PCR-RFLP identification of prohibited animal derived DNA in animal feed

A method for confirming identification of prohibited species tissue in animal feed has been developed on the basis of PCR-RFLP analysis. In Japan, to prevent the spread of BSE through animal feed, the use of animal protein in feed has been regulated. Species-specific PCR detection of prohibited species materials in feed has been used as one of a series of laboratory tests to ensure the proper implementation of the feed regulations. However, since the result of this PCR method is determined only by amplicon length, it is sometimes necessary to confirm whether or not the positive result is due to the effect of a non-specific reaction. For this purpose, DNA sequencing is the best way to confirm the test result but it is not suitable for routine analysis because of the required time and cost. In this study, we developed an easy and rapid method to confirm the species identification (mammals, ruminants and cattle) by using 4 restriction enzymes: SmlI, MboI, BlnI and Hpy188III. This PCR-RFLP method, which ensures identification of prohibited animal species in feed, is useful for enhancing the reliability of feed inspection for BSE prevention. This method will be added to the Official Methods of Feed Analysis.     

Sensitive and spatially multiplexed detection system based on dielectrophoretic manipulation of DNA-encoded particles used as immunoreactions platform

In this work, we designed a new immunodevice that combines competitive immunoreactions on the microparticles, accumulation of these particles by negative dielectrophoresis (n-DEP), and their subsequent capture through hybridization among single-stranded DNAs (ssDNAs). Two widely used pesticides, atrazine and bromopropylate, were used as target molecules to test the resulting simultaneous detection system. For sensing, we prepared two different sets of microparticles: one modified with atrazine-conjugated bovine serum albumin (BSA-2d) and ssDNA-J1(up) and the other with bromopropylate-conjugated aminodextran (AD-155) and ssDNA-J2(up). The microparticles were incubated in a mixture of analyte-specific antibody and analyte at different concentrations to trap the unreacted antibodies prior to being labeled with antibodies conjugated with a fluorescence molecule. A suspension containing both types of microparticles was introduced into an n-DEP device consisting of an interdigitated microarray (IDA) electrode and channel modified with ssDNA-J1(down) and ssDNA-J2(down), which are complementary to ssDNA-J1(up) and ssDNA-J2(up), respectively. The n-DEP force generated by applying AC voltage to the IDA electrode displaced the microparticles toward the encoded areas, causing them to rapidly accumulate on the upper surfaces. Hybridization allowed us to distinguish the microparticles and sense multiple analytes by spatial recognition in the DNA-encoded areas. The fluorescence intensity of the captured particles, which depends on analyte concentrations, was measured selectively by focusing on specific areas. The strategy is advantageous for sensitivity due to the equivalent trapping efficiency by DNA hybridization and large surface area of the microparticle for immunoreactions. The rapidity and simplicity were still supported by particle manipulation. Using this concept, we detect atrazine and bromopropylate simultaneously with limits of detection (LODs) of 0.2 μg·L(-1), which covered the maximum residue level (MRL) in food samples established the European Union (EU) and Japan Ministry of Health, Labor and Welfare (MHLW).     

Detection of surface antigens on living cells through incorporation of immunorecognition into the distinct positioning of cells with positive and negative dielectrophoresis

Rapid determination of surface antigens on cells is possible by immobilization of cells accumulated by positive dielectrophoresis (p-DEP) via effective surface immunoreactions and removal of unbound cells by negative DEP (n-DEP). The DEP device for cell manipulation comprises a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower ITO microband array electrode (band electrode) modified with an antibody. Cells with the surface antigen introduced into the channel immediately accumulated on the surface of the band electrode during p-DEP generated by the application of ac voltage between the ITO electrode and the band electrode to immobilize by the specific antibody. The removal of accumulated cells to the gap region during n-DEP was used for rapid estimation of the residual cells with a specific surface antigen. We demonstrate here that human promyelocytic leukemia cells with the surface antigen CD33 can be captured on a band electrode modified with anti-CD33 antibody. The time required for the determination of the surface antigen using this compelled accumulation of cells by p-DEP and the separation of unbound cells by n-DEP is decreased to 60 s compared to that required by a cell binding assay using microtiter plates (30 min). Furthermore, the present method for a novel cell binding assay does not require pretreatment such as target labeling or washing of unbound cells and thereby enhancing throughput in the clinic and in cytobiology studies.     

