[Chromosomal abnormalities in spontaneous miscarriage specimens detected by combinatorial probe anchor synthesis-based high-throughput low coverage whole genome sequencing].

Objective: To evaluate the application of combinatorial probe anchor synthesis (cPAS)-based high-throughput low coverage whole genome sequencing in chromosomal aberration detection in spontaneous miscarriage. Methods: From September 2015 to May 2017, spontaneous miscarriage samples were collected from Inner Mongolia Maternal and Child Health Care Hospital. Those samples were further analyzed with two independent methods, fluorescence in situ hybridization (FISH) and low coverage whole genome sequencing on the BGISEQ-500 high-throughput platform. The performance of low coverage whole genome sequencing was assessed by comparing to FISH results. Results: In 595 spontaneous miscarried specimens, low coverage whole genome sequencing revealed 144 cases (24.2%, 144/595) chromosomal abnormalities, of which a subset of 137 cases (23.0%, 137/595) were detected as aneuploidies, 2 cases (0.3%, 2/595) as mosaicisms and 5 cases (0.8%, 5/595) as copy number variation (≥5 Mb). Conclusion: cPAS-based high-throughput low coverage whole genome sequencing is a reliable method in detecting chromosomal aberrations inspontaneous abortion tissues, including chromosome aneuploidies, mosaicisms and copy number variation (≥5 Mb).

Effects of the Zhikang capsule on healing of the flap after radical breast cancer surgery.

This study aimed to investigate the effect and mechanism of trauma flap healing promoted by the Zhikang capsule after radical breast cancer surgery. The enrolled breast cancer patients were randomly divided into two groups: treatment and observation. The patients in the treatment group were treated with the Zhikang capsule in addition to the conventional dressing changes, while patients in the observation group underwent only the regular dressing changes. Serum samples of 98 breast cancer patients (with complete clinical data) who underwent modified radical mastectomy were collected and analyzed for expressions of transforming growth factor beta (TGF-ß) and basic fibroblast growth factor (bFGF). The drainage fluid amount and tissue necrosis rate were found to be lower in the treatment group than in the observation group. Moreover, bFGF expression in peripheral blood was higher in the treatment group than in the observation group. However, no significant difference was found between the two groups in the expression of TGF-ß in peripheral blood. In conclusion, Zhikang capsule is effective in promoting flap healing after radical breast cancer surgery, and the increase of bFGF expression in peripheral blood may be the underlying mechanism.

A promoter trap vector for knocking out bovine myostatin gene with high targeting efficiency.

With the development of gene targeting approaches, genomic mutation technologies in livestock animals such as gene trapping, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats and their associated systems have been improved. Although ZFNs have been used for gene targeting in many species, the off-target sites are still present. Using gene trapping, the workload of screening of targeted clones was decreased by generating a smaller number of drug-resistant clones. Determining whether the efficiency of gene trapping is lower than that of ZFNs for a specific gene has been challenging. In this study, to knock out the bovine myostatin gene, we constructed a promoter trap vector and compared its efficiency with that of ZFNs. The promoter trap vector contained a green fluorescent protein sequence without the promoter and a neomycin phosphotransferase (neo(R)) cassette driven by the phosphoglycerate kinase promoter. When the trapping vector was inserted downstream of the endogenous promoter, the fluorescent protein gene was expressed. The targeted-positive cell clones were identified based on green fluorescence and G418 double selection, followed by polymerase chain reaction analysis and sequencing. The targeting efficiency reached 5%. Compared with the efficiency of ZFN pairs (5.17 and 2.86%), the promoter trap vector PIII-myostatin could knock out the bovine myostatin gene. Therefore, gene trapping may be an effective tool for genomic modification.

Drosophila CBP is required for dorsal-dependent twist gene expression.

