Localization of Avian Influenza Virus in Formalin-Fixed, Paraffin-Embedded Chicken Tissues by In Situ Hybridization

In this study, in situ hybridization （ISH） was developed to detect avian influenza＇virus （AIV） in Madin-Darby canine kidney （MDCK） cells and formalin-fixed, paraffin-embedded chicken tissues. A cDNA probe corresponding to a region of AIV nucleoprotein （NP） gene was synthesized and labeled with digoxigenin. Probe specificity was determined by AIV infected MDCK cells in vitro and the results showed that strong cytoplasmic staining was only detected in AIV-infected cells. Various tissues were collected from 12 h to 35 days post-infection （PI） following inoculation with the H9N2 subtype A1V. AIV was localized in the epithelial cells of the duodenum and cartilage of the throat and trachea at 12 h PI. Tissues from uninfected chickens were negative. The finding of this study indicated ISH was a sensitive and specific technique to detect and localize AIV as well as to study AIV pathogenesis.

Detection of Embryo Sex Chromosome by Dual Color Fluorescent In-Situ Hybridization

In order to evaluate the effects of sex chromosomal mosaicism on the accuracy of single-cell gender diagnosis, sex chromosomes of 21 normal fertilized embryos were detected by dual color fluorescent in-situ hybridization (FISH). The results showed that 4 embryos had sex chromosomal mosaicism (19%) and the remaining 17 showed uniformly XX or XY signals in all blastomeres. In conclusion, identification of sex by dual color FISH analysis of a single cell was accurate and efficient , and sex chromosomal mosaicism would not affect preimplantation gender diagnosis.

Matrix Induced Synthesis of Eu^(3+) Doped Zn_3(PO_4)_2 and LaPO_4 Phosphors by in-Situ Composing Hybrid Precursors

Using rare earth and zinc coordination polymers with aromatic carboxylic acids as the precursors, composing with the polyethylene glycol (PEG) as the dispersing media, micro crystalline phosphors Zn_3(PO_4)_2∶Eu 3+ and LaPO_4∶Eu 3+ were synthesized by in-situ co-precipitation method. X-ray diffraction and scanning electronic micrograph were used to characterize the resultant samples, whose particle size are in the range of micrometer. The emission spectra of Zn_3(PO_4)_2∶Eu 3+ (λ_ ex=245 nm) and LaPO_4∶Eu 3+ (λ_ ex=390 nm) shows that the emission for Eu 3+ in Zn_3(PO_4)_2 is dominated by the 5D_0→7F_1 (592 nm) magnetic-dipole transition,While the dominant emission for Eu 3+ in LaPO_4 is the typical hypersensitive transition 5D_0→7F_2 (618 nm).

Preparation and mechanical properties of TLCP/ UP / GF in-situ hybrid composites

One kind of novel reactive thermotropic liquid crystalline polymer-methacryloyl copolymer (LCMC) containing polyester mesogenic units was synthesized. Its structure, morphology and properties were investigated systemically by Ubbelohde viscometer, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), wide-angle X-ray diffractometry (WAXD) and polarizing optical microscopy (POM). The results indicate that it is one kind of nematic thermotropic liquid crystal polymer (TLCP). The impact strength, bending strength and the morphologies of impact fracture surface of LCMC, unsaturated polyester (UP) and glass fiber (GF) in-situ hybrid composites were studied by Izod impact tester, universal testing machine and scanning electron microscopy (SEM), respectively. The results show that the impact and bending strength of composites containing LCMC are improved, especially the composites containing 5% LCMC increases most obviously. These results with SEM results reveal that LCMC plays an important role in the improvement of interfacial adhesive between matrix and fiber.