Crack growth characteristics of carbon nanotube-based polymer composites subjected to cyclic loading

This paper presents the characterization of crack growth in carbon nanotube (CNT)-based polymer composites under fatigue loading. Fatigue crack growth tests were performed on single-edge cracked plate specimens of CNT/polycarbonate composites at room temperature and liquid nitrogen temperature (77 K). An elastic–plastic finite element analysis was also conducted to determine the J-integral range. The crack growth rate data were expressed in terms of the J-integral range, and the effect of nanotube addition on the fatigue crack growth behavior was examined. In addition, possible mechanisms of the crack growth in the nanocomposites are discussed based on microscopic observations of the specimen fracture surfaces.     

A Simple Pneumatic Loading System Controlling Stress and Strain Rates for One-Dimensional Compression of Clay

A simple but automated pneumatic loading system that can control the stress and strain rates for one-dimensional (1D) compression of clay was developed. The rate-dependency of stress-strain behaviour due to the viscous property of clay was investigated by 1D compression tests on standard-size specimens by various loading methods: 1) Standard Consolidation Tests (SCTs), stepwise increasing the axial stress two times every one day; 2) ordinary Constant-Rate-of-Strain (CRS) tests at different strain rates; 3) special CRS tests including unloading and reloading cycles with different stress amplitudes at strain rates of which the absolute value was either kept constant throughout respective tests or changed at the start of reloading; and 4) special CRS tests including a number of sustained loading (SL) during otherwise primary loading or unloading or reloading at constant strain rate. Sufficiently low strain rates were employed to ensure essentially fully drained condition. The followings were found. Despite that the newly developed pneumatic loading system is rather simple, 1D compression tests following such various loading histories as above can be performed on four types of clay rather accurately. The stress-strain behaviour of clay is significantly rate-dependent, exhibiting significant creep strains at SL stages. The creep strain rate is significantly different whether SL starts during otherwise primary loading or unloading or reloading, controlled by the magnitude and sign of the initial strain rate at the start of SL. The whole observed trends of rate-dependent stress-strain behaviour can be qualitatively explained by the non-linear three-component elasto-viscoplastic model extended to cyclic loading conditions.     

Thin reduced graphene oxide interlayer with a conjugated block copolymer for high performance non-volatile ferroelectric polymer memory

Polymer ferroelectric-gate field effect transistors (Fe-FETs) employing ferroelectric polymer thin films as gate insulators are highly attractive as a next-generation non-volatile memory. For minimizing gate leakage current of a device which arises from electrically defective ferroelectric polymer layer in particular at low operation voltage, the materials design of interlayers between the ferroelectric insulator and gate electrode is essential. Here, we introduce a new solution-processed interlayer of conductive reduced graphene oxides (rGOs) modified with a conjugated block copolymer, poly(styrene-block-paraphenylene) (PS-b-PPP). A FeFET with a solution-processed p-type oligomeric semiconducting channel and ferroelectric poly(vinylidene fluoride-co-trifluoroethylene) (PVDF-TrFE) insulator exhibited characteristic source–drain current hysteresis arising from ferroelectric polarization switching of a PVDF-TrFE insulator. Our PS-b-PPP modified rGOs (PMrGOs) with conductive moieties embedded in insulating polymer matrix not only significantly reduced the gate leakage current but also efficiently lowered operation voltage of the device. In consequence, the device showed large memory gate voltage window and high ON/OFF source–drain current ratio with excellent data retention and read/write cycle endurance. Furthermore, our PMrGOs interlayers were successfully employed to FeFETs fabricated on mechanically flexible substrates with promising non-volatile memory performance under repetitive bending deformation.     

