Publications by authors named "Takeshi Noda"

201 Publications

CP100356 Hydrochloride, a P-Glycoprotein Inhibitor, Inhibits Lassa Virus Entry: Implication of a Candidate Pan-Mammarenavirus Entry Inhibitor.

Viruses 2021 Sep 3;13(9). Epub 2021 Sep 3.

Laboratory of Ultrastructural Virology, Institute for Frontier Life and Medical Sciences, Kyoto University, Shogoin-Kawahara-cho 53, Sakyo-ku, Kyoto 606-8507, Japan.

Lassa virus (LASV)-a member of the family -causes Lassa fever in humans and is endemic in West Africa. Currently, no approved drugs are available. We screened 2480 small compounds for their potential antiviral activity using pseudotyped vesicular stomatitis virus harboring the LASV glycoprotein (VSV-LASVGP) and a related prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV). Follow-up studies confirmed that CP100356 hydrochloride (CP100356), a specific P-glycoprotein (P-gp) inhibitor, suppressed VSV-LASVGP, LCMV, and LASV infection with half maximal inhibitory concentrations of 0.52, 0.54, and 0.062 μM, respectively, without significant cytotoxicity. Although CP100356 did not block receptor binding at the cell surface, it inhibited low-pH-dependent membrane fusion mediated by arenavirus glycoproteins. P-gp downregulation did not cause a significant reduction in either VSV-LASVGP or LCMV infection, suggesting that P-gp itself is unlikely to be involved in arenavirus entry. Finally, our data also indicate that CP100356 inhibits the infection by other mammarenaviruses. Thus, our findings suggest that CP100356 can be considered as an effective virus entry inhibitor for LASV and other highly pathogenic mammarenaviruses.
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http://dx.doi.org/10.3390/v13091763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8473031PMC
September 2021

Isoflurane induces Art2-Rsp5-dependent endocytosis of Bap2 in yeast.

FEBS Open Bio 2021 Sep 18. Epub 2021 Sep 18.

Center of Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Japan.

Although general anesthesia is indispensable during modern surgical procedures, the mechanism by which inhalation anesthetics act on the synaptic membrane at the molecular and cellular level is largely unknown. In this study, we used yeast cells to examine the effect of isoflurane, an inhalation anesthetic, on membrane proteins. Bap2, an amino acid transporter localized on the plasma membrane, was endocytosed when yeast cells were treated with isoflurane. Depletion of RSP5, an E3 ligase, prevented this endocytosis and Bap2 was ubiquitinated in response to isoflurane, indicating an ubiquitin-dependent process. Screening all the Rsp5 binding adaptors showed that Art2 plays a central role in this process. These results suggest that isoflurane affects Bap2 via an Art2-Rsp5-dependent ubiquitination system.
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http://dx.doi.org/10.1002/2211-5463.13302DOI Listing
September 2021

Antiviral Face Mask Functionalized with Solidified Hand Soap: Low-Cost Infection Prevention Clothing against Enveloped Viruses Such as SARS-CoV-2.

ACS Omega 2021 Sep 2;6(36):23495-23503. Epub 2021 Sep 2.

Biomaterials and Bioengineering Laboratory, Centro de Investigación Traslacional San Alberto Magno, Universidad Católica de Valencia San Vicente Mártir, c/Guillem de Castro 94, Valencia 46001, Spain.