Although CREB-binding protein (CBP) functions as a co-activator of many transcription factors, relatively little is known about the physiological role of CBP. Mutations in the human CBP gene are associated with Rubinstein-Taybi syndrome, a haplo-insufficiency disorder characterized by abnormal pattern formation. Recently, we isolated a Drosophila CBP (dCBP) mutant, and found dCBP to be maternally expressed, suggesting that it plays a role in early embryogenesis. Mesoderm formation is one of the most important events during early embryogenesis. To initiate the differentiation of the mesoderm in Drosophila, multiple zygotic genes such as twist (twi) and snail (sna), which encode a basic-helix-loop-helix and a zinc finger transcription factor, respectively, are required. The transcription of these genes is induced by maternal dorsal (dl) protein (Dl; refs 8-10), a transcription factor that is homologous to the NF-kappa B family of proteins. The activity of dl is negatively regulated by cactus (cact), a Drosophila homologue of I kappa B. Here, we show that dCBP mutants fail to express twi and generate twisted embryos. This is explained by results showing that dCBP is necessary for dl-mediated activation of the twi promoter.

Trans-activation by the Drosophila myb gene product requires a Drosophila homologue of CBP.

Attempts to demonstrate trans-activation activity by the Drosophila myb gene product (D-Myb) have been unsuccessful so far. We demonstrate that co-transfection of Schneider cells with a plasmid expressing the Drosophila homologue of transcriptional co-activator CBP (dCBP) results in transactivation by D-Myb. Using this assay system, the functional domains of D-Myb were analyzed. Two domains located in the N-proximal region, one of which is required for DNA binding and the other for dCBP binding, are both necessary and sufficient for trans-activation. In this respect, D-Myb is similar to c-Myb and A-Myb, but different from mammalian B-Myb. These results shed light on how the myb gene diverged during the course of evolution.

Transcriptional regulation of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase in murine hepatoma cells by 6-(methylsufinyl)hexyl isothiocyanate, an active principle of wasabi (Eutrema wasabi Maxim).

Wasabi is a very popular pungent spice in Japan. This study examined the ability of 6-(methylsufinyl)hexyl isothiocyanate (6-MITC), an active principle of wasabi, to induce the cellular expression of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase (QR) in Hepa 1c1c7 cells. The cells were treated with various concentrations of 6-MITC, and were then assessed for cell growth, QR activity and QR mRNA expression. The induction of QR activity and QR mRNA expression was time- and dose-responsive over a narrow range of 0.1-5 microM, with declining induction at higher concentrations due to cell toxicity. Furthermore, transfection studies demonstrated that the induction of transcription of the QR gene by 6-MITC involved an antioxidant/electrophile-responsive element (ARE/EpRE) activation. Our results suggest a novel mechanism by which dietary wasabi 6-MITC may be implicated in cancer chemoprevention.

Fisetin induces transcription of NADPH:quinone oxidoreductase gene through an antioxidant responsive element-involved activation.

Induction of phase II enzymes such as NADPH:quinone oxidoreductase (QR) can reduce carcinogen-induced mutagenesis and tumor formation. In our search for novel dietary anticarcinogens, fisetin, a flavonol widely distributed in fruits and vegetables was found to induce QR activity in murine hepatoma 1c1c7 cells. The cells were treated with various concentrations of fisetin, and then were assessed for cell growth, QR activity, QR mRNA expression and transcription activation of the QR gene. The results showed that fisetin induced QR activity in time- and dose-dependent manner in the concentration range of 0.1 to 10 microM, and the activity induction was associated with QR mRNA expression as detected by reverse transcription-PCR. Furthermore, transfection studies using a human QR antioxidant/electrophile-response element (ARE/EpRE) reporter construct demonstrated that fisetin activated the ARE/EpRE. These results show that fisetin increases QR activity by transcriptional activation of the ARE/EpRE, suggesting a novel mechanism by which dietary fisetin may be implicated in cancer chemoprevention.

Induction of NADPH:quinone oxidoreductase in murine hepatoma cells by methylsulfinyl isothiocyanates: methyl chain length-activity study.