Green Approach for <i>In-Situ</i>Growth of CdS Nanorods in Low Band Gap Polymer Network for Hybrid Solar Cell Applications

In-situ growth of CdS nanorods (NRs) has been demonstrated via solvothermal, in a low band gap polymer, poly [[4,8-bis[(2-ethylhexyl)oxy] benzo [1,2-b:4,5-b’] dithiophene-2,6-diyl] [3-fluoro-2-[(2-ethylhexyl) carbonyl] thieno [3,4-b] thiophenediyl]] (PTB7). It is a high yielding, green approach as it removes use of volatile and hazardous chemicals such as pyridine as ligand which are conventionally used to synthesize precursors of CdS (NRs). Moreover the solvothermal process is a zero emission process being a close vessel synthesis and hence no material leaching into the atmosphere during the synthesis. The PTB7:CdS nanocomposite has been characterized by SEM, XRD, FTIR, UV-visible spectroscopy techniques. The photoluminescence (PL) spectroscopy study of PTB7 with CdS NRs has shown significant PL quenching by the incorporation of CdS NRs in PTB7;this shows that CdS NRs are efficient electron acceptors with the PTB7. The PTB7:CdS is used as active layer in the fabrication of hybrid solar cells (HSC) as donor-acceptor combination in the bulk heterojunction (BHJ) geometry. The HSCs fabricated using this active layer without any additional supporting fullerene based electron acceptor has given power conversion efficiency of above 1%.

The Distribution of Repetitive DNAs Along Chromosomes in Plants Revealed by Self-genomic in situ Hybridization

The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） procedure, fluorescence in situ hybridization （FISH） with genomic DNA to their own chromosomes （called self-genomic in situ hybridization, self-GISH） was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions （NORs）. The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata, and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with Cot-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with repetitive DNAs in plants.

Detection of Differentiation Among BB, CC and EE Genomes in the Genus Oryza by Two-probe Genomic in situ Hybridization (GISH)

The genus Oryza consists of two cultivated species (O. sativa L. and O. glaberrima Steud.) and approximately 20 wild relative species widely distributed in the pan-tropics. These species have been classified into four complexes following the Vaughan's taxonomic system([1]). The O. officinalis complex is the largest complex in the genus, which includes ten species, having BE, CC, on, and EE genomes in the diploids as well as BBCC and CCDD genomes in the tetraploids. The relationships among the BE, CC, and EE genomes still remain unclear, although previous studies have indicated certain affinities of these genomes([2-4]). Genomic in situ hybridization (GISH) is a powerful technique to detect the relationships among the related genomes at chromosome and DNA levels. The objective of the present study was to investigate the relationships among the BE, CC and EE genomes in the genus Oryza by the two-probe GISH.

六氰合铁酸铜钴薄膜修饰电极的in-situ FTIR研究

本文用循环伏安法在铂电极的表面电沉积了六氰合铁酸铜钴薄膜 ,结合电化学方法 ,研究了修饰膜氧化过程中的现场红外光谱。

In situ hybridization assay of androgen receptor gene in hepatocarcinogenesis

AIM To determine the correlation between expression of androgen receptor (AR) gene and hepatocarcinogenesis. METHODS Male SD rats were used as experimental animals and the animal model of experimental hepatocarcinoma was established by means of 3′ me DAB administration. Androgen receptor mRNA was detected by a non radioactive in situ hybridization assay in neoplastic and non neoplastic liver tissues. RESULTS The expression of androgen receptor mRNA was observed only in neoplastic cells and some atypical hyperplastic cells. In the liver tissue of control animal and the remaining normal liver cells adjacent to the carcinoma tissue, no positive signal was seen. CONCLUSION Androgen has an important correlation with hepatocarcinogenesis and the expression of androgen receptor gene might be a mark event during hepatocarcinogenesis.

Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modem lily cultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the FI hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC1 progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic （LA） hybrids in Lilium. Therefore, the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research. The results showed that： at metaphase I, the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell （PMC）, and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva- lents were disjoined and most univalents were divided. Both the disjoined bivalents （half-bivalents） and the divided univalents （sister chromatids） moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents, were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the Fl LA hybrids and the variation of their BCx progenies, but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.

Chromosome analysis of esophageal squamous cell carcinoma cell line KYSE 410-4 by repetitive multicolor fluorescence in situ hybridization

Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization （M-FISH） for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermediate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chromosomes in the whole genome of human tumors.