Infection prevention clothing is becoming an essential protective tool in the current pandemic, especially because now we know that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can easily infect humans in poorly ventilated indoor spaces. However, commercial infection prevention clothing is made of fabrics that are not capable of inactivating the virus. Therefore, viral infections of symptomatic and asymptomatic individuals wearing protective clothing such as masks can occur through aerosol transmission or by contact with the contaminated surfaces of the masks, which are suspected as an increasing source of highly infectious biological waste. Herein, we report an easy fabrication method of a novel antiviral non-woven fabric containing polymer filaments that were coated with solidified hand soap. This extra protective fabric is capable of inactivating enveloped viruses such as SARS-CoV-2 and phage Φ6 within 1 min of contact. In this study, this antiviral fabric was used to fabricate an antiviral face mask and did not show any cytotoxic effect in human keratinocyte HaCaT cells. Furthermore, this antiviral non-woven fabric could be used for the fabrication of other infection prevention clothing such as caps, scrubs, shirts, trousers, disposable gowns, overalls, hoods, aprons, and shoe covers. Therefore, this low-cost technology could provide a wide range of infection-protective tools to combat COVID-19 and future pandemics in developed and underdeveloped countries.
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http://dx.doi.org/10.1021/acsomega.1c03511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8424690PMC
September 2021

Antimicrobial Face Shield: Next Generation of Facial Protective Equipment against SARS-CoV-2 and Multidrug-Resistant Bacteria.

Int J Mol Sci 2021 Sep 1;22(17). Epub 2021 Sep 1.

Biomaterials and Bioengineering Lab., Centro de Investigación Traslacional San Alberto Magno, Universidad Católica de Valencia San Vicente Mártir, c/Guillem de Castro 94, 46001 Valencia, Spain.

Transparent materials used for facial protection equipment provide protection against microbial infections caused by viruses and bacteria, including multidrug-resistant strains. However, transparent materials used for this type of application are made of materials that do not possess antimicrobial activity. They just avoid direct contact between the person and the biological agent. Therefore, healthy people can become infected through contact of the contaminated material surfaces and this equipment constitute an increasing source of infectious biological waste. Furthermore, infected people can transmit microbial infections easily because the protective equipment do not inactivate the microbial load generated while breathing, sneezing or coughing. In this regard, the goal of this work consisted of fabricating a transparent face shield with intrinsic antimicrobial activity that could provide extra-protection against infectious agents and reduce the generation of infectious waste. Thus, a single-use transparent antimicrobial face shield composed of polyethylene terephthalate and an antimicrobial coating of benzalkonium chloride has been developed for the next generation of facial protective equipment. The antimicrobial coating was analyzed by atomic force microscopy and field emission scanning electron microscopy with elemental analysis. This is the first facial transparent protective material capable of inactivating enveloped viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one minute of contact, and the methicillin-resistant and . Bacterial infections contribute to severe pneumonia associated with the SARS-CoV-2 infection, and their resistance to antibiotics is increasing. Our extra protective broad-spectrum antimicrobial composite material could also be applied for the fabrication of other facial protective tools such as such as goggles, helmets, plastic masks and space separation screens used for counters or vehicles. This low-cost technology would be very useful to combat the current pandemic and protect health care workers from multidrug-resistant infections in developed and underdeveloped countries.
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http://dx.doi.org/10.3390/ijms22179518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8431192PMC
September 2021

A CRISPR/Cas9-based method for seamless N-terminal protein tagging in Saccharomyces cerevisiae.

Yeast 2021 Aug 31. Epub 2021 Aug 31.

Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Japan.

Protein tagging is an effective method for characterizing a gene of interest. Tagging can be accomplished in vivo in Saccharomyces cerevisiae by chromosomal integration of a PCR-amplified cassette. However, common tagging cassettes are not suitable for in situ N-terminal tagging when we aim to preserve the gene's endogenous promoter. Existing methods require either two rounds of homologous recombination or a relatively complex cloning process to construct strains with N-terminal protein tags. Here, we describe a simple CRISPR/Cas9-based method for seamless N-terminal tagging of yeast genes that preserves their endogenous promoter. This method enables the generation of N-terminally tagged strains by introducing an expression vector containing the cas9 gene and a specific gRNA for cleaving the 5' end of the target gene's protein-coding sequence, along with donor DNA containing the tag sequence and homology arms. gRNA cloning was executed by inverse PCR instead of the conventional method. After verifying the tag, the Cas9 and gRNA expression plasmids were eliminated without using antibiotic-containing medium. By this method, we generated strains that express N-terminally tagged subunits of the TORC1 protein kinase complex and found that these strains are comparable to strains made by conventional methods. Thus, our method provides a cost-effective alternative for seamless N-terminal tagging in baker's yeast.
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http://dx.doi.org/10.1002/yea.3666DOI Listing
August 2021

Quercetin in Tartary Buckwheat Induces Autophagy against Protein Aggregations.

