Publications by authors named "Masahiro Sato"

211 Publications

Therapeutic potential of d-cysteine against in vitro and in vivo models of spinocerebellar ataxia.

Exp Neurol 2021 Sep 19;343:113791. Epub 2021 Jun 19.

Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan. Electronic address:

Spinocerebellar ataxia (SCA) is a group of autosomal-dominantly inherited ataxia and is classified into SCA1-48 by the difference of causal genes. Several SCA-causing proteins commonly impair dendritic development in primary cultured Purkinje cells (PCs). We assume that primary cultured PCs expressing SCA-causing proteins are available as in vitro SCA models and that chemicals that improve the impaired dendritic development would be effective for various SCAs. We have recently revealed that D-cysteine enhances the dendritic growth of primary cultured PCs via hydrogen sulfide production. In the present study, we first investigated whether D-cysteine is effective for in vitro SCA models. We expressed SCA1-, SCA3-, and SCA21-causing mutant proteins to primary cultured PCs using adeno-associated viral serotype 9 (AAV9) vectors. D-Cysteine (0.2 mM) significantly ameliorated the impaired dendritic development commonly observed in primary cultured PCs expressing these three SCA-causing proteins. Next, we investigated the therapeutic effect of long-term treatment with D-cysteine on an in vivo SCA model. SCA1 model mice were established by the cerebellar injection of AAV9 vectors, which express SCA1-causing mutant ataxin-1, to ICR mice. Long-term treatment with D-cysteine (100 mg/kg/day) significantly inhibited the progression of motor dysfunction in SCA1 model mice. Immunostaining experiments revealed that D-cysteine prevented the reduction of mGluR1 and glial activation at the early stage after the onset of motor dysfunction in SCA1 model mice. These findings strongly suggest that D-cysteine has therapeutic potential against in vitro and in vivo SCA models and may be a novel therapeutic agent for various SCAs.
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http://dx.doi.org/10.1016/j.expneurol.2021.113791DOI Listing
September 2021

RNA analysis based on a small number of manually isolated fixed cells (RNA-snMIFxC) to profile stem cells from human deciduous tooth-derived dental pulp cells.

Biol Proced Online 2021 Jun 11;23(1):12. Epub 2021 Jun 11.

Department of Genome Medicine, National Center for Child Health and Development, 2-10-1, Tokyo, 157-8535, Japan.

Background: Expression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette.

Results: Two or more colored cells (~ 10), after staining with a chromogen such a 3,3'-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as OCT3/4, SOX2, and ALP) was confined to positively stained cells.

Conclusion: These findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.
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http://dx.doi.org/10.1186/s12575-021-00149-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8194139PMC
June 2021

Resource partitioning is not coupled with assortative mating in sympatrically divergent ricefish in a Wallacean ancient lake.

J Evol Biol 2021 Jul 15;34(7):1133-1143. Epub 2021 Jun 15.

Tropical Biosphere Research Center, University of the Ryukyus, Okinawa, Japan.

Sympatric speciation is considered to be difficult without the coupling between ecological traits that allow resource partitioning and reproductive traits that allow assortative mating. Such "magic traits" are known to be involved in most of the compelling examples of sympatric speciation. In this study, we report a possible case of sympatric speciation without magic traits. Three species of ricefish (genus Oryzias) are suggested to have diverged sympatrically within Lake Poso, an ancient lake in Sulawesi. An analysis of genome-wide single-nucleotide polymorphisms showed that these three species are reproductively isolated from each other throughout the lake. Stable isotope analyses revealed that the three species use different food resources, which reflect differences in their feeding morphologies (gill rakers and digestive tracts) and feeding sites. Field and laboratory observations showed that O. nebulosus and O. orthognathus share a mating habitat of cobbles, where they scatter fertilized eggs, whereas this site is never used by O. nigrimas, indicating that assortative mating is partly achieved by spatial isolation. The small, less-adhesive eggs of O. nebulosus and O. orthognathus probably reflect their adaptation to spawning on cobble beaches. Laboratory mating experiments showed strong prezygotic isolation between O. nebulosus and O. orthognathus, which is achieved by strong species recognition presumably by both sexes based on species-specific mating dances and nuptial coloration. In summary, the assortative mating of O. nebulosus and O. orthognathus is probably not coupled to resource partitioning. We discussed how sympatric speciation among these species might have been achieved even without magic traits.
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http://dx.doi.org/10.1111/jeb.13874DOI Listing
July 2021

Induced Tissue-Specific Stem Cells (iTSCs): Their Generation and Possible Use in Regenerative Medicine.

Pharmaceutics 2021 May 23;13(6). Epub 2021 May 23.

Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.