Dual fluorescence in situ hybridization in detection of HER-2 oncogene amplification in primary hepatocellular carcinoma

BACKGROUND: Molecular cytogenetics of oncogene HER-2 amplification in primary hepatocellular carcinoma (HCC) is still unknown. The aim of this study was to in vestigate the frequency of HER-2 oncogene amplification in primary HCC and its relations to clinicopathological pa rameters and prognosis. METHODS: Forty-two surgical samples from patients with primary HCC were detected for their HER-2 oncogene am plification. The number of chromosome 17 and their ratio were tested by dual fluorescence in situ hybridization (FISH) technique, and then the correlations between HER-2 amplification, clinicopathological characteristics and prog nosis were analyzed statistically. RESULTS: HER-2 oncogene amplification was detected in 9 (21.4%) of the 42 primary HCCs, including 4 patient with high copy (HC) (9.5%) and 5 patients with low copy (LC) (11.9%). HER-2 amplification was associated signifi cantly with tumor size and postoperative survival time o HCC patients (P<0.05), and the presence of HER-2 gene amplification was correlated with postoperative relapse (P— 0.257), but not related to sex, age, AFP level, HBV infec tion, histopathological grading and clinical staging of HCC patients (P>0.05). The HER-2 oncogene copy was exa mined in 31 (73.8%) of the 42 primary HCCs, consisting of 9 patients with HER-2 amplification (21.4%) and 22 pa tients with aneuploidy (52.4%). No significant relation were observed between the HER-2 oncogene copy, patien sex, tumor size, histopathological grading, clinical stag ing, postoperative relapse and survival time (P >0.05); bu the HER-2 oncogene copy was correlated significantly to age, AFP level and HBV infection (P <0.05). CONCLUSIONS: There are a lower frequency of HER-2 oncogene amplification and a higher frequency of chromo- some 17 aneuploidy in primary HCC. HER-2 oncogene amplification may be involved in the development and pro- gression of large HCC in some patients, and seems to be a valuably independent prognostic factor predicting the re- currence and poor survival in patients with large HCC.

Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H pylori infection and determination of clarithromycin resistance in H pylori eradication therapy

H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradicati-on therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue secti-ons. This technique is helpful for determining the bac-terial density and the results of treatment where clarith-romycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.

Comparison of Fluorescence in situ Hybridization and Immunohistochemistry for Assessment of HER-2 Status in Breast Cancer Patients

The accurate assessment ofa proto-oncogene, human epidermal growth factor receptor-2 gene （HER-2）, is extremely important for the therapy and prognosis of breast cancer. Currently, immunohistochemistry （IHC） is the method widely used for the detection of HER-2 protein. Fluorescence in situ hybridization （FISH） has been suggested to be a golden standard assay for HER-2 amplification. This study examined the expression and amplification of HER-2 in paraffin-embedded sections of breast cancer tissues, and compared the two methods on the measurement of HER-2 status. HER-2 gene and protein were determined in breast cancer samples from 52 Chinese women by FISH and IHC respectively. The findings indicated that the HER-2 gene amplification was found in 18 cases （34.6%） by FISH and the HER-2 protein over-expression （score 3＋） in 15 cases （28.8%） by IHC. hnmunohistochemically, 28.6% of the cases scored as 2＋ and 93.3% of the cases scored as 3＋ were HER-2-positive by FISH. There was a significant correlation between the HER-2 gene amplification and HER-2 protein over-expression in breast cancer （P〈0.005）. No correlation was noted between the HER-2 gene amplification and any of the clinicopathological parameters examined, including age, menopausal status, menarche age, tumor size, histological tumor type, histological grade, lymph node status, and the expression of ER and PR. It was concluded that the detection of HER-2 gene amplification in breast cancer by FISH is valuable and can compare with HER-2 protein detection by IHC.

