Optimization of QuEChERS Extraction for Determination of Carotenoids, Polyphenols, and Sterols in Orange Juice Using Design of Experiments and Response Surface Methodology.

Several compounds with different physical properties are present in foods, biological components, and environmental samples, and there are cases in which these must be analyzed simultaneously. However, it is difficult to extract compounds with different physical properties from the same sample using a single method. In the present study, we examined the optimal conditions for the QuEChERS extraction of several kinds of compounds from orange juice using design of experiments (DoE) and response surface methodology (RSM) to determine the optimal ratio of organic solvent to sodium chloride. We determined the optimal extraction conditions, which were within the design space, using 100% tetrahydrofuran (THF) as the extraction organic solvent and NaCl:MgSO4 = 75:25 as the salt. The developed LC/MS/MS method using QuEChERS extraction achieved specific detection and precise quantification. Finally, we measured the polyphenols, sterols, and carotenoids in citrus juice using the optimized QuEChERS extraction method before LC/MS/MS analysis. Most of the analytes were quantifiable in orange juice. The optimized method achieved ease of operation, the extraction of analytes from food samples in a short time (within 30 min), minimization of analytical residues, and reliability. The DoE and RSM approach may contribute to better optimization of the extraction conditions for the lowest number of experiments.

Photosynthetic 1,8-cineole production using cyanobacteria.

Terpenoid is an important group of compounds not only as biocomponents but also as useful secondary metabolites. A volatile terpenoid 1,8-cineole, which is used as a food additive, flavoring agent, cosmetic, etc., is also attracting attention from a medical perspective due to its antiinflammation and antioxidation. The 1,8-cineole fermentation using a recombinant Escherichia coli strain has been reported, although a carbon source supplement is necessary for a high-yield 1,8-cineole production. We constructed the 1,8-cineole-producing cyanobacteria toward a carbon-free and sustainable 1,8-cineole production. cnsA, the 1,8-cineole synthase gene in Streptomyces clavuligerus ATCC 27064, was introduced and overexpressed in the cyanobacterium Synechococcus elongatus PCC 7942. We succeeded in producing an average of 105.6 µg g-1 wet cell weight of 1,8-cineole in S. elongatus 7942 without supplementing any carbon source. Using the cyanobacteria expression system is an efficient approach to producing 1,8-cineole by photosynthesis.

Mechanical Properties of Thermoplastic Composites Made of Commingled Carbon Fiber/Nylon Fiber.

Commingled yarns consisting of thermoplastic nylon fibers and carbon fibers can be used to produce superior carbon fiber reinforced thermoplastics (CFRTP) by applying fiber spreading technology after commingling. In this study, we examined whether spread commingled carbon fiber/nylon fiber yarns could reduce the impregnation distance, as there are few reports on this. From this study, the following are revealed. The impregnation speed of the nylon resin on the carbon fiber was very fast, less than 1 min. As the molding time increased, the tensile strength and tensile fracture strain slightly decreased, and the nylon resin deteriorated. The effects of molding time on flexural strength, flexural modulus, and flexural fracture strain were negligible. From the cross-sectional observation conducted to confirm the impregnation state of the matrix resin, no voids were observed in the molded products, regardless of molding time or molding pressure, indicating that resin impregnation into the carbon fiber bundle of the spread commingled yarn fabric was completed at a molding pressure of 5 MPa and a molding time of 5 min.

Regression of viral pathogen indicators due to improvement of hygiene protocols on boots in a bovine farm.

Rotavirus A (RVA), bovine torovirus (BToV), bovine enterovirus (BEV) and bovine coronavirus (BCV) at a bovine farm in Ibaraki prefecture were monitored by one-step multiplex reverse transcription polymerase chain reaction (RT-PCR), with the aim of confirming the reduction of "viral pathogen indicators". A total of 960 bovine fecal samples were collected from calves less than 2 month-old within the period from October 2016 to October 2018 every 2 months at the bovine farm. In each sampling, 40 samples were taken from calves 3 week-old or less, and 40 samples from calves over 3 week-old, in principle. At the end of September 2017, the farm introduced improvement of hygiene protocols on boots by exchanging boots and appropriate usage of a footbath at the entrance of calf sheds. In the comparison of the virus detection by RT-PCR, prevalence of all 4 viruses was significantly reduced (P<0.01) in calves 3 week-old or less after the improvement. The mortality of calves less than 2 month-old was also significantly reduced after the improvement of hygiene protocols. These data suggest that the proper control of boots at calf sheds is important, perhaps even vital, for rearing hygiene measures at bovine farms so as to attain substantial decrease in the prevalence of pathogens.