Methylsulfinyl isothiocyanates (MITC) are a class of isothiocyanates occurring in a variety of cruciferous vegetables showing anticarcinogenic activity. To develop analogues of methylsulfinyl isothiocyanate with less toxicity and better biological activity, four types of methyl chain length (designated as 2-, 4-, 6- and 8-MITC) were synthesized. The murine hepatoma cells (Hepa 1c1c7) were treated with various concentrations of MITC, and then assessed for cell growth, enzyme activity and mRNA expression of the detoxifying enzyme NADPH:quinone oxidoreductase (QR). All of four MITC augmented the induction of QR activity and the expression of QR mRNA in a dose-dependent manner. In the non-toxic concentration range, an increase in the methyl chain length resulted in a higher QR induction in both activity and mRNA expression. However, increasing cytotoxicity was also observed by an increase in the methyl chain length. Our results suggested that 4- and 6-MITC as QR inducers appeared to be less toxic and even more potent.

Nuclear factor-kappaB sensitizes to benzyl isothiocyanate-induced antiproliferation in p53-deficient colorectal cancer cells.

Benzyl isothiocyanate (BITC), a dietary isothiocyanate derived from cruciferous vegetables, inhibits the proliferation of colorectal cancer cells, most of which overexpress ß-catenin as a result of mutations in the genes for adenomatous polyposis coli or mutations in ß-catenin itself. Because nuclear factor-κB (NF-κB) is a plausible target of BITC signaling in inflammatory cell models, we hypothesized that it is also involved in BITC-inhibited proliferation of colorectal cancer cells. siRNA-mediated knockdown of the NF-κB p65 subunit significantly decreased the BITC sensitivity of human colorectal cancer HT-29 cells with mutated p53 tumor suppressor protein. Treating HT-29 cells with BITC induced the phosphorylation of IκB kinase, IκB-α and p65, the degradation of IκB-α, the translocation of p65 to the nucleus and the upregulation of NF-κB transcriptional activity. BITC also decreased ß-catenin binding to a positive cis element of the cyclin D1 promoter and thus inhibited ß-catenin-dependent cyclin D1 transcription, possibly through a direct interaction between p65 and ß-catenin. siRNA-mediated knockdown of p65 confirmed that p65 negatively affects cyclin D1 expression. On the other hand, when human colorectal cancer HCT-116 cells with wild-type p53 were treated with BITC, translocation of p65 to the nucleus was inhibited rather than enhanced. p53 knockout increased the BITC sensitivity of HCT-116 cells in a p65-dependent manner, suggesting that p53 negatively regulates p65-dependent effects. Together, these results identify BITC as a novel type of antiproliferative agent that regulates the NF-κB pathway in p53-deficient colorectal cancer cells.

Jun dimerization protein 2 is a critical component of the Nrf2/MafK complex regulating the response to ROS homeostasis.

Oxidative stress and reactive oxygen species (ROS) are associated with diseases such as cancer, cardiovascular complications, inflammation and neurodegeneration. Cellular defense systems must work constantly to control ROS levels and to prevent their accumulation. We report here that the Jun dimerization protein 2 (JDP2) has a critical role as a cofactor for transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) and small Maf protein family K (MafK) in the regulation of the antioxidant-responsive element (ARE) and production of ROS. Chromatin immunoprecipitation-quantitative PCR (qPCR), electrophoresis mobility shift and ARE-driven reporter assays were carried out to examine the role of JDP2 in ROS production. JDP2 bound directly to the ARE core sequence, associated with Nrf2 and MafK (Nrf2-MafK) via basic leucine zipper domains, and increased DNA-binding activity of the Nrf2-MafK complex to the ARE and the transcription of ARE-dependent genes. In mouse embryonic fibroblasts from Jdp2-knockout (Jdp2 KO) mice, the coordinate transcriptional activation of several ARE-containing genes and the ability of Nrf2 to activate expression of target genes were impaired. Moreover, intracellular accumulation of ROS and increased thickness of the epidermis were detected in Jdp2 KO mice in response to oxidative stress-inducing reagents. These data suggest that JDP2 is required to protect against intracellular oxidation, ROS activation and DNA oxidation. qPCR demonstrated that several Nrf2 target genes such as heme oxygenase-1, glutamate-cysteine ligase catalytic and modifier subunits, the notch receptor ligand jagged 1 and NAD(P)H dehydrogenase quinone 1 are also dependent on JDP2 for full expression. Taken together, these results suggest that JDP2 is an integral component of the Nrf2-MafK complex and that it modulates antioxidant and detoxification programs by acting via the ARE.