Induced tissue-specific stem cells (iTSCs) are partially reprogrammed cells which have an intermediate state, such as progenitors or stem cells. They originate from the de-differentiation of differentiated somatic cells into pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), or from the differentiation of undifferentiated cells. They show a limited capacity to differentiate and a morphology similar to that of somatic cell stem cells present in tissues, but distinct from that of iPSCs and ESCs. iTSCs can be generally obtained 7 to 10 days after reprogramming of somatic cells with Yamanaka's factors, and their fibroblast-like morphology remains unaltered. iTSCs can also be obtained directly from iPSCs cultured under conditions allowing cellular differentiation. In this case, to effectively induce iTSCs, additional treatment is required, as exemplified by the conversion of iPSCs into naïve iPSCs. iTSCs can proliferate continuously in vitro, but when transplanted into immunocompromised mice, they fail to generate solid tumors (teratomas), implying loss of tumorigenic potential. The low tendency of iTSCs to elicit tumors is beneficial, especially considering applications for regenerative medicine in humans. Several iTSC types have been identified, including iTS-L, iTS-P, and iTS-D, obtained by reprogramming hepatocytes, pancreatic cells, and deciduous tooth-derived dental pulp cells, respectively. This review provides a brief overview of iPSCs and discusses recent advances in the establishment of iTSCs and their possible applications in regenerative medicine.
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http://dx.doi.org/10.3390/pharmaceutics13060780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8224740PMC
May 2021

Novel reporter mouse models useful for evaluating gene editing and for optimization of methods of delivering genome editing tools.

Mol Ther Nucleic Acids 2021 Jun 5;24:325-336. Epub 2021 Mar 5.

Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Kanagawa, Japan.

The clustered regularly interspersed palindromic repeats (CRISPR) system is a powerful genome-editing tool to modify genomes, virtually in any species. The CRISPR tool has now been utilized in many areas of medical research, including gene therapy. Although several proof-of-concept studies show the feasibility of gene therapy applications for correcting disease-causing mutations, and new and improved tools are constantly being developed, there are not many choices of suitable reporter models to evaluate genome editor tools and their delivery methods. Here, we developed and validated reporter mouse models containing a single copy of disrupted (ΔEGFP) via frameshift mutations. We tested several delivery methods for validation of the reporters, and we demonstrated their utility to assess both non-homologous end-joining (NHEJ) and via homology-directed repair (HDR) processes in embryos and in somatic tissues. With the use of the reporters, we also show that hydrodynamic delivery of ribonucleoprotein (RNP) with (Sp)Cas9 protein mixed with synthetic guide RNA (gRNA) elicits better genome-editing efficiencies than the plasmid vector-based system in mouse liver. The reporters can also be used for assessing HDR efficiencies of the (As)Cas12a nuclease. The results suggest that the ΔEGFP mouse models serve as valuable tools for evaluation of genome editing.
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http://dx.doi.org/10.1016/j.omtn.2021.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020343PMC
June 2021

Electric-field-induced second-harmonic generation using high-intensity femtosecond laser pulses over the observable optical breakdown threshold.

Opt Lett 2021 Jan;46(2):238-241

We investigated the performance of electric-field-induced second-harmonic generation (E-FISHG) by spectroscopic measurement using high-intensity femtosecond laser pulses. The second-harmonic intensity increased quadratically versus the applied electric field, as expected from the theory, up to 15 kV/cm with the laser energy up to 2.5 mJ, which is ∼5 times higher than the observable optical breakdown threshold. In addition, when the laser energy was 2.8 mJ, ∼80 times signal intensity at 0.23 mJ was obtained. These results suggest that the electric-field measurement by E-FISHG with high-intensity second harmonics is expected by using high-intensity laser pulses above the observable optical breakdown threshold. Spectroscopic measurement shows no E-FISHG of white light generated by self-phase modulation in laser-induced filament.
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http://dx.doi.org/10.1364/OL.412856DOI Listing
January 2021

Volar transfer of the lateral band with transverse retinacular ligament is effective for the correction of swan-neck deformity caused by volar plate injury of the PIP joint.

Mod Rheumatol Case Rep 2020 01 24;4(1):152-155. Epub 2019 Oct 24.

Department of Orthopaedic Surgery, School of Medicine, Keio University, Tokyo, Japan.

We introduced a technique with a volar transfer of the lateral band using the transverse retinacular ligament for swan-neck deformity caused by volar plate injury of the PIP joint. A 61-year-old woman injured her 5th finger and was diagnosed with a volar plate injury of the PIP joint. She presented with snapping of the finger together with the appearance of a swan-neck deformity, and surgery was performed. Dorsally located lateral bands were transferred towards the volar aspect of the finger, and their position was maintained using the transverse retinacular ligament. Improvements in the snapping and swan-neck deformities were confirmed by intraoperative active motion of the finger. One year postoperatively, the deformity had not recurred, and there was no contracture of the finger. Surgical transfer of the lateral band using the transverse retinacular ligament is effective for swan-neck deformity caused by volar plate injury of the PIP joint.
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http://dx.doi.org/10.1080/24725625.2019.1681636DOI Listing
January 2020

Periodontal surgery using rhFGF-2 with deproteinized bovine bone mineral or rhFGF-2 alone: 2-year follow-up of a randomized controlled trial.

J Clin Periodontol 2021 01 12;48(1):91-99. Epub 2020 Nov 12.

Department of Periodontology, Tokyo Dental College, Tokyo, Japan.

Aim: To compare outcomes of rhFGF-2 + DBBM therapy with rhFGF-2 alone in the treatment of intrabony defects. This study provides 2-year follow-up results from the previous randomized controlled trial.

Materials And Methods: Defects were randomly allocated to receive rhFGF-2 + DBBM (test) or rhFGF-2 (control). Treated sites were re-evaluated at 2 years postoperatively, using original clinical and patient-centred measures.