Antioxidants (Basel) 2021 Jul 29;10(8). Epub 2021 Jul 29.

Center for Frontier of Oral Sciences, Graduate School of Dentistry, Osaka University, Osaka 5650871, Japan.

Tartary buckwheat is used as an ingredient in flour and tea, as well as in traditional Chinese medicine for its antioxidant effects. Here, we found that an ethanol extract of tartary buckwheat (TBE) potently induced autophagy flux in HeLa cells by suppressing mTORC1 activity, as revealed by dephosphorylation of the mTORC1 substrates Ulk1, S6K, and 4EBP, as well as by the nuclear translocation of transcriptional factor EB. In addition to non-selective bulk autophagy, TBE also induced aggrephagy, which is defined as autophagy against aggregated proteins. Quercetin is a flavonol found at high levels in TBE. We showed that quercetin induced both non-selective bulk autophagy and aggrephagy. These effects were also observed in Huh-7 cells derived from hepatocytes. Thus, aggrephagy induction by TBE and quercetin may relieve alcoholic hepatitis, which is closely linked to the accumulation of protein aggregations called Mallory-Denk bodies.
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http://dx.doi.org/10.3390/antiox10081217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8388858PMC
July 2021

Acetylation of the influenza A virus polymerase subunit PA in the N-terminal domain positively regulates its endonuclease activity.

FEBS J 2021 Jul 16. Epub 2021 Jul 16.

Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Japan.

The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity. Specifically, the full-length PA subunit expressed in cultured 293T cells was found to be strongly acetylated. Moreover, the partial recombinant protein of the PA N-terminal region containing the endonuclease domain was also acetylated by PCAF and GCN5 in vitro, which facilitated its endonuclease activity. Mass spectrometry analyses identified K19 as a candidate acetylation target in the PA N-terminal region. Notably, the substitution of the lysine residue at position 19 with glutamine, a mimic of the acetyl-lysine residue, enhanced its endonuclease activity in vitro; this point mutation also accelerated influenza A virus RNA-dependent RNA polymerase activity in the cell. Our findings suggest that PA acetylation is important for the regulation of the endonuclease and RNA polymerase activities of the influenza A virus.
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http://dx.doi.org/10.1111/febs.16123DOI Listing
July 2021

Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis.

Commun Biol 2021 07 9;4(1):858. Epub 2021 Jul 9.

Laboratory of Ultrastructural Virology, Department of Virus Research, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.

The single-stranded, negative-sense, viral genomic RNA (vRNA) of influenza A virus is encapsidated by viral nucleoproteins (NPs) and an RNA polymerase to form a ribonucleoprotein complex (vRNP) with a helical, rod-shaped structure. The vRNP is responsible for transcription and replication of the vRNA. However, the vRNP conformation during RNA synthesis is not well understood. Here, using high-speed atomic force microscopy and cryo-electron microscopy, we investigated the native structure of influenza A vRNPs during RNA synthesis in vitro. Two distinct types of vRNPs were observed in association with newly synthesized RNAs: an intact, helical rod-shaped vRNP connected with a folded RNA and a deformed vRNP associated with a looped RNA. Interestingly, the looped RNA was a double-stranded RNA, which likely comprises a nascent RNA and the template RNA detached from NPs of the vRNP. These results suggest that while some vRNPs keep their helical structures during RNA synthesis, for the repeated cycle of RNA synthesis, others accidentally become structurally deformed, which likely results in failure to commence or continue RNA synthesis. Thus, our findings provide the ultrastructural feature of vRNPs during RNA synthesis.
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http://dx.doi.org/10.1038/s42003-021-02388-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8271009PMC
July 2021

Resistance of SARS-CoV-2 variants to neutralization by antibodies induced in convalescent patients with COVID-19.