Inhibitory effect of grapefruit seed extract (GSE) on avian pathogens.

The inhibitory activities of grapefruit seed extract (GSE) on avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), Salmonella Infantis (SI) and Escherichia coli (EC) were evaluated. Original GSE contained 0.24% benzalkonium chloride (BZC), however, 0.0025% BZC solution could not inactivate bacteria. The activity of diluted GSE (×100, ×500 and ×1,000 with redistilled water) against selected viruses and bacteria was evaluated in this study. The GSE solutions were incubated with the pathogens over a period of time after which the remaining viruses were titrated and the bacterial colonies were counted. In the presence of organic material-5% fetal bovine serum (FBS), the test solutions were sprayed at 1 cm and 30 cm distances to test the efficacy of GSE in a spray form. Furthermore, the efficacy of GSE against bacteria on clothes was tested using non-woven cloth. GSE×100 reduced the viral titer of both AIV and NDV even in 5% FBS condition. IBDV showed high resistance to GSE. GSE×1,000 inactivated both SI and EC within 5 sec, even in the presence of 5% FBS. The disinfectant was able to maintain its efficacy in the spray form at 30 cm distance. GSE was also effective against SI and EC inoculated on fabric. GSE is a potential novel disinfectant against viruses and bacteria, effective even within a short contact time.

Virucidal Efficacy of a Quaternary Ammonium Compound with Food Additive-Grade Calcium Hydroxide Toward Avian Influenza Virus and Newcastle Disease Virus on Abiotic Carriers.

The virucidal efficacies of a 0.2% food additive-grade calcium hydroxide [FdCa(OH)2] solution, a quaternary ammonium compound (QAC) diluted at 1:500 (QACx500), and their mixture [Mix500; FdCa(OH)2 powder added at a final concentration of 0.2% to QACx500] were investigated as fomites for avian influenza virus (AIV) and Newcastle disease virus (NDV) on abiotic carriers (steel, rubber, and plastic) at two different temperatures (room temperature [RT; 25 ± 2 C] and 2 C). These viruses were seeded on coupons (5 cm×5 cm) of rubber, steel, or plastic with 5% fetal bovine serum. After complete drying, the coupons were covered with the test solutions at RT or 2 C. After fixed incubation periods, viruses were recovered from the coupons and titrated. At RT, Mix500 required a short time (3 min) to inactivate AIV and NDV to effective levels (≥3 log virus reduction) on rubber, steel, and plastic carriers compared with QAC or FdCa(OH)2. At low temperature, QACx500 inactivated AIV on steel and plastic carriers to effective levels within 60 min, whereas Mix500 did so within 10 min. QACx500 and FdCa(OH)2 solutions could inactivate NDV on steel and plastic carriers within 20 and 10 min, respectively, and Mix500 could do so within 3 min. Viruses on the carriers required longer incubation periods for inactivation at 2 C than at 25 C. These results demonstrate desirable synergistic virucidal effects of Mix500 for important poultry viruses on abiotic carriers, while indicating high applicability within poultry farming.

