Biology subjects
Publications and source records attributed to Okada, S..
Small mammals harbor various zoonotic viruses and are natural reservoirs for emerging viruses. Here, we identified a novel orthonairovirus, which is genetically close to the virus suggested the association with human neural diseases. The virus was found in 24.6% of the small mammals captured in Gabon, Central Africa.
Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing and their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a software to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in both insertion of gene fragments to essential genes and complete deletion of an open reading frame. The backbone vectors with the software would thus provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.
Recent advances in omics studies have enabled analysis at the single-cell level; however, methods for analyzing the whole cell of large organs and tissues remain challenging. Here, we developed a method named tsChIL to understand the diverse cellular dynamics at the tissue level using high-depth epigenomic data. tsChIL allowed the analysis of a single tissue section and could reproducibly acquire epigenomic profiles from several types of tissues, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation of cells in regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analysis by tsChIL can elucidate in vivo cell-type dynamics of tissues.
Embryonic development occurs through commitment of pluripotent stem cells to differentiation programs that require highly coordinated changes in gene expression. Chromatin remodeling of gene regulatory elements is a critical component of how such changes are achieved. While many factors controlling chromatin dynamics are known, mechanisms of how different chromatin regulators are orchestrated during development are not well understood. Here, we describe LINKED (LINKage-specific-deubiquitylation-deficiency-induced Embryonic Defects) syndrome, a novel multiple congenital anomaly disorder caused by hypomorphic hemizygous missense variants in the deubiquitylase OTUD5/DUBA. Studying LINKED mutations in vitro, in mouse, and in models of neuroectodermal differentiation of human pluripotent stem cells, we uncover a novel regulatory circuit that coordinates chromatin remodeling pathways during early differentiation. We show that the K48-linkage-specific deubiquitylation activity of OTUD5 is essential for murine and human development and, if reduced, leads to aberrant cell-fate specification. OTUD5 controls differentiation through preventing the degradation of multiple chromatin regulators including ARID1A/B and HDAC2, mutation of which underlie developmental syndromes that exhibit phenotypic overlap with LINKED patients. Accordingly, loss of OTUD5 during early differentiation leads to less accessible chromatin at neural and neural crest enhancers and thus aberrant rewiring of gene expression networks. Our work identifies a novel mechanistic link between phenotypically related developmental disorders and an essential function for linkagespecific ubiquitin editing of substrate groups (i.e. chromatin remodeling complexes) during early cellfate decisions - a regulatory concept, we predict to be a general feature of embryonic development.
Industrial nitrogen fertilizer is intrinsic to modern agriculture yet expensive and environmentally harmful. We aim to reconstitute bacterial nitrogenase function within plant mitochondria to reduce nitrogen fertilizer usage. Many nitrogen fixation (Nif) proteins are required for biosynthesis and function of the mature nitrogenase enzyme, and these will need to be correctly processed and soluble within mitochondria as a pre-requisite for function. Here we present our workflow that assessed processing, solubility and relative abundance of 16 Klebsiella oxytoca Nif proteins targeted to the plant mitochondrial matrix using an Arabidopsis mitochondrial targeting peptide (MTP). The functional consequence of the N-terminal modifications required for mitochondrial targeting of Nif proteins was tested using bacterial nitrogenase assays. We found that despite the use of the same constitutive promoter and MTP, MTP::Nif processing and relative abundance in plant leaf varied considerably. Assessment of solubility for all MTP::Nif proteins found NifF, M, N, S, U, W, X, Y and Z were soluble, while NifB, E, H, J, K, Q and V were mostly insoluble. Although most Nif proteins tolerated the N-terminal extension as a consequence of mitochondrial processing, this extension in NifM reduced nitrogenase activity to 10% of controls. Using proteomics, we detected a [~]50-fold increase in the abundance of NifM when it contained the N-terminal MTP extension, which may account for this reduction seen in nitrogenase activity. Based on plant mitochondrial processing and solubility, and retention of function in a bacterial assay, our workflow has identified that NifF, N, S, U, W, Y and Z satisfied all these criteria. Future work can now focus on improving these parameters for the remaining Nif components to assemble a complete set of plant-ready Nif proteins for reconstituting nitrogen fixation in plant mitochondria.
Incorporating deep learning in the image analysis pipeline has opened the possibility of introducing precision phenotyping in the field of agriculture. However, to train the neural network, a sufficient amount of training data must be prepared, which requires a time-consuming manual data annotation process that often becomes the limiting step. Here, we show that an instance segmentation neural network (Mask R-CNN) aimed to phenotype the barley seed morphology of various cultivars, can be sufficiently trained purely by a synthetically generated dataset. Our attempt is based on the concept of domain randomization, where a large amount of image is generated by randomly orienting the seed object to a virtual canvas. After training with such a dataset, performance based on recall and the average Precision of the real-world test dataset achieved 96% and 95%, respectively. Applying our pipeline enables extraction of morphological parameters at a large scale, enabling precise characterization of the natural variation of barley from a multivariate perspective. Importantly, we show that our approach is effective not only for barley seeds but also for various crops including rice, lettuce, oat, and wheat, and thus supporting the fact that the performance benefits of this technique is generic. We propose that constructing and utilizing such synthetic data can be a powerful method to alleviate human labor costs needed to prepare the training dataset for deep learning in the agricultural domain.
To engineer Mo dependent nitrogenase function in plants expression of proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in E. coli identified a subset that retained function, including a single amino acid (Y100Q) variant. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in E. coli based nitrogenase assays, demonstrating this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.
HIV-1 causes the loss of CD4+ T cells via depletion or impairment of their production. The latter involves infection of thymocytes, but the involvement of other cells including haematopoietic CD34+ cells remains unclear even though HIVpositive patients frequently manifest myelosuppression. In this study, we utilised the OP9-DL1 coculture system, which supports in vitro T-lineage differentiation of human haematopoietic stem/progenitor cells. Cord-derived CD34+ cells were infected with CXCR4-tropic HIV-1NL4-3 and cocultured. HIV-infected cocultures exhibited sustained viral replication for 5 weeks, as well as reduced CD4+ T-cell growth at weeks 3-5. We further revealed that CD34+CD7+CXCR4+ cells can be quickly depleted as early as in 1 week after infection of the subset, and this was accompanied by the emergence of CD34+CD7+CD4+ cells. These results indicate that CXCR4-tropic HIV-1 strains may disrupt CD34+CD7+ lymphoid progenitor cell pools, presumably leading to impaired T-cell production potential.
Human immunodeficiency virus (HIV) causes damage, directly or indirectly, to the whole hematopoietic system including CD34+ hematopoietic stem/progenitor cells (HSPC). CXCR4-tropic strains of HIV-1 may be potent to affect the function of CD34+CXCR4+ progenitor cells either by infecting the cells or by modifying the dynamics of more differentiated hematopoietic cells. However, CD34+ cells are known for the resistance to HIV-1 infection in vitro, restricting the detailed analysis of the impact of HIV upon HSPC. Here the authors report a use of RetroNectin, a recombinant fibronectin fragment used for gene transfer with lentiviral vectors, to overcome the limitation.