Biology subjects

Tsutsui, H.

Publications and source records attributed to Tsutsui, H..

7 recordsLinked to original sources

bioRxiv2024

Rapid colorimetric detection of citrus tristeza virus combining portable sample preparation and reverse transcription-loop mediated isothermal amplification

AbstractsA sensing platform combining semi-automated sample preparation protocol and one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) is reported for rapid colorimetric detection of citrus tristeza virus (CTV) in a greenhouse. An OmniLyse micro-homogenizer and cellulose paper disks were integrated for quick sample preparation of total nucleic acids (<15 min). RT-LAMP assays were optimized in terms of primers concentrations and minimization of false positives for both CTV and cytochrome oxidase (COX) detections. Specifically, the optimal reaction time for lab-based RT-LAMP assays was determined as 40 minutes with the detection limits of CTV and COX as 43 copies/L (equivalent to 86 copies/mg of tissue) and 5 copies/L (equivalent to 10 copies/mg of tissue), respectively. Additionally, an in-greenhouse colorimetric RT-LAMP assay with lyophilized reaction mix for endpoint CTV detection was successfully conducted in 35 minutes without a false response in either colorimetric or fluorometric assays. Overall, this quick sample preparation protocol integrated with the lyophilized RT-LAMP assays showed high efficiency and reliability in plant pathogen detection in a greenhouse. This strategy holds great potential to be integrated into a portable, autonomous system and be universally adopted for in-field diagnosis of different pathogens.

bioengineering↗

bioRxiv2024

A 3D-printed handheld device for quick citrus tissue lysis and nucleic acid extraction

AbstractsA 3D-printed handheld device has been developed for rapid and efficient sample preparation from citrus leaves, aimed at streamlining protocols traditionally reliant on mortar and pestle. With its high-speed motor, knurled lysis chamber for rapid sample lysis, and quick nucleic acid extraction using paper disks, this device can yield ready-to-use extracts in just 12 minutes, significantly reducing the time required for sample preparation. The device was optimized for maximum sample lysis by evaluating operation voltages and chamber features. The results showed that the lysis chamber with internal knurling and the motor operated at 7.5 V was sufficient for effective sample lysis in 1 minute, achieving total RNA concentrations up to 87.6% of those obtained with a mortar and pestle. Furthermore, concerns regarding heat generation and resin release during the lysis process were found to not impact sample quality. To further facilitate in-field diagnosis, the capability of in-device sample preparation was verified with citrus sources infected with citrus tristeza virus and Spiroplasma citri in qPCR-based assays, where low assay variations were demonstrated (< 3.8%). Overall, the in-device sample preparation integrated with the paper disks showed good reliability and compatibility across different pathogens for downstream analysis. An eco-friendly sterilization protocol using household bleach and vitamin C solution was also developed to safely reuse the device for in-field deployment.

bioengineering↗

bioRxiv2023

Microfluidic device for simple diagnosis of plant growth condition by detecting miRNAs from filtered plant extracts

Plants are exposed to a variety of environmental stress and starvation of inorganic phosphorus can be a major constraint in crop production. In plants, in response to phosphate deficiency in soil, miR399, a type of microRNA (miRNA), is upregulated. By detecting miR399, the early diagnosis of phosphorus deficiency stress in plants can be accomplished. However, general miRNA detection methods require complicated experimental manipulations. Therefore, simple and rapid miRNA detection methods are required for early plant nutritional diagnosis. For the simple detection of miR399, microfluidic technology is suitable for point-of-care applications because of its ability to detect target molecules in small amounts in a short time and with simple manipulation. In this study, we developed a microfluidic device to detect miRNAs from filtered plant extracts for the easy diagnosis of plant growth conditions. To fabricate the microfluidic device, verification of the amine-terminated glass as the basis of the device and the DNA probe immobilization method on the glass was conducted. In this device, the target miRNAs were detected by fluorescence of sandwich hybridization in a microfluidic channel. For plant stress diagnostics using a microfluidic device, we developed a protocol for miRNA detection by validating the sample preparation buffer, filtering, and signal amplification. Using this system, endogenous sly-miR399 in tomatoes, which is expressed in response to phosphorus deficiency, was detected before the appearance of stress symptoms. This early diagnosis system of plant growth conditions has a potential to improve food production and sustainability through cultivation management.