Cell Rep 2021 07 25;36(2):109385. Epub 2021 Jun 25.

Division of Clinical Retrovirology, Joint Research Center for Human Retrovirus infection, Kumamoto University, Kumamoto 860-0811, Japan. Electronic address:

Administration of convalescent plasma or neutralizing monoclonal antibodies (mAbs) is a potent therapeutic option for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, SARS-CoV-2 variants with mutations in the spike protein have emerged in many countries. To evaluate the efficacy of neutralizing antibodies induced in convalescent patients against emerging variants, we isolate anti-spike mAbs from two convalescent COVID-19 patients infected with prototypic SARS-CoV-2 by single-cell sorting of immunoglobulin-G-positive (IgG) memory B cells. Anti-spike antibody induction is robust in these patients, and five mAbs have potent neutralizing activities. The efficacy of most neutralizing mAbs and convalescent plasma samples is maintained against B.1.1.7 and mink cluster 5 variants but is significantly decreased against variants B.1.351 from South Africa and P.1 from Brazil. However, mAbs with a high affinity for the receptor-binding domain remain effective against these neutralization-resistant variants. Rapid spread of these variants significantly impacts antibody-based therapies and vaccine strategies against SARS-CoV-2.
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http://dx.doi.org/10.1016/j.celrep.2021.109385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226103PMC
July 2021

Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells.

iScience 2021 May 16;24(5):102428. Epub 2021 Apr 16.

Center for iPS Cell Research and Application (CiRA), Kyoto University, Shogoin Kawaharacho 53, Sakyo-ku, Kyoto 606-8507, Japan.

Genetic differences are a primary reason for differences in the susceptibility and severity of COVID-19. As induced pluripotent stem (iPS) cells maintain the genetic information of the donor, they can be used to model individual differences in SARS-CoV-2 infection . We found that human iPS cells expressing the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) (ACE2-iPS cells) can be infected w SARS-CoV-2. In infected ACE2-iPS cells, the expression of SARS-CoV-2 nucleocapsid protein, budding of viral particles, and production of progeny virus, double membrane spherules, and double-membrane vesicles were confirmed. We performed SARS-CoV-2 infection experiments on ACE2-iPS/ embryonic stem (ES) cells from eight individuals. Male iPS/ES cells were more capable of producing the virus compared with female iPS/ES cells. These findings suggest that ACE2-iPS cells can not only reproduce individual differences in SARS-CoV-2 infection but also are a useful resource to clarify the causes of individual differences in COVID-19 due to genetic differences.
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http://dx.doi.org/10.1016/j.isci.2021.102428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8051014PMC
May 2021

Amplification-free RNA detection with CRISPR-Cas13.

Commun Biol 2021 04 19;4(1):476. Epub 2021 Apr 19.

Molecular Physiology Laboratory, Cluster for Pioneering Research, RIKEN, Saitama, Japan.

CRISPR-based nucleic-acid detection is an emerging technology for molecular diagnostics. However, these methods generally require several hours and could cause amplification errors, due to the pre-amplification of target nucleic acids to enhance the detection sensitivity. Here, we developed a platform that allows "CRISPR-based amplification-free digital RNA detection (SATORI)", by combining CRISPR-Cas13-based RNA detection and microchamber-array technologies. SATORI detected single-stranded RNA targets with maximal sensitivity of ~10 fM in <5 min, with high specificity. Furthermore, the simultaneous use of multiple different guide RNAs enhanced the sensitivity, thereby enabling the detection of the SARS-CoV-2 N-gene RNA at ~5 fM levels. Therefore, we hope SATORI will serve as a powerful class of accurate and rapid diagnostics.
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http://dx.doi.org/10.1038/s42003-021-02001-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055673PMC
April 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Vacuolar protein Tag1 and Atg1-Atg13 regulate autophagy termination during persistent starvation in .