Eficacia virucida de un compuesto de amonio cuaternario con hidróxido de calcio con grado aditivo alimentario contra el virus de la influenza aviar y el virus de la enfermedad de Newcastle en vehículo abióticos. Las eficacias virucidas de una solución de hidróxido de calcio [FdCa(OH)2] con grado aditivo alimentario al 0.2%, de un compuesto de amonio cuaternario (QAC) diluido a 1:500 (QACx500) y su mezcla [Mix500; FdCa(OH)2 en polvo agregado en una concentración final de 0.2% en QACx500] se investigaron en forma de fómites para el virus de la influenza aviar (AIV) y para el virus de la enfermedad de Newcastle (NDV) en vehículos abióticos (acero, hule y plástico) a dos temperaturas diferentes (temperatura ambiente [RT; 25 ± 2 C] y 2 C). Estos virus se sembraron en placas (5 cm x 5 cm) de hule, acero o plástico con suero bovino fetal al 5%. Después del secado completo, los cupones se cubrieron con las soluciones bajo ensayo a temperatura ambiente o 2 C. Después de los períodos de incubación fijos, los virus se recuperaron de las placas y se titularon. A temperatura ambiente, la mezcla Mix500 requirió un corto tiempo (tres minutos) para desactivar a los virus de influenza y de Newcastle a niveles efectivos (reducción igual o mayor de tres logaritmos en el título viral) en las placa de hule, acero y plástico en comparación con el tratamiento QAC o FdCa(OH)2. A baja temperatura, el tratamiento QACx500 inactivó al virus de la influenza en placas de acero y plástico a niveles efectivos dentro de 60 minutos, mientras que el tratamiento Mix500 lo hizo en 10 minutos. Las soluciones QACx500 y FdCa(OH)2 pudieron inactivar al virus de Newcastle en las placas de acero y plástico dentro de 20 y 10 minutos, respectivamente, y el tratamiento Mix500 pudo hacerlo dentro de tres minutos. Los virus en los vehículos requirieron períodos de incubación más prolongados para la inactivación a 2 C que a 25 C. Estos resultados demuestran los efectos virucidas sinérgicos deseables del tratamiento Mix500 para virus aviares importantes en vehículos abióticos, además de que indican una alta aplicabilidad dentro de la avicultura.

Planarian D-amino acid oxidase is involved in ovarian development during sexual induction.

To elucidate the molecular mechanisms underlying switching from asexual to sexual reproduction, namely sexual induction, we developed an assay system for sexual induction in the hermaphroditic planarian species Dugesia ryukyuensis. Ovarian development is the initial and essential step in sexual induction, and it is followed by the formation of other reproductive organs, including the testes. Here, we report a function of a planarian D-amino acid oxidase, Dr-DAO, in the control of ovarian development in planarians. Asexual worms showed significantly more widespread expression of Dr-DAO in the parenchymal space than did sexual worms. Inhibition of Dr-DAO by RNAi caused the formation of immature ovaries. In addition, we found that feeding asexual worms 5 specific D-amino acids could induce the formation of immature ovaries that are similar to those observed in Dr-DAO knockdown worms, suggesting that Dr-DAO inhibits the formation of immature ovaries by degrading these D-amino acids. Following sexual induction, Dr-DAO expression was observed in the ovaries. The knockdown of Dr-DAO during sexual induction delayed the maturation of the other reproductive organs, as well as ovary. These findings suggest that Dr-DAO acts to promote ovarian maturation and that complete sexual induction depends on the production of mature ovaries. We propose that Dr-DAO produced in somatic cells prevents the onset of sexual induction in the asexual state, and then after sexual induction, the female germ cells specifically produce Dr-DAO to induce full maturation. Therefore, Dr-DAO produced in somatic and female germline cells may play different roles in sexual induction.

A complementary-metal-oxide-semiconductor-field-effect-transistor-compatible atomic force microscopy tip fabrication process and integrated atomic force microscopy cantilevers fabricated with this process.

A complementary-metal-oxide-semiconductor-field-effect-transistor-compatible process for the fabrication of atomic force microscopy cantilevers with integrated tips has been developed. For the first time, the tips are fabricated after the completion of the regular complementary metal-oxide-semiconductor-field-effect-transistor fabrication process sequence. On-chip circuit components, such as piezoresistive deflection sensors, deflection actuators, and amplifiers, are fabricated on the mirror-polished surface of the wafer, ensuring optimal performance. The tip fabrication process is based on anisotropic silicon etching at low temperature using a tetramethylammonium hydroxide solution. The anisotropic etching process has been optimized to ensure process controllability. Using the described process, complementary-metal-oxide-semiconductor-field-effect-transistor-based cantilevers with piezoresistive deflection sensors and integrated tips have been successfully fabricated. Force-distance curves and scanning images in constant-force mode have been recorded.