Photooxidative degradation of polyethylene containing flame retardants by monochromatic light

Photodegradation of incombustible polymer materials [high-density (HD) and low-density (LD) polyethylene (PE) containing 0.5 to 2.0 phr of decabromodiphenyl ether (DBDE) or tetrabromobisphenol A (TBA) as a flame retardant] were studied using an Okazaki Large Spectrograph (OLS). Samples were irradiated in air at 23°C with monochromatic light of wavelengths at 260, 280, 300, 320, 340, and 360 nm. Ultraviolet and Fourier transform infrared (FTIR) spectra were taken to estimate the chemical changes caused by photoirradiation. Molecular weight change was followed by gel permeation chromatography (GPC) measurements. It was found that the photostability of PE samples was reduced by the addition of flame retardants. The threshold wavelengths of photodegradation are 320 nm and 360 nm for PE–TBA samples and PE–DBDE samples, respectively. Main-chain scission is favored when the irradiation was carried out with the light of wavelength 300 nm for HDPE–DBDE and HDPE–TBA samples. The most effective irradiation wavelengths for crosslinking are found to be 300 nm and 280 nm for LDPE–DBDE and LDPE–TBA samples, respectively. 1995 John Wiley & Sons, Inc.     

Suppression by carotenoids of microtenoids of microcystin-induced morphological changes in mouse hepatocytes

Microcystin-LR is a liver tumor promoter in the okadaic acid class, a group of potent inhibitors of protein phosphatases 1 and 2A. Because of inhibition of protein phosphatases, microcystin-LR induces hyperphosphorylation of cellular proteins, including cytoskeletal proteins—cytokeratins 8 and 18—and causes morphological changes in mouse hepatocytes in primary culture. We studied the effects of carotenoids to antagonize microcystin-LR-induced morphological changes in hepatocytes. β-Carotene (100 nM to 100 μM), suppressed the morphological changes induced by 100 nM microcystin-LR in a dose-dependent manner. Other carotenoids tested exerted similar suppressive effects, although retinoids, such as all-trans retinol, all-trans retinoic acid, and 9-cis retinoic acid, were only weakly suppressive. The relative potency of the suppression correlated significantly with the number of conjugated double bonds in thetrans configuration. β-Carotene strongly suppressed the hyperphosphorylation of cellular proteins induced by microcystin-LR without significant changes in the basal phosphorylation level. Other antioxidants, such as α-tocopherol, did not protect the cells against microcystin-LR. Taken together, the antagonistic effects of carotenoids against microcystin-LR are difficult to explain by their antioxidant or provitamin A activities. Suppression of the hyperphosphorylation of cellular proteins may be a novel mechanism by which carotenoids inhibit tumor promotion.     

Synthesis of N-Doped ZnO by grinding and subsequent heating ZnO-urea mixture

Synthesis of nitrogen (N) doped zinc oxide (ZnO) has been attempted by a serial operation of co-grinding the mixture of ZnO and urea (CO(NH₂)₂), followed by calcining the ground product at 400 °C. The prepared ZnO sample has been characterized by X-ray diffraction (XRD) analysis, infrared spectroscopy (IR), Raman spectroscopy. All spectra from these techniques are completely different from those of the hand ground mixture sample, confirming the mechanochemical effect of N-doping. The prepared N-doped ZnO sample exhibits light absorbance in the visible light wavelength region, and has high photo-catalytic ability in an anti-bacterial test.     

Presence of Streptococcus anginosus DNA in esophageal cancer, dysplasia of esophagus, and gastric cancer

We recently reported cloning of Streptococcus anginosus (S. anginosus) DNA fragments containing the 16S ribosomal gene from DNA samples of surgical specimens of gastric cancers. To investigate the specificity of S. anginosus infection, Southern blot analysis with S. anginosus 16S ribosomal DNA probe and PCR analysis with S. anginosus-specific primers were performed in DNA samples prepared from 15 esophageal cancers, 43 gastric cancers, 16 lung cancers, 10 cervical cancers, 14 renal cell carcinomas, 10 colorectal cancers, and 19 bladder cancers. We frequently found S. anginosus DNA sequences in DNA samples from esophageal cancer and gastric cancer tissues, as well as in those from dysplasia of the esophagus of esophageal cancer patients. No S. anginosus DNA bands were detected by Southern blot analysis on DNAs from the noncancerous portions of the esophagus or the stomach. By PCR analysis with 35 cycles, only 7% of the noncancerous portion of the esophagus was shown to contain S. anginosus sequences. No S. anginosus sequences were found in DNAs from cancers in lung, cervix, and kidney, but they were found in 1 of 10 colon cancers.