J Cell Sci 2021 02 26;134(4). Epub 2021 Feb 26.

Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Yamadaoka 1-8, Suita, Osaka 565-0871, Japan

Under starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast , autophagy is terminated after 10-12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δ mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.
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http://dx.doi.org/10.1242/jcs.253682DOI Listing
February 2021

A novel aqueous extract from rice fermented with Aspergillus oryzae and Saccharomyces cerevisiae possesses an anti-influenza A virus activity.

PLoS One 2021 15;16(1):e0244885. Epub 2021 Jan 15.

Faculty of Pharmaceutical Sciences, Laboratory of Biochemistry, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan.

Human influenza virus infections occur annually worldwide and are associated with high morbidity and mortality. Hence, development of novel anti-influenza drugs is urgently required. Rice Power® extract developed by the Yushin Brewer Co. Ltd. is a novel aqueous extract of rice obtained via saccharization and fermentation with various microorganisms, such as Aspergillus oryzae, yeast [such as Saccharomyces cerevisiae], and lactic acid bacteria, possessing various biological and pharmacological properties. In our previous experimental screening with thirty types of Rice Power® extracts, we observed that the 30th Rice Power® (Y30) extract promoted the survival of influenza A virus-infected Madin-Darby canine kidney (MDCK) cells. Therefore, to identify compounds for the development of novel anti-influenza drugs, we aimed to investigate whether the Y30 extract exhibits anti-influenza A virus activity. In the present study, we demonstrated that the Y30 extract strongly promoted the survival of influenza A H1N1 Puerto Rico 8/34 (A/PR/8/34), California 7/09, or H3N2 Aichi 2/68 (A/Aichi/2/68) viruses-infected MDCK cells and inhibited A/PR/8/34 or A/Aichi/2/68 viruses infection and growth in the co-treatment and pre-infection experiments. The pre-treatment of Y30 extract on MDCK cells did not induce anti-influenza activity in the cell. The Y30 extract did not significantly affect influenza A virus hemagglutination, and neuraminidase and RNA-dependent RNA polymerase activities. Interestingly, the electron microscopy experiment revealed that the Y30 extract disrupts the integrity of influenza A virus particles by permeabilizing the viral membrane envelope, suggesting that Y30 extract has a direct virucidal effect against influenza A virus. Furthermore, we observed that compared to the ethyl acetate (EtOAc) extract, the water extract of Y30 extract considerably promoted the survival of cells infected with A/PR/8/34 virus. These results indicated that more anti-influenza components were present in the water extract of Y30 extract than in the EtOAc extract. Our results highlight the potential of a rice extract fermented with A. oryzae and S. cerevisiae as an anti-influenza medicine and a drug source for the development of anti-influenza compounds.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0244885PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810313PMC
May 2021

Protective Face Mask Filter Capable of Inactivating SARS-CoV-2, and Methicillin-Resistant and .

Polymers (Basel) 2021 Jan 8;13(2). Epub 2021 Jan 8.

Biomaterials and Bioengineering Lab, Centro de Investigación Traslacional San Alberto Magno, Universidad Católica de Valencia San Vicente Mártir, c/Guillem de Castro 94, 46001 Valencia, Spain.