Detection of H.pylori DNA in gastric epithelial cells by in situ hybridization

AIM: To investigate the presence of H.pylori DNA within gastric epithelial cells in patients with H.pylori infection and its possible carcinogenic mechanism. METHODS: Total 112 patients, with pathologically confirmed chronic superficial gastritis, chronic atrophic gastritis, intestinal metaplasia, atypical hyperplasia or gastric cancer were studied. Among them, 28 were H.pylori negative and 84 H.pylori positive. H.pylori DNA in gastric epithelial cells was detected by GenPoint catalyzed signal amplification system for in situ hybridization. RESULTS: In the H.pylori positive group, zero out of 24 chronic superficial gastritis (0.0%), four out of 25 precancerous changes (16.0%) and thirteen out of 35 gastric cancers (37.1%) showed H.pylori DNA in the nucleus of gastric epithelial cells, the positive rates of H.pylori DNA in the nucleus of gastric epithelial cells were progressively increased in chronic superficial gastritis, precancerous changes and gastric cancer groups (chi(2)=12.56, P=0.002); One out of 24 chronic superficial gastritis (4.2%), eleven out of 25 precancerous changes (44.0%) and thirteen out of 35 gastric cancers (37.1%) showed H.pylori DNA in the cytoplasm of gastric epithelial cells (chi(2)=10.86, P=0.004). In the H.pylori negative group, only one patient with gastric cancer was found H.pylori DNA in the nucleus of gastric epithelial cells; Only two patients, one patient with precancerous changes and another with gastric cancer, showed H.pylori DNA in the cytoplasm of gastric epithelial cells. Furthermore, H.pylori DNA must have been in the cytoplasm as long as it existed in the nucleus of gastric epithelial cells. CONCLUSION: H.pylori DNA exists both in the nucleus and the cytoplasm of gastric epithelial cells in patients with H.pylori infections. The pathological progression from chronic superficial gastritis, precancerous changes to gastric cancer is associated with higher positive rates of H.pylori DNA presence in the nucleus of gastric epithelial cells.

DETECTION OF WHITE SPOT SYNDROME VIRUS(WSSV) OF PENAEUS CHINENSIS BY IN SITU HYBRIDIZATION

White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I digested fragments of the WSSV genome were cloned; three of these fragments were used as non radioactive probes labeled with DIG 11 dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp.

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization

The genomic structures of Oryza sativa （A genome） and O. meyeriana （G genome） were comparatively studied using bicolor genomic in situ hybridization （GISH）. GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa （A-A）, O. sativa-O, meyeriana （A-G） and O. meyeriana-O, meyeriana （G-G）, were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.

THE IMMUNOHISTOCHEMISTRY AND IN SITU cDNA-mRNA HYBRIDIZATION OF CARBAMYL PHOSPHATE SYNTHETASE I IN ENZYME-ALTERED LIVER CELLS DURING CARCINOGENESIS

The changes of carbamyl phosphate synthetase I(CPS 1)in diethylnitrosamine-(DEN)-inducedenzyme-altered liver cells were studied by means of immunohistochemical(PAP)and in situcDNA-mRNA hybridization methods.The experimental rats were treated with DEN,2-acetylaminofluorene(2-AAF)and 2/3 hepatectomy according to Solt-Farber’s protocol andwere further promoted by oral daily administration of 0.05% phenobarbital in drinking water.The results showed that the average number of lesions showing abnormal expression of CPS1 was relatively constant over the course of the experiment(8 months),while the numberof normally expressing lesions gradually decreased.The former lesions were also largerin volume than the latter ones.We conclude that in DEN-initiated lesions the abnormallyexpressed CPS 1 lesions may grow continuously,thus leading to the formation of largernodules.We also suspect that some of these lesions have increased tendencies to developinto tumors.

Preparation and characterization of biodegradable aliphatic-aromatic copolyesters/nano-SiO_2 hybrids via in situ melt polycondensation

In situ melt polycondensation was proposed to prepare biodegradable aliphatic-aromatic copolyesters/nano-SiO2 hybrids based on terephthalic acid （TPA）, poly（L-lactic acid） oligomer （OLLA）, 1,4-butanediol （BDO） and nano-SiO2. TEM and FT-IR characterizations confirmed that TPA, OLLA and BDO copolymerized to obtain biodegradable copolyesters, poly（butylene terepbthalate-co-lactate） （PBTL）, and the abundant hydroxyl groups on the surface of nano-SiO2 provided potential sites for in situ grafting with the simultaneous resulted PBTL. The nano-SiO2 particles were chemically wrapped with PBTL to form PBTL/nano- SiO2 hybrids. Due to the good dispersion and interfacial adhesion of nano-SiO2 particles with the copolyester matrix, the tensile strength and the Young＇s modulus increased from 5.4 and 5.6 MPa for neat PBTL to 16 and 390 MPa for PBTL/nano-SiO2 hybrids with 5 wt.% nano-SiO2, respectively. The mechanical properties of PBTL/nano-SiO2 hybrids were substantially improved.