Results: Thirty-eight sites were available for re-evaluation. At 2 years, both groups showed a significant improvement in clinical attachment level (CAL) from baseline. A gain in CAL of 3.4 ± 1.3 mm in the test group and 3.1 ± 1.5 mm in the control group was found. No significant inter-group difference was noted. Both groups showed a progressive increase in radiographic bone fill (RBF). The test treatment yielded greater RBF (56%) compared with the control group (41%). The control treatment performed better in contained defects in terms of CAL and RBF. There was no significant difference in patient-reported outcomes between groups.

Conclusions: At 2-year follow-up, the test and cotrol treatments were similarly effective in improving CAL, whereas the test treatment achieved a significantly greater RBF. In both treatments, favourable clinical, radiographic, and patient-reported outcomes can be sustained for at least 2 years.

Trial Registration: The University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) 000025257.
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http://dx.doi.org/10.1111/jcpe.13385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984167PMC
January 2021

Lactose-appended β-cyclodextrin as an effective nanocarrier for brain delivery.

J Control Release 2020 12 28;328:722-735. Epub 2020 Sep 28.

Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan. Electronic address:

The blood-brain barrier (BBB) prevents the permeability of drugs into the brain, and as such limits the management of various brain diseases. To overcome this barrier, drug-encapsulating nanoparticles or vesicles, drug conjugates, and other types of drug delivery systems (DDSs) have been developed. However, the brain-targeting ability of nanoparticles or vesicles is still insufficient. Recently, among the various brain-targeting ligands previously studied for facilitating transcellular BBB transport, several sugar-appended nanocarriers for brain delivery were identified. Meanwhile, cyclodextrins (CyDs) have been used as nanocarriers for drug delivery since they can encapsulate hydrophobic compounds with high biocompatibility. Therefore, in this study, we created various sugar-appended β-cyclodextrins (β-CyDs) to discover novel brain-targeting ligands. As a result, of the six sugar-appended CyDs, lactose-appended β-CyD (Lac-β-CyD) showed greater cellular uptake in hCMEC/D3 cells, human brain microvascular endothelial cells, than other sugar-appended β-CyDs did. In addition, the permeability of Lac-β-CyD within the in vitro human BBB model was greater than that of other sugar-appended β-CyDs. Moreover, Lac-β-CyD significantly accumulated in the mouse brain after intravenous administration. Thus, Lac-β-CyD efficiently facilitated the accumulation of the model drug into the mouse brain. These findings suggest that Lac-β-CyD has the potential to be a novel carrier for drugs across the BBB.
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http://dx.doi.org/10.1016/j.jconrel.2020.09.043DOI Listing
December 2020

General description for nonequilibrium steady states in periodically driven dissipative quantum systems.

Sci Adv 2020 Jul 3;6(27). Epub 2020 Jul 3.

Department of Physics, Ibaraki University, Mito, Ibaraki 310-8512, Japan.

Laser technology has developed and accelerated photo-induced nonequilibrium physics, from both the scientific and engineering viewpoints. Floquet engineering, i.e., controlling material properties and functionalities by time-periodic drives, is at the forefront of quantum physics of light-matter interaction. However, it is limited to ideal dissipationless systems. Extending Floquet engineering to various materials requires understanding of the quantum states emerging in a balance of the periodic drive and energy dissipation. Here, we derive a general description for nonequilibrium steady states (NESSs) in periodically driven dissipative systems by focusing on systems under high-frequency drive and time-independent Lindblad-type dissipation. Our formula correctly describes the time average, fluctuation, and symmetry properties of the NESS, and can be computed efficiently in numerical calculations. This approach will play fundamental roles in Floquet engineering in a broad class of dissipative quantum systems from atoms and molecules to mesoscopic systems, and condensed matter.
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http://dx.doi.org/10.1126/sciadv.abb4019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458437PMC
July 2020

Drug-Induced Naïve iPS Cells Exhibit Better Performance than Primed iPS Cells with Respect to the Ability to Differentiate into Pancreatic β-Cell Lineage.

J Clin Med 2020 Sep 2;9(9). Epub 2020 Sep 2.

Division of Pediatric Dentistry, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8514, Japan.

Pluripotent stem cells are classified as naïve and primed cells, based on their in vitro growth characteristics and potential to differentiate into various types of cells. Human-induced pluripotent stem cells (iPSCs, also known as epiblast stem cells [EpiSCs]) have limited capacity to differentiate and are slightly more differentiated than naïve stem cells (NSCs). Although there are several in vitro protocols that allow iPSCs to differentiate into pancreatic lineage, data concerning generation of β-cells from these iPSCs are limited. Based on the pluripotentiality of NSCs, it was hypothesized that NSCs can differentiate into pancreatic β-cells when placed under an appropriate differentiation induction condition. We examined whether NSCs can be efficiently induced to form potentially pancreatic β cells after being subjected to an in vitro protocol. Several colonies resembling in vitro-produced β-cell foci, with β-cell-specific marker expression, were observed when NSC-derived embryoid bodies (EBs) were induced to differentiate into β-cell lineage. Conversely, EpiSC-derived EBs failed to form such foci in vitro. Intrapancreatic grafting of the in vitro-formed β-cell foci into nude mice (BALB/c-nu/nu) generated a cell mass containing insulin-producing cells (IPCs), without noticeable tumorigenesis. These NSCs can be used as a promising resource for curing type 1 diabetes.
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http://dx.doi.org/10.3390/jcm9092838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564489PMC
September 2020

Author Correction: Nonequilibrium Magnetic Oscillation with Cylindrical Vector Beams.