Experimental study on low pulse energy processing with femtosecond lasers for glaucoma treatment.

The feasibility of low energy processing in ocular tissues with femtosecond laser sources was investigated in this research. One laser source was a femtosecond amplifier, and the other was a femtosecond oscillator. The amplifier used in this experiment was a CPA-2001 (Clark-MXR, Inc), with 150 fs pulse duration and 1 kHz repetition rate. The femtosecond oscillator (model 900-B Mira) produced a 200 fs pulse duration and a 76 MHz repetition rate. Both these two laser systems operated at 800 nm wavelengths. Firstly, the pulse intensity thresholds in water produced by the two laser sources were compared. The optical breakdown probability analysis shows that the pulse energy threshold achieved by the oscillator was less than 10% of that achieved by the amplifier. Then, the non-linear propagation of the femtosecond pulses in the ocular tissues was studied with the femtosecond oscillator. The results showed a potential for pulse energy processing at the nanojoule level with a femtosecond oscillator in glaucoma treatment.

Femtosecond laser for glaucoma treatment: a study on ablation energy in pig iris.

Safe and effective laser ophthalmic surgery requires a fine balance between the efficiency of laser delivered and the degree of collateral side damage. The laser-ocular tissue interaction process is reliant on three main variables, namely, wavelength, pulse duration, and deposited energy. A certain amount of energy is needed to achieve ablation, while too much energy can result in unwanted collateral thermal damage. In our work the relationship between energy deposition and ablation effect is studied by an in-vitro experiment using an 800 nm wavelength 150 fs-pulse-duration laser system. This experiment aims to validate the probability of decreasing the supplied energy during glaucoma surgery by femtosecond laser. Our results show that less energy is needed using femtosecond laser than that using a longer pulse laser.

Structural difference of chymotrypsinogens forming chymotrypsin variants in Japanese quail.

A chymotrypsinogen showing the phenotype AA of chymotrypsin was purified from quail pancreas, and its chromatographic behavior, molecular weight, and electrophoretic mobility were very similar to those of another chymotrypsinogen, showing the aa phenotype of chymotrypsin. However, after activation, this chymotrypsinogen from AA showed band 5 of chymotrypsin, while the other chymotrypsinogen from aa did not. Peptide mapping demonstrated that the molecular structures of the two chymotrypsinogens are different. It was recognized that chymotrypsin variants result from activated products of different molecular structures of chymotrypsinogens.

Purification and characterization of chymotrypsinogen from pancreas of Japanese quail (Coturnix coturnix japonica).

1. A chymotrypsinogen from pancreas of Japanese quail (Coturnix coturnix japonica) was purified by acid extraction, salt fractionation and chromatographic separation on CM-cellulose and Sephadex G-100, and gave a single protein band on SDS-PAGE. 2. Quail chymotrypsinogen had a mol. wt of 26,100 calculated from amino acid composition data, an isoelectric point of 7.68, a Km of 3.1 mM and K0 of 40.7 sec-1 for tyrosine ester substrate. 3. The activated chymotrypsinogen of quail had a maximum activity at pH 7.0-8.0 and at 45 degrees C, and was stable at pH 4.0-6.0 below 55 degrees C. 4. Comparison of quail and bovine chymotrypsinogens indicates that the activities of the enzymes from quail and bovine are more constant than their physical characteristics.

Primary culture of chicken hepatocytes as an in vitro model for determining the influence of dioxin.

An easy method for primary culture of chicken hepatocytes was developed to study the influence of dioxin on birds. Chicken hepatocytes could maintain gene expression and protein secretion of albumin for a long period in serum-free medium with free atmosphere exchange at 37 degrees C. Moreover, the cells showed a sensitive response to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) by monitoring the expression of P450 1A, theta GST (theta-GST) and albumin genes.

