicable to older nursing infants.

Fluorescence in situ hybridization with X and Y chromosome probes for cytogenetic studies on bone marrow cells after opposite sex transplantation.

We investigated the efficacy of fluorescence-labelled chromosome probes (CEP-X/Y) for the X and Y chromosomes to study patients who have had opposite sex BMT. These probes hybridize to the centromere region of the X chromosome and nearly the entire long arm of the Y chromosome. These probes are direct-labelled and produce X and Y signals that can be simultaneously viewed and readily distinguished from each other by color and size after only five brief washes. We investigated BM specimens from 20 normal donors and 16 patients who had undergone an opposite sex BMT. We found no significant interinvestigator differences with respect to scoring XX or XY interphase cells. The 'normal range' for XX cells in males was up to 0.628% and for XY cells in females it was up to 0.299%. Each of the specimens from the patients who underwent BMT had a significant number of donor cells compared with normal range. We suggest that an economical, rapid and accurate cytogenetic test can be achieved by using these probes as an adjunct to conventional cytogenetics.

Glucocorticoids and histone deacetylase inhibitors cooperate to block the invasiveness of basal-like breast cancer cells through novel mechanisms.

Aggressive cancers often express E-cadherin in cytoplasmic vesicles rather than on the plasma membrane and this may contribute to the invasive phenotype of these tumors. Therapeutic strategies are not currently available that restore the anti-invasive function of E-cadherin in cancers. MDA-MB-231 cells are a frequently used model of invasive triple-negative breast cancer, and these cells express low levels of E-cadherin that is mislocalized to cytoplasmic vesicles. MDA-MB-231 cell lines stably expressing wild-type E-cadherin or E-cadherin fused to glutathione S-transferase or green fluorescent protein were used as experimental systems to probe the mechanisms responsible for cytoplasmic E-cadherin localization in invasive cancers. Although E-cadherin expression partly reduced cell invasion in vitro, E-cadherin was largely localized to the cytoplasm and did not block the invasiveness of the corresponding orthotopic xenograft tumors. Further studies indicated that the glucocorticoid dexamethasone and the highly potent class I histone deacetylase (HDAC) inhibitor largazole cooperated to induce E-cadherin localization to the plasma membrane in triple-negative breast cancers, and to suppress cellular invasion in vitro. Dexamethasone blocked the production of the cleaved form of the CDCP1 (that is, CUB domain-containing protein 1) protein (cCDCP1) previously implicated in the pro-invasive activities of CDCP1 by upregulating the serine protease inhibitor plasminogen activator inhibitor-1. E-cadherin preferentially associated with cCDCP1 compared with the full-length form. In contrast, largazole did not influence CDCP1 cleavage, but increased the association of E-cadherin with γ-catenin. This effect on E-cadherin/γ-catenin complexes was shared with the nonisoform selective HDAC inhibitors trichostatin A (TSA) and vorinostat (suberoylanilide hydroxamic acid, SAHA), although largazole upregulated endogenous E-cadherin levels more strongly than TSA. These results demonstrate that glucocorticoids and HDAC inhibitors, both of which are currently in clinical use, cooperate to suppress the invasiveness of breast cancer cells through novel, complementary mechanisms that converge on E-cadherin.

Overexpression of Syk tyrosine kinase in peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) are fatal in the majority of patients and novel treatments, such as protein tyrosine kinase (PTK) inhibition, are needed. The recent finding of SYK/ITK translocations in rare PTCLs led us to examine the expression of Syk PTK in 141 PTCLs. Syk was positive by immunohistochemistry (IHC) in 133 PTCLs (94%), whereas normal T cells were negative. Western blot on frozen tissue (n=6) and flow cytometry on cell suspensions (n=4) correlated with IHC results in paraffin. Additionally, western blot demonstrated that Syk-positive PTCLs show tyrosine (525/526) phosphorylation, known to be required for Syk activation. Fluorescence in situ hybridization showed no SYK/ITK translocation in 86 cases. Overexpression of Syk, phosphorylation of its Y525/526 residues and the availability of orally available Syk inhibitors suggest that Syk merits further evaluation as a candidate target for pharmacologic PTK inhibition in patients with PTCL.