Sci Rep 2020 Sep 1;10(1):14663. Epub 2020 Sep 1.

Department of Physics, Ibaraki University, Mito, Ibaraki, 310-8512, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-71839-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459284PMC
September 2020

Development of Novel Heparin/Protamine Nanoparticles Useful for Delivery of Exogenous Proteins In Vitro and In Vivo.

Nanomaterials (Basel) 2020 Aug 12;10(8). Epub 2020 Aug 12.

Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan.

We previously reported that heparin/protamine particles (LHPPs) produced as nanoparticles through simple mixing of raw materials exhibit sustained protein release and can be retained in cells. In the present study, we modified LHPPs without employing any organic synthetic approach. The resulting LHPPs were re-named as improved LHPPs (-LHPPs) and have the ability to retain cell-penetrating peptides (GRKKRRQRRRPPQ) based on electrostatic interactions. We examined whether -LHPPs can introduce exogenous proteins (i.e., lacZ protein encoding bacterial β-galactosidase) into cultured cells in vitro, or into murine hepatocytes in vivo through intravenous injection to anesthetized mice. We found an accumulation of the transferred protein in both in vitro cultured cells and in vivo hepatocytes. To the best of our knowledge, reports of successful in vivo delivery to hepatocytes are rare. The -LHPP-based protein delivery technique will be useful for in vivo functional genetic modification of mouse hepatocytes using Cas9 protein-mediated genome editing targeting specific genes, leading to the creation of hepatic disease animal models for research that aims to treat liver diseases.
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http://dx.doi.org/10.3390/nano10081584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466629PMC
August 2020

Ataxic phenotype and neurodegeneration are triggered by the impairment of chaperone-mediated autophagy in cerebellar neurons.

Neuropathol Appl Neurobiol 2021 02 9;47(2):198-209. Epub 2020 Aug 9.

Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.

Aims: Chaperone-mediated autophagy (CMA) is a pathway involved in the autophagy lysosome protein degradation system. CMA has attracted attention as a contributing factor to neurodegenerative diseases since it participates in the degradation of disease-causing proteins. We previously showed that CMA is generally impaired in cells expressing the proteins causing spinocerebellar ataxias (SCAs). Therefore, we investigated the effect of CMA impairment on motor function and the neural survival of cerebellar neurons using the micro RNA (miRNA)-mediated knockdown of lysosome-associated protein 2A (LAMP2A), a CMA-related protein.

Methods: We injected adeno-associated virus serotype 9 vectors, which express green fluorescent protein (GFP) and miRNA (negative control miRNA or LAMP2A miRNA) under neuron-specific synapsin I promoter, into cerebellar parenchyma of 4-week-old ICR mice. Motor function of mice was evaluated by beam walking and footprint tests. Immunofluorescence experiments of cerebellar slices were conducted to evaluate histological changes in cerebella.

Results: GFP and miRNA were expressed in interneurons (satellite cells and basket cells) in molecular layers and granule cells in the cerebellar cortices, but not in cerebellar Purkinje cells. LAMP2A knockdown in cerebellar neurons triggered progressive motor impairment, prominent loss of cerebellar Purkinje cells, interneurons, granule cells at the late stage, and astrogliosis and microgliosis from the early stage.

Conclusions: CMA impairment in cerebellar interneurons and granule cells triggers the progressive ataxic phenotype, gliosis and the subsequent degeneration of cerebellar neurons, including Purkinje cells. Our present findings strongly suggest that CMA impairment is related to the pathogenesis of various SCAs.
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http://dx.doi.org/10.1111/nan.12649DOI Listing
February 2021

Hydrodynamics-Based Transplacental Delivery as a Useful Noninvasive Tool for Manipulating Fetal Genome.

Cells 2020 07 21;9(7). Epub 2020 Jul 21.

Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan.

We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α () gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in . Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses.
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http://dx.doi.org/10.3390/cells9071744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409276PMC
July 2020

Strategy of landfilled waste reduction by a distributed materials recovery facility system in Surabaya, Indonesia.

Waste Manag Res 2020 Oct 29;38(10):1142-1152. Epub 2020 Jun 29.

Graduate School of Engineering, 12810Hokkaido University, Sapporo, Hokkaido, Japan.

Slow progress in municipal waste reduction and landfill space scarcity lead to numerous environmental problems in Indonesia and developing countries. Surabaya, the role model of an environmental management city in Indonesia and other countries, is no exception. Despite the situation, Surabaya's initiative of deploying a distributed materials recovery facility (MRF) and its performance in recovering recyclables show a potential to be developed for addressing the landfill waste reduction issues. This study proposes a new strategy with small-sized distributed MRFs to achieve 30% landfilled waste reduction and reducing greenhouse gas (GHG) emissions, focusing on Surabaya as the case study. Scenario 1 merged three pairs of transfer stations which shows some indistinguishable optimizations and failed to meet the target. Both Scenario 2 and Scenario 3 added six years of landfill lifetime for reaching the target. However, the distributed MRF system and different transportation systems in Scenario 3 accomplished the goal with only 24 new MRFs, whereas Scenario 2 needs to upgrade 48 transfer stations into MRFs. Scenario 3 decreased the GHG emissions generation by 29%, possibly contributing to Indonesia's GHG emissions target of 0.2%.
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http://dx.doi.org/10.1177/0734242X20932217DOI Listing
October 2020

Effects of intermittent treatment with parathyroid hormone (PTH) on osteoblastic differentiation and mineralization of mouse induced pluripotent stem cells in a 3D culture model.