Face masks have globally been accepted to be an effective protective tool to prevent bacterial and viral transmission, especially against indoor aerosol transmission. However, commercial face masks contain filters that are made of materials that are not capable of inactivating either SARS-CoV-2 or multidrug-resistant bacteria. Therefore, symptomatic and asymptomatic individuals can infect other people even if they wear them because some viable viral or bacterial loads can escape from the masks. Furthermore, viral or bacterial contact transmission can occur after touching the mask, which constitutes an increasing source of contaminated biological waste. Additionally, bacterial pathogens contribute to the SARS-CoV-2-mediated pneumonia disease complex, and their resistance to antibiotics in pneumonia treatment is increasing at an alarming rate. In this regard, herein, we report the development of a non-woven face mask filter fabricated with a biofunctional coating of benzalkonium chloride that is capable of inactivating more than 99% of SARS-CoV-2 particles in one minute of contact, and the life-threatening methicillin-resistant and (normalized antibacterial of 0.52 ± 0.04 and 0.72 ± 0.04, respectively). Nonetheless, despite the results obtained, further studies are needed to ensure the safety and correct use of this technology for the mass production and commercialization of this broad-spectrum antimicrobial face mask filter. Our novel protective non-woven face mask filter would be useful for many healthcare workers and researchers working in this urgent and challenging field.
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http://dx.doi.org/10.3390/polym13020207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827663PMC
January 2021

Optimal Expression of the Envelope Glycoprotein of Orthobornaviruses Determines the Production of Mature Virus Particles.

J Virol 2020 Dec 2. Epub 2020 Dec 2.

Laboratory of RNA Viruses, Department of Virus Research, Institute for Frontier Life and Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan

An RNA virus-based episomal vector (REVec) whose backbone is Borna disease virus 1 (BoDV-1) can provide long-term gene expression in transduced cells. To improve the transduction efficiency of REVec, we evaluated the role of the viral envelope glycoprotein (G) of the genus , including that of BoDV-1, in the production of infectious particles. By using G-pseudotype assay in which the lack of G in G-deficient REVec (ΔG-REVec) was compensated for expression of G, we found that excess expression of BoDV-1-G does not affect particle production itself but results in uncleaved and aberrant mature G expression in the cells, leading to the production of REVec particles with low transduction titers. We revealed that the expression of uncleaved G in the cells inhibits the incorporation of mature G and vgRNA into the particles. This feature of G was conserved among mammalian and avian orthobornaviruses; however, the cleavage efficacy of canary bornavirus 1 (CnBV-1)-G was exceptionally not impaired by its excess expression, which led to the production of the pseudotype ΔG-REVec with the highest titer. Chimeric G proteins between CnBV-1 and -2 revealed that the signal peptide of CnBV-1-G was responsible for the cleavage efficacy through the interaction with intracellular furin. We showed that CnBV-1 G leads to the development of pseudotyped REVec with high transduction efficiency and a high-titer recombinant REVec. Our study demonstrated that the restricted expression of orthobornavirus G contributes to the regulation of infectious particle production, the mechanism of which can improve the transduction efficiency of REVec. Most viruses causing persistent infection produce few infectious particles from the infected cells. Borna disease virus 1, a member of the genus , is an RNA virus that persistently infects the nucleus and has been applied to vectors for long-term gene expression. In this study, we showed that, common among orthobornaviruses, excessive G expression does not affect particle production itself but reduces the production of infectious particles with mature G and genomic RNA. This result suggested that limited G expression contributes to suppressing abnormal viral particle production. On the other hand, we found that canary bornavirus 1 has an exceptional G maturation mechanism and produces a high-titer virus. Our study will contribute to not only understanding the mechanism of infectious particle production but also improving the vector system of orthobornaviruses.
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http://dx.doi.org/10.1128/JVI.02221-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092845PMC
December 2020

Cryo-EM Structure of the Prostaglandin E Receptor EP4 Coupled to G Protein.

Structure 2021 03 1;29(3):252-260.e6. Epub 2020 Dec 1.

Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Kyoto 606-8501, Japan; Department of Medical Chemistry, Kansai Medical University, Hirakata, Osaka 573-1010, Japan. Electronic address:

Prostaglandin E receptor EP4, a class A G protein-coupled receptor (GPCR), is a common drug target in various disorders, such as acute decompensated heart failure and ulcerative colitis. Here, we report the cryoelectron microscopy (cryo-EM) structure of the EP4-heterotrimeric G protein (Gs) complex with the endogenous ligand at a global resolution of 3.3 Å. In this structure, compared with that in the inactive EP4 structure, the sixth transmembrane domain is shifted outward on the intracellular side, although the shift is smaller than that in other class A GPCRs bound to Gs. Instead, the C-terminal helix of Gs is inserted toward TM2 of EP4, and the conserved C-terminal hook structure formsthe extended state. These structural features are formed by the conserved residues in prostanoid receptors (Phe54 and Trp327). These findings may be important for the thorough understanding of the G protein-binding mechanism of EP4 and other prostanoid receptors.
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http://dx.doi.org/10.1016/j.str.2020.11.007DOI Listing
March 2021

Autophagosome formation in relation to the endoplasmic reticulum.

J Biomed Sci 2020 Oct 22;27(1):97. Epub 2020 Oct 22.

Center for Frontier Oral Sciences, Graduate School of Dentistry, Osaka University Graduate School, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.
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http://dx.doi.org/10.1186/s12929-020-00691-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7579975PMC
October 2020

Selective Genome Packaging Mechanisms of Influenza A Viruses.

Authors:
Takeshi Noda

Cold Spring Harb Perspect Med 2021 Jul 1;11(7). Epub 2021 Jul 1.

Laboratory of Ultrastructural Virology, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.

The genome of influenza A virus (IAV) comprises eight segmented, single-stranded, negative-sense RNAs. The genome packaging mechanism of IAV was a long-standing enigma, but it is now widely accepted that IAV packages one copy of each of the eight viral RNA (vRNA) segments in a selective manner. Accumulating evidence over the last decade suggests that the eight unique vRNAs are selected via intersegment interactions mediated by their segment-specific genome packaging signals; however, the characteristics of these RNA-based interactions largely remain unknown. This review summarizes our current knowledge of IAV selective genome packaging and the possible mechanisms underlying the selection of the eight unique vRNAs.
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http://dx.doi.org/10.1101/cshperspect.a038497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8247559PMC
July 2021

STEEP mediates STING ER exit and activation of signaling.

Nat Immunol 2020 08 20;21(8):868-879. Epub 2020 Jul 20.

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.
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http://dx.doi.org/10.1038/s41590-020-0730-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610351PMC
August 2020

Influenza A virus NS1 optimises virus infectivity by enhancing genome packaging in a dsRNA-binding dependent manner.

Virol J 2020 07 16;17(1):107. Epub 2020 Jul 16.

Laboratory of Molecular Genetics, Institute for Frontier Life and Medical Science, Kyoto University, Kyoto, Japan.

Background: The non-structural protein 1 (NS1) of influenza A virus (IAV) is a key player in inhibiting antiviral response in host cells, thereby facilitating its replication. However, other roles of NS1, which are independent of antagonising host cells' antiviral response, are less characterised.

Methods: To investigate these unidentified roles, we used a recombinant virus, which lacks NS1 expression, and observed its phenotypes during the infection of antiviral defective cells (RIG-I KO cells) in the presence or absence of exogeneous NS1. Moreover, we used virus-like particle (VLP) production system to further support our findings.

Results: Our experiments demonstrated that IAV deficient in NS1 replicates less efficiently than wild-type IAV in RIG-I KO cells and this replication defect was complemented by ectopic expression of NS1. As suggested previously, NS1 is incorporated in the virion and participates in the regulation of viral transcription and translation. Using the VLP production system, in which minigenome transcription or viral protein production was unaffected by NS1, we demonstrated that NS1 facilitates viral genome packaging into VLP, leading to efficient minigenome transfer by VLP. Furthermore, the incorporation of NS1 and the minigenome into VLP were impaired by introducing a point mutation (R38A) in the double stranded RNA-binding domain of NS1.