Initial steps of aromatic ring formation in a laminar premixed fuel-rich cyclopentene flame.

A fuel-rich, nonsooting, premixed laminar cyclopentene flame (phi = 2.0) at 37.6 Torr (50 mbar) is investigated by flame-sampling photoionization molecular-beam mass spectrometry utilizing vacuum-ultraviolet synchrotron radiation. Mole fractions as a function of distance from the burner are measured for 49 intermediates with ion masses ranging from 2 (H2) to 106 (C8H10), providing a broad database for flame modeling studies. The isomeric composition is resolved for most species, and the identification of several C4Hx, C7H6, and C7H8 isomers is discussed in detail. The presence of C5H5CCH/C5H4CCH2 and cycloheptatriene is revealed by comparisons between flame-sampled photoionization efficiency data and theoretical simulations, based on calculated ionization energies and Franck-Condon factors. This insight suggests a new potential molecular- weight growth mechanism that is characterized by C5-C7 ring enlargement reactions.

Utility of multicolor fluorescent in situ hybridization in clinical cytogenetics.

PURPOSE: Multicolor FISH (M-FISH) was introduced in 1996 to scan all 24 chromosomes in different fluorescent colors by use of a specific filter set and computer software. However, the clinical utility of M-FISH has been limited because of the lack of commercial availability of reagents and hardware. We have evaluated M-FISH for identification of markers, derivative chromosomes, and complex karyotypes. METHODS: We present our findings based on a representative sample of one normal and six abnormal cases from a variety of tissue types. The results of M-FISH were confirmed by other well-established FISH probes. RESULTS: M-FISH analyses were successful in all six cases. The derivative chromosomes, ring, and a complex karyotype were resolved. CONCLUSIONS: We find M-FISH to be an invaluable tool for a high degree of accuracy and efficiency for chromosome identification. The limitations similar to spectral karyotyping system (SKY) include the inability to detect intrachromosomal anomalies, abnormalities involving the p-arms of acrocentrics and areas rich in highly repetitive DNA. In addition, there are some concerns of misinterpretation due to overlap of fluorophore combinations of different chromosomes, especially for subtle insertional translocations.

Maternal cell contamination of buccal smear samples in nursing neonates.

Buccal smear analysis is a non-invasive method which is being popularized by new fluorescence in situ hybridization (FISH) techniques. It is frequently used for gender identification and detection of sex chromosome aneuploidy in neonates. We attempted to determine whether or not buccal smears of nursing infants can be contaminated by maternal cells from breast feeding. FISH involving centromere specific directly labeled, multicolor probes for chromosomes X, Y and 18 were used for analysis of buccal smear samples. Buccal smear samples from 22 breast fed and 20 formula fed male neonates were analyzed in a blinded fashion. Twenty-seven percent of samples from breast fed infants had some (0.5-2.5%) XX signal pattern while the samples from formula fed infants had no XX signal pattern (difference statistically significant, p < 0.02, at 95% confidence interval). Our results indicate that breast feeding can cause maternal cell contamination of buccal smear samples that can lead to misinterpretation of results involving FISH analysis or other DNA based diagnostic studies. We have also modified the FISH technique to suit the neonates.

Detection of diagnostically critical, often hidden, anomalies in complex karyotypes of haematological disorders using multicolour fluorescence in situ hybridization.

Multicolour fluorescence in situ hybridization (M-FISH) simultaneously detects all 24 human chromosomes in unique fluorescent colours. The identification of diagnostically critical gene rearrangement(s) in complex karyotypes of haematological disorders continues to be a challenge. We present five cases in which t(9;11), complex t(8;22), t(12;21) and t(11;14) were detected primarily using M-FISH and were confirmed using locus-specific probes. We conclude that M-FISH can be effective in complete characterization of critical gene rearrangements in haematological disorders.