J Periodontal Res 2020 Oct 25;55(5):734-743. Epub 2020 Jun 25.

Oral Health Science Center, Tokyo Dental College, Tokyo, Japan.

Background/objectives: PTH plays an important role in bone remodeling, and different actions have been reported depending on its administration method. iPSCs are promising as a cell source for regeneration of periodontal tissue due to their ability of proliferation and pluripotency. However, the effects of PTH on iPSCs remain mostly unknown. The purpose of this study was to investigate in vitro effects of parathyroid hormone (PTH) on osteoblastic differentiation of induced pluripotent stem cells (iPSCs) in a 3D culture model.

Materials And Methods: Following embryoid body (EB) induction from mouse iPSCs (miPSCs), dissociated cells (miPS-EB-derived cells) were seeded onto atelocollagen sponge (ACS) in osteoblast differentiation medium (OBM). Cell-ACS constructs were divided into three groups: continuous treatment with human recombinant PTH (1-34) (PTH-C), intermittent PTH treatment (PTH-I) or OBM control. To confirm the expression of PTH receptor-1(PTH1R), the expression of Pth1r and cAMP production over time were assessed. Real-time PCR was used to assess the expression of genes encoding osterix (Sp7), runt-related transcription factor 2 (Runx2), collagen type 1 (Col1a1), and osteocalcin (Bglap) at different time points. Mineralization was assessed by von Kossa staining. Histochemical staining was used to analyze alkaline phosphatase (ALP) activity, and immunolocalization of SP7 and BGLAP was analyzed by confocal laser scanning microscopy (CLSM).

Results: On days 7 and 14, expression of the Pth1r in miPS-EB-derived cells was increased in all groups. Production of cAMP, the second messenger of the PTH1R, tended to increase in the PTH-I group compared with PTH-C group on day 14. Expression of Col1a1 in the PTH-I group on day 14 was significantly higher than other groups. There was a time-dependent increase in the expression of Sp7 in all groups. On day 14, the expression level of Sp7 in the PTH-I group was significantly higher than other groups. In von Kossa staining, the PTH-I group showed higher level of staining compared with other groups on day 14, whereas the level was slightly attenuated in the PTH-C group. In histochemical staining, ALP-positive cells were significantly increased in the PTH-I group compared with other groups on day 14. In CLSM analysis, the numbers of SP7- and BGLAP-positive cells showed a gradual increase over time, and on day 14, a significantly greater SP7 expression was observed in the PTH-I group than other groups.

Conclusion: These results suggested that the intermittent PTH treatment promotes osteoblastic differentiation and mineralization of miPSCs in the ACS scaffold.
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http://dx.doi.org/10.1111/jre.12762DOI Listing
October 2020

Low fraction of methane in landfill gas emissions in an industrial waste landfill containing incineration ash and gypsum board waste under anaerobic conditions.

Waste Manag Res 2020 Oct 22;38(10):1101-1109. Epub 2020 Jun 22.

Center for Material Cycles and Waste Management Research, 13585National Institute for Environmental Studies, Japan.

The behaviour of carbon dioxide (CO) and methane (CH) emissions at the surface and below the soil cover in an industrial waste landfill under anaerobic operating conditions was evaluated for six years. This landfill contained gypsum board waste and incineration ash - a practice currently allowed because of a change in Japanese regulations. The CO and CH fluxes decreased throughout the six years of the survey. Almost all of the survey points exhibited fractions of CH in landfill gas emissions of <0.5 (mean values: 0.0-0.1 [surface], 0.0-0.3 [subsurface]) under anaerobic conditions. In addition, a relatively high first-order reaction rate constant for the landfill gas emissions (0.3 year) was observed. The landfill leachate showed a relatively high sulphate ion (SO ) concentration, although other environmental conditions, such as the pH, oxidation-reduction potential and ammonium concentration, were not at levels that could have inhibited CH production. These findings suggest that the low fractions could have been related to the lower amounts of CH generation caused by competition between methanogens and sulphate-reducing bacteria (SRB). Therefore, SRB could play a major role in the degradation of organic carbon in the landfill.
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http://dx.doi.org/10.1177/0734242X20931939DOI Listing
October 2020

Glucocorticoids negatively regulates chaperone mediated autophagy and microautophagy.

Biochem Biophys Res Commun 2020 07 30;528(1):199-205. Epub 2020 May 30.

Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan. Electronic address:

Glucocorticoids are released from the adrenal cortex and are important for regulating various physiological functions. However, a persistent increase in glucocorticoids due to chronic stress causes various dysfunctions in the central nervous system which can lead to mental disorders such as depression. Macroautophagy, one of the pathways of the autophagy-lysosome protein degradation system, is dysregulated in psychiatric disorders, implicating a disturbance of protein degradation in the pathogenesis of psychiatric disorders. In the present study, we investigated whether glucocorticoids affect the activity of chaperone-mediated autophagy (CMA) and microautophagy (mA), the other two pathways of the autophagy-lysosome system. Treatment of human-derived AD293 cells and primary cultured rat cortical neurons with dexamethasone, a potent glucocorticoid receptor agonist, and endogenous glucocorticoids decreased both CMA and mA activities. However, this decrease was significantly suppressed by treatment with RU-486, a glucocorticoid receptor antagonist. In addition, dexamethasone significantly decreased lysosomal Hsc70. These findings suggest that glucocorticoids negatively regulate CMA and mA in a glucocorticoid receptor-dependent manner, and provide evidence for CMA and mA as novel therapeutic targets for depression.
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http://dx.doi.org/10.1016/j.bbrc.2020.04.132DOI Listing
July 2020

Genetic and antigenic characterization of H5 and H7 avian influenza viruses isolated from migratory waterfowl in Mongolia from 2017 to 2019.

Virus Genes 2020 Aug 19;56(4):472-479. Epub 2020 May 19.

Laboratory of Microbiology, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, 060-0818, Japan.

The circulation of highly pathogenic avian influenza viruses (HPAIVs) of various subtypes (e.g., H5N1, H5N6, H5N8, and H7N9) in poultry remains a global concern for animal and public health. Migratory waterfowls play important roles in the transmission of these viruses across countries. To monitor virus spread by wild birds, active surveillance for avian influenza in migratory waterfowl was conducted in Mongolia from 2015 to 2019. In total, 5000 fecal samples were collected from lakesides in central Mongolia, and 167 influenza A viruses were isolated. Two H5N3, four H7N3, and two H7N7 viruses were characterized in this study. The amino acid sequence at hemagglutinin (HA) cleavage site of those isolates suggested low pathogenicity in chickens. Phylogenetic analysis revealed that all H5 and H7 viruses were closely related to recent H5 and H7 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds in Asia and Europe. Antigenicity of H7Nx was similar to those of typical non-pathogenic avian influenza viruses (AIVs). While HPAIVs or A/Anhui/1/2013 (H7N9)-related LPAIVs were not detected in migratory waterfowl in Mongolia, sporadic introductions of AIVs including H5 and H7 viruses into Mongolia through the wild bird migration were identified. Thus, continued monitoring of H5 and H7 AIVs in both domestic and wild birds is needed for the early detection of HPAIVs spread into the country.
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http://dx.doi.org/10.1007/s11262-020-01764-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235438PMC
August 2020

Ultraviolet Irradiation Enhances the Microbicidal Activity of Silver Nanoparticles by Hydroxyl Radicals.

Int J Mol Sci 2020 Apr 30;21(9). Epub 2020 Apr 30.

Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan.

It is known that silver has microbicidal qualities; even at a low concentration, silver is active against many kinds of bacteria. Silver nanoparticles (AgNPs) have been extensively studied for a wide range of applications. Alternately, the toxicity of silver to human cells is considerably lower than that to bacteria. Recent studies have shown that AgNPs also have antiviral activity. We found that large amounts of hydroxyl radicals-highly reactive molecular species-are generated when AgNPs are irradiated with ultraviolet (UV) radiation with a wavelength of 365 nm, classified as ultraviolet A (UVA). In this study, we used electron spin resonance direct detection to confirm that UV irradiation of AgNPs produced rapid generation of hydroxyl radicals. As hydroxyl radicals are known to degrade bacteria, viruses, and some chemicals, the enhancement of the microbicidal activity of AgNPs by UV radiation could be valuable for the protection of healthcare workers and the prevention of the spread of infectious diseases.
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http://dx.doi.org/10.3390/ijms21093204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247328PMC
April 2020

Modification of -GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain.

Cells 2020 04 13;9(4). Epub 2020 Apr 13.

Laboratory Animal Facilities & Services, Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan.

Improved genome editing via oviductal nucleic acid delivery (-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. -GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins via the oviductal wall of pregnant females at 0.7 days post-coitum, followed by in vivo electroporation (EP). Unlike outbred Institute of Cancer Research (ICR) and hybrid mouse strains, genome editing of the most widely used C57BL/6J (B6) strain with -GONAD has been considered difficult but, recently, setting a constant current of 100 mA upon EP enabled successful -GONAD in this strain. Unfortunately, the most widely used electroporators employ a constant voltage, and thus we explored conditions allowing the generation of a 100 mA current using two electroporators: NEPA21 (Nepa Gene Co., Ltd.) and GEB15 (BEX Co., Ltd.). When the current and resistance were set to 40 V and 350-400 Ω, respectively, the current was fixed to 100 mA. Another problem in using B6 mice for -GONAD is the difficulty in obtaining pregnant B6 females consistently because estrous females often fail to be found. A single intraperitoneal injection of low-dose pregnant mare's serum gonadotrophin (PMSG) led to synchronization of the estrous cycle of these mice. Consequently, approximately 51% of B6 females had plugs upon mating with males 2 days after PMSG administration, which contrasts with the case (≈26%) when B6 females were subjected to natural mating. -GONAD performed on PMSG-treated pregnant B6 females under conditions of average resistance of 367 Ω and average voltage of 116 mA resulted in the production of pregnant females at a rate of 56% (5/9 mice), from which 23 fetuses were successfully delivered. Nine (39%) of these fetuses exhibited successful genome editing at the target locus.
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http://dx.doi.org/10.3390/cells9040957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226992PMC
April 2020

Potential Role of Nonneutralizing IgA Antibodies in Cross-Protective Immunity against Influenza A Viruses of Multiple Hemagglutinin Subtypes.