plant biology↗

bioRxiv2023

Brefeldin A and M-COPA block the export of RTKs from the endoplasmic reticulum via simultaneous inactivation of ARF1, ARF4, and ARF5

Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, which ARFs among AFR family proteins are inhibited by BFA or M-COPA, that is, which ARFs are involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.

cell biology↗

bioRxiv2019

Micrografting device for testing environmental conditions for grafting and systemic signaling in Arabidopsis

SummaryGrafting techniques have been applied in studies of systemic, long-distance signaling in several model plants. Seedling grafting in Arabidopsis, known as micrografting, enables investigation of the molecular mechanisms of systemic signaling between shoots and roots. However, conventional micrografting requires a high level of skill, limiting its use. Thus, an easier user-friendly method is needed. Here, we developed a silicone microscaled device, the micrografting chip, to obviate the need for training and to generate less stressed and more uniformly grafted seedlings. The chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro-path with pairs of pillars for hypocotyl holding. Grafting, including seed germination, micrografting manipulation, and establishment of tissue reunion, is performed on the chip. Using the micrografting chip, we evaluated the effect of temperature and the carbon source on grafting and showed that a temperature of 27{degrees}C and a sucrose concentration of 0.5% were optimal. We also used the chip to investigate the mechanism of systemic signaling of iron status using a quadruple nicotianamine synthase (nas) mutant. The constitutive iron-deficiency response in the nas mutant because of aberrant partitioning was significantly rescued by grafting of wild-type shoots or roots, suggesting that shoot-and root-ward translocation of nicotianamine-iron complexes is essential for iron mobilization. Thus, our micrografting chip will promote studies of long-distance signaling in plants. Significance StatementA number of micrografting studies on systemic, long-distance signaling have been performed, but the technique is not yet used widely. Here, we developed a silicone-based micrografting chip to improve the ease-of-use, efficiency, and success rate of micrografting, even for untrained users.

plant biology↗

bioRxiv2018

mitoNEET Regulates Mitochondrial Iron Homeostasis Interacting with Transferrin Receptor

Iron is an essential trace element for regulation of redox and mitochondrial function, and then mitochondrial iron content is tightly regulated in mammals. We focused on a novel protein localized at the outer mitochondrial membrane. Immunoelectron microscopy revealed transferrin receptor (TfR) displayed an intimate relationship with the mitochondria, and mass spectrometry analysis also revealed mitoNEET interacted with TfR in vitro. Moreover, mitoNEET was endogenously coprecipitated with TfR in the heart, which indicates that mitoNEET also interacts with TfR in vivo. We generated mice with cardiac-specific deletion of mitoNEET (mitoNEET-knockout). Iron contents in isolated mitochondria were significantly increased in mitoNEET-knockout mice compared to control mice. Mitochondrial reactive oxygen species (ROS) were higher, and mitochondrial maximal capacity and reserve capacity were significantly decreased in mitoNEET-knockout mice, which was consistent with cardiac dysfunction evaluated by echocardiography. The complex formation of mitoNEET with TfR may regulate mitochondrial iron contents via an influx of iron. A disruption of mitoNEET could thus be involved in mitochondrial ROS production by iron overload in the heart.

cell biology↗

bioRxiv2018

Adaptive Reduction of Male Gamete Number in a Selfing Species

The number of male gametes produced is critical for reproductive success and varies greatly between and within species1-3. Evolutionary reduction of male gamete production has been widely reported in plants as a hallmark of the selfing syndrome, as well as in humans. Such a reduction may simply represent deleterious decay4-7, but evolutionary theory predicts that breeding systems could act as a major selective force on male gamete number: while large numbers of sperm should be produced in highly promiscuous species because of male-male gamete competition1, reduced sperm numbers may be advantageous at lower outcrossing rates because of the cost of gamete production. Here we used genome-wide association study (GWAS) to show a signature of polygenic selection on pollen number in the predominantly selfing plant Arabidopsis thaliana. The top associations with pollen number were significantly more strongly enriched for signatures of selection than those for ovule number and 107 phenotypes analyzed previously, indicating polygenic selection8. Underlying the strongest association, responsible for 20% of total pollen number variation, we identified the gene REDUCED POLLEN NUMBER 1 affecting cell proliferation in the male germ line. We validated its subtle but causal allelic effects using a quantitative complementation test with CRISPR-Cas9-generated null mutants in a nonstandard wild accession. Our results support polygenic adaptation underlying reduced male gamete numbers.

evolutionary biology↗