Isolation of human chromosome 21-specific cosmids and their uses in mapping of cosmid contigs on chromosomal subregions.

A cosmid library of 3 x 10(5) clones has been constructed from a human x hamster hybrid cell line, 153E9a3, which contains human chromosome 21 (HC21) as the only human chromosome. From 56,500 clones of this library, 229 HC21-specific cosmids have been isolated by their hybridization to total human DNA and by their failure to hybridize to total Chinese hamster DNA. The cosmids isolated were then characterized, of these, 28 cosmids (12.2% of those tested) contained Not1 site(s), and 41 cosmids were localized on the eight subregions of HC21 by differential hybridization with Alu-PCR products obtained from a hybrid mapping panel. The cosmids localized were further integrated into the existing contigs using the end-specific probes of the clone insert. Therefore, they provided useful anchor points for contig mapping and walking.

Genetic studies on pancreatic proteinase in Japanese quail.

A genetic variation was found in pancreatic proteinase of Japanese quail. In eight bands of proteinase isozymes, the variation of band 5 (presence or absence) was detected among quails. Band 5 was identified as a chymotrypsin. The presence and absence of band 5 are controlled by a pair of allelic genes (Prt-5A and Prt-5a) on an autosomal locus, and gene Prt-5A, causing expression of band 5, is dominant to gene Prt-5a, a null allele for band 5. Zymograms of proteinases and their zymogens were also compared; no variation of chymotrypsinogen; corresponding to chymotrypsin Prt 5 were detected. It is suggested that the Prt-5 variant of chymotrypsin may be formed during activation of chymotrypsinogen.

Expression of cell adhesion molecule and albumin genes in primary culture of rat hepatocytes.

Changes in the expression of cell adhesion molecule and albumin genes were investigated in primary cultures of rat hepatocytes with and without poly- N-p -vinylbenzyl-D-lactonamide (PVLA) coating of the dishes. In PVLA-coated cultures, hepatocytes aggregated into spheroids and expressed liver cadherin and albumin mRNAs at higher levels. In uncoated cultures, hepatocytes revealed low levels of cadherin and albumin mRNAs, but higher levels of integrin alpha-1 mRNA. The changes in mRNA levels of liver cadherin and integrin alpha-1 coordinated well with those in spheroid and monolayer formation of hepatocytes, respectively. These results suggest that, in the PVLA-coated culture, hepatocytes expressed cadherin at higher levels to promote cell-cell adhesion and further maintain the differentiated function, such as albumin secretion, for prolonged times.

Phenotypic conversion of SV40-immortalized human diploid fibroblasts to senescing cells by introduction of an antisense gene for SV40-T antigen.

Normal human lung fibroblast diploid cells, WI-38, become senescent after a definite number of divisions. VA-13 is a line of immortalized cells established by transformation of WI-38 cells by SV40 virus. To determine whether SV40 large T (SV40-T) antigen is essential for this immortalization of WI-38 cells we introduced an antisense gene for T antigen into VA-13. Two morphologically different types of antisense transformant (VA-AS5-8 and VA-AS37-8) were obtained. In both antisense transformants the expression of T antigen was reduced by more than 70% as compared to that in the parent cells. The morphology of the antisense transformants indicated a partial conversion to the senescent phenotype of WI-38. The relative number of cells in the S phase of the antisense transformants was decreased as compared to that in cultures of VA-13 and about 50% of cells were at G1/0. The doubling time of the transformants was prolonged to close to the doubling time of WI-38. The level of expression of retinoblastoma protein (pRB) complexed with SV40-T antigen of the antisense transformants was significantly decreased although the level of total pRB was much higher than that in VA-13. The pRB was present exclusively in the underphosphorylated form. Thus, the decreased level of formation of the complex between SV40-T and pRB or the underphosphorylation of pRB may explain the suppression of growth of antisense transformants. Together, these results show that an antisense gene for SV40-T antigen can efficiently block the cell proliferation and the cell immortalization of VA-13 cells.