J Virol 2020 06 1;94(12). Epub 2020 Jun 1.

Division of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan

IgA antibodies on mucosal surfaces are known to play an important role in protection from influenza A virus (IAV) infection and are believed to be more potent than IgG for cross-protective immunity against IAVs of multiple hemagglutinin (HA) subtypes. However, in general, neutralizing antibodies specific to HA are principally HA subtype specific. Here, we focus on nonneutralizing but broadly cross-reactive HA-specific IgA antibodies. Recombinant IgG, monomeric IgA (mIgA), and polymeric secretory IgA (pSIgA) antibodies were generated based on the sequence of a mouse anti-HA monoclonal antibody (MAb) 5A5 that had no neutralizing activity but showed broad binding capacity to multiple HA subtypes. While confirming that there was no neutralizing activity of the recombinant MAbs against IAV strains A/Puerto Rico/8/1934 (H1N1), A/Adachi/2/1957 (H2N2), A/Hong Kong/483/1997 (H5N1), A/shearwater/South Australia/1/1972 (H6N5), A/duck/England/1/1956 (H11N6), and A/duck/Alberta/60/1976 (H12N5), we found that pSIgA, but not mIgA and IgG, significantly reduced budding and release of most of the viruses from infected cells. Electron microscopy demonstrated that pSIgA deposited newly produced virus particles on the surfaces of infected cells, most likely due to tethering of virus particles. Furthermore, we found that pSIgA showed significantly higher activity to reduce plaque sizes of the viruses than IgG and mIgA. These results suggest that nonneutralizing pSIgA reactive to multiple HA subtypes may play a role in intersubtype cross-protective immunity against IAVs. Mucosal immunity represented by pSIgA plays important roles in protection from IAV infection. Furthermore, IAV HA-specific pSIgA antibodies are thought to contribute to cross-protective immunity against multiple IAV subtypes. However, the mechanisms by which pSIgA exerts such versatile antiviral activity are not fully understood. In this study, we generated broadly cross-reactive recombinant IgG and pSIgA having the same antigen-recognition site and compared their antiviral activities These recombinant antibodies did not show "classical" neutralizing activity, whereas pSIgA, but not IgG, significantly inhibited the production of progeny virus particles from infected cells. Plaque formation was also significantly reduced by pSIgA, but not IgG. These effects were seen in infection with IAVs of several different HA subtypes. Based on our findings, we propose an antibody-mediated host defense mechanism by which mucosal immunity may contribute to broad cross-protection from IAVs of multiple HA subtypes, including viruses with pandemic potential.
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http://dx.doi.org/10.1128/JVI.00408-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307104PMC
June 2020

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ Cells, Embryos, and Fetuses in Mice.

Cells 2020 03 26;9(4). Epub 2020 Mar 26.

Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan.

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of efficient and simple methods to produce genome-edited (GE) animals would accelerate research in this field. The CRISPR/Cas9 system was initially employed in early embryos, utilizing classical gene delivery methods such as microinjection or electroporation, which required ex vivo handling of zygotes before transfer to recipients. Recently, novel in vivo methods such as genome editing via oviductal nucleic acid delivery (GONAD), improved GONAD (-GONAD), or transplacental gene delivery for acquiring genome-edited fetuses (TPGD-GEF), which facilitate easy embryo manipulation, have been established. Studies utilizing these techniques employed pregnant female mice for direct introduction of the genome-editing components into the oviduct or were dependent on delivery via tail-vein injection. In mice, embryogenesis occurs within the oviducts and the uterus, which often hampers the genetic manipulation of embryos, especially those at early postimplantation stages (days 6 to 8), owing to a thick surrounding layer of tissue called decidua. In this review, we have surveyed the recent achievements in the production of GE mice and have outlined the advantages and disadvantages of the process. We have also referred to the past achievements in gene delivery to early postimplantation stage embryos and germ cells such as primordial germ cells and spermatogonial stem cells, which will benefit relevant research.
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http://dx.doi.org/10.3390/cells9040799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226049PMC
March 2020

Bac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs.

Pharmaceutics 2020 Mar 19;12(3). Epub 2020 Mar 19.

Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan.

In vivo gene delivery involves direct injection of nucleic acids (NAs) into tissues, organs, or tail-veins. It has been recognized as a useful tool for evaluating the function of a gene of interest (GOI), creating models for human disease and basic research targeting gene therapy. Cargo frequently used for gene delivery are largely divided into viral and non-viral vectors. Viral vectors have strong infectious activity and do not require the use of instruments or reagents helpful for gene delivery but bear immunological and tumorigenic problems. In contrast, non-viral vectors strictly require instruments (i.e., electroporator) or reagents (i.e., liposomes) for enhanced uptake of NAs by cells and are often accompanied by weak transfection activity, with less immunological and tumorigenic problems. Chromosomal integration of GOI-bearing transgenes would be ideal for achieving long-term expression of GOI. Bac (PB), one of three transposons (PB, (SB), and ) found thus far, has been used for efficient transfection of GOI in various mammalian cells in vitro and in vivo. In this review, we outline recent achievements of PB-based production of genetically modified animals and organs and will provide some experimental concepts using this system.
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http://dx.doi.org/10.3390/pharmaceutics12030277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7151002PMC
March 2020

Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice.