Conclusion: These results suggest a novel function of NS1 in improving genome packaging in a dsRNA binding-dependent manner. Taken together, NS1 acts as an essential pro-viral regulator, not only by antagonizing host immunity but also by facilitating viral replication and genome packaging.
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http://dx.doi.org/10.1186/s12985-020-01357-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367362PMC
July 2020

ERdj8 governs the size of autophagosomes during the formation process.

J Cell Biol 2020 08;219(8)

Institute for Protein Dynamics, Kyoto Sangyo University, Kyoto, Japan.

In macroautophagy, membrane structures called autophagosomes engulf substrates and deliver them for lysosomal degradation. Autophagosomes enwrap a variety of targets with diverse sizes, from portions of cytosol to larger organelles. However, the mechanism by which autophagosome size is controlled remains elusive. We characterized a novel ER membrane protein, ERdj8, in mammalian cells. ERdj8 localizes to a meshwork-like ER subdomain along with phosphatidylinositol synthase (PIS) and autophagy-related (Atg) proteins. ERdj8 overexpression extended the size of the autophagosome through its DnaJ and TRX domains. ERdj8 ablation resulted in a defect in engulfing larger targets. C. elegans, in which the ERdj8 orthologue dnj-8 was knocked down, could perform autophagy on smaller mitochondria derived from the paternal lineage but not the somatic mitochondria. Thus, ERdj8 may play a critical role in autophagosome formation by providing the capacity to target substrates of diverse sizes for degradation.
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http://dx.doi.org/10.1083/jcb.201903127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401821PMC
August 2020

Highly Reproducible and Efficient FASnI Perovskite Solar Cells Fabricated with Volatilizable Reducing Solvent.

J Phys Chem Lett 2020 Apr 31;11(8):2965-2971. Epub 2020 Mar 31.

State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, China.

Lead-free tin halide perovskite solar cells (PSCs) have attracted great attention because of their low toxicity, ideal band gap, and high carrier mobilities. However, the efficiency and reproducibility of tin halide PSCs has been limited because of the facile oxidation of Sn to Sn. Herein, liquid formic acid (LFA) was introduced as a reducing solvent in the FASnI (FA: formamidinium) perovskite precursor solution. Unlike solid reducing additives, the LFA solvent is volatile, so no residual LFA remained in the FASnI perovskite film. Use of the LFA solvent resulted in production of the FASnI perovskite film with high crystallinity, low Sn content, reduced background doping, and low electronic trap density. As a result, an efficiency of over 10% was obtained for lead-free tin halide PSCs with improved reproducibility.
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http://dx.doi.org/10.1021/acs.jpclett.0c00923DOI Listing
April 2020

Finding cell-specific expression patterns in the early Ciona embryo with single-cell RNA-seq.

Sci Rep 2020 03 18;10(1):4961. Epub 2020 Mar 18.

Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, 904-0495, Japan.

Single-cell RNA-seq has been established as a reliable and accessible technique enabling new types of analyses, such as identifying cell types and studying spatial and temporal gene expression variation and change at single-cell resolution. Recently, single-cell RNA-seq has been applied to developing embryos, which offers great potential for finding and characterising genes controlling the course of development along with their expression patterns. In this study, we applied single-cell RNA-seq to the 16-cell stage of the Ciona embryo, a marine chordate and performed a computational search for cell-specific gene expression patterns. We recovered many known expression patterns from our single-cell RNA-seq data and despite extensive previous screens, we succeeded in finding new cell-specific patterns, which we validated by in situ and single-cell qPCR.
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http://dx.doi.org/10.1038/s41598-020-61591-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080732PMC
March 2020

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

Nat Commun 2020 03 13;11(1):1334. Epub 2020 Mar 13.

Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.

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.
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http://dx.doi.org/10.1038/s41467-020-14957-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070030PMC
March 2020

Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan.

PLoS One 2020 5;15(3):e0230156. Epub 2020 Mar 5.

Center for Frontier Oral Science, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca2+ dependent manner. Cytosolic Ca2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca2+ increase, thereby suppressing induction of autophagy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230156PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058311PMC
June 2020
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