Chloroplast cold-resistance is mediated by the acidic domain of the RNA binding protein CP31A

Chloroplast RNA metabolism is characterized by long-lived mRNAs that undergo a multitude of post-transcriptional processing events. Chloroplast RNA accumulation responds to environmental cues, foremost light and temperature. A large number of nuclear-encoded RNA-binding proteins (RBPs) are required for chloroplast RNA metabolism, but we do not yet know how chloroplast RBPs convert abiotic signals into gene expression changes. Previous studies showed that the chloroplast ribonucleoprotein 31A (CP31A) is required for the stabilization of multiple chloroplast mRNAs in the cold, and that the phosphorylation of CP31A at various residues within its N-terminal acidic domain (AD) can alter its affinity for RNA in vitro. Loss of CP31A leads to cold sensitive plants that exhibit bleached tissue at the center of the vegetative rosette. Here, by applying RIP-Seq, we demonstrated that CP31A shows increased affinity for a large number of chloroplast RNAs in vivo in the cold. Among the main targets of CP31A were RNAs encoding subunits of the NDH complex and loss of CP31A lead to reduced accumulation of ndh transcripts. Deletion analyses revealed that cold-dependent RNA binding and cold resistance of chloroplast development both depend on the AD of CP31A. Together, our analysis established the AD of CP31A as a key mediator of cold acclimation of the chloroplast transcriptome

Autonomous clustering using rough set theory

This paper proposes a clustering technique that minimises the need for subjective human intervention and is based on elements of rough set theory. The proposed algorithm is unified in its approach to clustering and makes use of both local and global data properties to obtain clustering solutions. It handles single-type and mixed attribute data sets with ease and results from three data sets of single and mixed attribute types are used to illustrate the technique and establish its efficiency

Expression Profiling of PBMC-based Diagnostic Gene Markers Isolated from Vasculitis Patients

Vasculitis (angiitis) is a systemic autoimmune disease that often causes fatal symptoms. We aimed to isolate cDNA markers that would be useful for diagnosing not only vasculitis but also other autoimmune diseases. For this purpose, we used stepwise subtractive hybridization and cDNA microarray analyses to comprehensively isolate the genes whose expressions are augmented in peripheral blood mononuclear cells (PBMCs) pooled from vasculitis patients. Subsequently, we used quantitative real-time polymerase chain reaction (qRT–PCR) to examine the mRNA levels of each candidate gene in individual patients. These analyses indicated that seven genes exhibit remarkably augmented expression in many vasculitis patients. Of these genes, we analyzed G0/G1 switch gene 2 (G0S2) further because G0S2 expression is also enhanced in the PBMCs of patients with systemic lupus erythematodes (SLE). We generated G0S2 transgenic mice that ubiquitously overexpress human G0S2. Although we did not observe any obvious vasculitis-related histopathologic findings in these mice, these mice are unhealthy as they produce only few offspring and showed elevated serum levels of two autoimmunity-related antibodies, anti-nuclear antibody, and anti-double strand DNA antibody. Thus, our large-scale gene profiling study may help finding sensitive and specific DNA markers for diagnosing autoimmune diseases including vasculitis and SLE

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond

LeukoCatch, a quick and efficient tool for the preparation of leukocyte extracts from blood

<p>Abstract</p> <p>Background</p> <p>Whole-protein extracts from peripheral blood leukocytes are ideal for basic and clinical research. However, lack of a simple preparation technique has limited the use of such extracts. The aim of this study is to develop a simple and easy system that can selectively obtain leukocyte extracts without hemoglobin.</p> <p>Methods</p> <p>A filter that captures the leukocytes but not RBCs was set at the bottom of a 10-mL medical syringe by sandwiching it between plastic stoppers. The capturing efficiency of leukocytes with this tool, called LeukoCatch, was examined using human macrophage cells (MONO-MAC-6). The abilities of LeukoCatch system to capture the leukocyte proteins and to remove the hemoglobin from RBCs were tested by western blot analysis using human blood samples.</p> <p>Results</p> <p>This study presents the development of LeukoCatch, a novel tool that allows the preparation of leukocyte extracts from blood samples within 3 min without centrifugation. Tissue-cultured human macrophage cells were tested to determine the optimal filter numbers and pass-through frequencies of LeukoCatch, which was then applied to 2-mL blood samples. Samples were passed 2~5 times through a LeukoCatch equipped with 5 filters, washed twice with phosphate-buffered saline for red cell removal, and leukocyte proteins were extracted with 0.5 mL of elution buffer. Western blot analysis of the purified extract indicated that more than 90% of hemoglobin was removed by the LeukoCatch and that the protein recovery rate of leukocytes was at least 4 times better than that of the conventional centrifugation method.</p> <p>Conclusion</p> <p>We conclude that LeukoCatch is useful not only for diagnosis at the bedside but also for basic research using blood samples or tissue culture cells.</p

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige–Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T0 generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T1 seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T1 generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use

Targeted plant improvement through genome editing: from laboratory to field

This review illustrates how far we have come since the emergence of GE technologies and how they could be applied to obtain superior and sustainable crop production. The main challenges of today's agriculture are maintaining and raising productivity, reducing its negative impact on the environment, and adapting to climate change. Efficient plant breeding can generate elite varieties that will rapidly replace obsolete ones and address ongoing challenges in an efficient and sustainable manner. Site-specific genome editing in plants is a rapidly evolving field with tangible results. The technology is equipped with a powerful toolbox of molecular scissors to cut DNA at a pre-determined site with different efficiencies for designing an approach that best suits the objectives of each plant breeding strategy. Genome editing (GE) not only revolutionizes plant biology, but provides the means to solve challenges related to plant architecture, food security, nutrient content, adaptation to the environment, resistance to diseases and production of plant-based materials. This review illustrates how far we have come since the emergence of these technologies and how these technologies could be applied to obtain superior, safe and sustainable crop production. Synergies of genome editing with other technological platforms that are gaining significance in plants lead to an exciting new, post-genomic era for plant research and production. In previous months, we have seen what global changes might arise from one new virus, reminding us of what drastic effects such events could have on food production. This demonstrates how important science, technology, and tools are to meet the current time and the future. Plant GE can make a real difference to future sustainable food production to the benefit of both mankind and our environment.European Cooperation in Science and Technology (COST) CA18111info:eu-repo/semantics/publishedVersio