Cells 2020 02 26;9(3). Epub 2020 Feb 26.

Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan.

Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4-1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4-1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called "sequential i-GONAD (si-GONAD)."
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http://dx.doi.org/10.3390/cells9030546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140409PMC
February 2020

Thy1 promoter activity in the Rosa26 locus in mice: lessons from Dre-rox conditional expression system.

Exp Anim 2020 Aug 11;69(3):287-294. Epub 2020 Feb 11.

Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.

The pronuclear injection (PI)-based targeted transgenesis (PITT) method allows the generation of targeted transgenic (Tg) mice wherein a single copy of a transgene is integrated into the Rosa26 locus following PI. The Rosa26 locus allows unbiased ubiquitous expression of integrated transgenes; however, it remains little known whether tissue-specific promoters retain their functional properties when placed at the Rosa26 locus. We evaluated tissue-specific activity and reproducibility of exogenous tissue-specific promoters targeted to the Rosa26 locus by generating Thy1-Dre/Dre reporter mice using PITT and assessed spatial expression patterns of the transgenes. The Thy1 promoter targeted to the Rosa26 locus appeared active in virtually all Purkinje cells in the cerebellum and hippocampus. However, mosaic expression of the transgene under the Thy1 promoter was observed in many other organs. This phenomenon was consistent in all the Tg lines generated by PITT, indicating a high degree of reproducibility for this experiment.
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http://dx.doi.org/10.1538/expanim.20-0002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445056PMC
August 2020

Production of genetically engineered mice with higher efficiency, lower mosaicism, and multiplexing capability using maternally expressed Cas9.

Sci Rep 2020 01 23;10(1):1091. Epub 2020 Jan 23.

Department of Life Innovation, Institute for Biomedical Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan.

The CRISPR/Cas9 system is widely used to generate gene-edited animals. Here, we developed an efficient system for generating genetically modified mice using maternal Cas9 from Cas9 transgenic mice. Using this system, we achieved lower mosaicism and higher rates of knock-in success, gene-editing, and birth compared to the similar parameters obtained using exogenously administered Cas9 (mRNA/protein) system. Furthermore, we successfully induced simultaneous mutations at multiple loci (a maximum of nine). Our novel gene-editing system based on maternal Cas9 could potentially facilitate the generation of mice with single and multiple gene modifications.
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http://dx.doi.org/10.1038/s41598-020-57996-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978307PMC
January 2020

Niemann-Pick C1 Heterogeneity of Bat Cells Controls Filovirus Tropism.

Cell Rep 2020 01;30(2):308-319.e5

Division of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo 001-0020, Japan; Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo 001-0020, Japan; Department of Disease Control, School of Veterinary Medicine, University of Zambia, Lusaka 10101, Zambia. Electronic address:

Fruit bats are suspected to be natural hosts of filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV). Interestingly, however, previous studies suggest that these viruses have different tropisms depending on the bat species. Here, we show a molecular basis underlying the host-range restriction of filoviruses. We find that bat-derived cell lines FBKT1 and ZFBK13-76E show preferential susceptibility to EBOV and MARV, respectively, whereas the other bat cell lines tested are similarly infected with both viruses. In FBKT1 and ZFBK13-76E, unique amino acid (aa) sequences are found in the Niemann-Pick C1 (NPC1) protein, one of the cellular receptors interacting with the filovirus glycoprotein (GP). These aa residues, as well as a few aa differences between EBOV and MARV GPs, are crucial for the differential susceptibility to filoviruses. Taken together, our findings indicate that the heterogeneity of bat NPC1 orthologs is an important factor controlling filovirus species-specific host tropism.
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http://dx.doi.org/10.1016/j.celrep.2019.12.042DOI Listing
January 2020

Transplacental Gene Delivery (TPGD) as a Noninvasive Tool for Fetal Gene Manipulation in Mice.

Int J Mol Sci 2019 Nov 25;20(23). Epub 2019 Nov 25.

Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan.

Transplacental gene delivery (TPGD) is a technique for delivering nucleic acids to fetal tissues via tail-vein injections in pregnant mice. After transplacental transport, administered nucleic acids enter fetal circulation and are distributed among fetal tissues. TPGD was established in 1995 by Tsukamoto et al., and its mechanisms, and potential applications have been further characterized since. Recently, discoveries of sequence specific nucleases, such as zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9), have revolutionized genome editing. In 2019, we demonstrated that intravenous injection of plasmid DNA containing CRISPR/Cas9 produced indels in fetal myocardial cells, which are comparatively amenable to transfection with exogenous DNA. In the future, this unique technique will allow manipulation of fetal cell functions in basic studies of fetal gene therapy. In this review, we describe developments of TPGD and discuss their applications to the manipulation of fetal cells.
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http://dx.doi.org/10.3390/ijms20235926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928727PMC
November 2019
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