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Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1, 000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies., 気管支オルガノイドを用いた新型コロナウイルス研究とその創薬応用. 京都大学プレスリリース. 2022-05-30., COVID-19 Research Using Bronchial Organoids and Drug Discovery Applications. 京都大学プレスリリース. 2022-06-02.

In the ongoing COVID-19 pandemic, rapid and sensitive diagnosis of viral infection is a critical deterrent to the spread of SARS-CoV-2. To this end, we developed an automated amplification-free digital RNA detection platform using CRISPR-Cas13a and microchamber device (opn-SATORI), which automatically completes a detection process from sample mixing to RNA quantification in clinical specimens within ~9 min. Using the optimal Cas13a enzyme and magnetic beads technology, opn-SATORI detected SARS-CoV-2 genomic RNA with a LoD of, 新型コロナウイルスの超高感度・全自動迅速検出装置の開発 --汎用的な感染症診断装置としての社会実装に期待--. 京都大学プレスリリース. 2022-05-27.

Tin-based perovskite solar cells (TPSCs) as the most promising candidate for lead-free PSCs have incurred extensive researches all over the world. However, the crystallization process of tin-based perovskite is too fast during the solution-deposited process, resulting in abundant pinholes and poor homogeneity that cause serious charge recombination in perovskite layer. Here, we employed the π-conjugated Lewis base molecules with high electron density to systematically control the crystallization rate of FASnI3 perovskite by forming stable intermediate phase with the Sn-I frameworks, leading to a compact and uniform perovskite film with large increase in the carrier lifetime. Meanwhile, the introduction of the π-conjugated systems also retards the permeation of moisture into perovskite crystal, which significantly suppresses the film degradation in air. These benefits contributed to a stabilizing power conversion efficiency (PCE) of 10.1% for the TPSCs and maintained over 90% of its initial PCE after 1000-h light soaking in air. Also, a steady-state efficiency of 9.2% was certified at the accredited test center.

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., Nakano et al. use high-speed AFM and cryo-EM to study influenza virus RNA synthesis in vitro. They observe the synthesis of two types of RNA: folded single stranded RNA associated to an intact viral ribonucleoprotein (vRNP) and looped, double stranded RNA associated with a partially rearranged vRNP structure.

RNA-modulating factors not only regulate multiple steps of cellular RNA metabolism, but also emerge as key effectors of the immune response against invading viral pathogens including human immunodeficiency virus type-1 (HIV-1). However, the cellular RNA-binding proteins involved in the establishment and maintenance of latent HIV-1 reservoirs have not been extensively studied. Here, we screened a panel of 62 cellular RNA-binding proteins and identified NEDD4-binding protein 1 (N4BP1) as a potent interferon-inducible inhibitor of HIV-1 in primary T cells and macrophages. N4BP1 harbours a prototypical PilT N terminus-like RNase domain and inhibits HIV-1 replication by interacting with and degrading viral mRNA species. Following activation of CD4+ T cells, however, N4BP1 undergoes rapid cleavage at Arg 509 by the paracaspase named mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1). Mutational analyses and knockout studies revealed that MALT1-mediated inactivation of N4BP1 facilitates the reactivation of latent HIV-1 proviruses. Taken together, our findings demonstrate that the RNase N4BP1 is an efficient restriction factor of HIV-1 and suggest that inactivation of N4BP1 by induction of MALT1 activation might facilitate elimination of latent HIV-1 reservoirs. The interferon-inducible RNA-binding protein N4BP1 binds to and degrades HIV-1 mRNAs, thus inhibiting viral degradation and promoting viral latency. However, metacaspase-mediated degradation of N4BP1 following activation of CD4+ T cells facilitates HIV-1 latency reversal.

In this letter, we have introduced copper chloride (CuCl2) as an additive in the CH3NH3PbI3 precursor solution to improve the surface morphology and crystallinity of CH3NH3PbI3 films in a single solvent system. Our optimized perovskite solar cells (PSCs) with 2.5 mol% CuCl2 additive showed best power conversion efficiency (PCE) of 15.22%. The PCE of the PSCs fabricated by CuCl2 (2.5 mol%) additive engineering was 56% higher than the PSC fabricated with pristine CH3NH3PbI3.

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein-nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein, and cryo-electron microscopy of nucleocapsid or nucleocapsid-like structures. There have been no high-resolution reconstructions of complete mononegavirus nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Autophagy is a bulk degradation process in eukaryotic cells and has fundamental roles in cellular homeostasis.The origin and source of autophagosomal membranes are long-standing questions in the field. Using electron microscopy, we show that, in mammalian culture cells, the endoplasmic reticulum (ER) associates with early autophagic structures called isolation membranes (IMs). Overexpression of an Atg4B mutant, which causes defects in autophagosome formation, induces the accumulation of ER-IM complexes. Electron tomography revealed that the ER-IM complex appears as a subdomain of the ER that formed a cradle encircling the IM, and showed that both ER and isolation membranes are interconnected.

Beclin 1, a protein essential for autophagy, binds to hVps34/Class III phosphatidylinositol-3-kinase and UVRAG. Here, we have identified two Beclin 1 associated proteins, Atg14L and Rubicon. Atg14L and UVRAG bind to Beclin 1 in a mutually exclusive manner, whereas Rubicon binds only to a subpopulation of UVRAG complexes; thus, three different Beclin 1 complexes exist. GFP-Atg14L localized to the isolation membrane and autophagosome, as well as to the ER and unknown puncta. Knockout of Atg14L in mouse ES cells caused a defect in autophagosome formation. GFP-Rubicon was localized at the endosome/lysosome. Knockdown of Rubicon caused enhancement of autophagy, especially at the maturation step, as well as enhancement of endocytic trafficking. These data suggest that the Beclin 1-hVps34 complex functions in two different steps of autophagy by altering the subunit composition.

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole1,2. Apg8/Aut7 is an essential factor for autophagy in yeast3,4,5. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus6. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane6. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy6.

In viruses, as in eukaryotes, elaborate mechanisms have evolved to protect the genome and to ensure its timely replication and reliable transmission to progeny. Influenza A viruses are enveloped, spherical or filamentous structures, ranging from 80 to 120 nm in diameter. Inside each envelope is a viral genome consisting of eight single-stranded negative-sense RNA segments of 890 to 2,341 nucleotides each. These segments are associated with nucleoprotein and three polymerase subunits, designated PA, PB1 and PB2; the resultant ribonucleoprotein complexes (RNPs) resemble a twisted rod (10-15 nm in width and 30-120 nm in length) that is folded back and coiled on itself. Late in viral infection, newly synthesized RNPs are transported from the nucleus to the plasma membrane, where they are incorporated into progeny virions capable of infecting other cells. Here we show, by transmission electron microscopy of serially sectioned virions, that the RNPs of influenza A virus are organized in a distinct pattern (seven segments of different lengths surrounding a central segment). The individual RNPs are suspended from the interior of the viral envelope at the distal end of the budding virion and are oriented perpendicular to the budding tip. This finding argues against random incorporation of RNPs into virions, supporting instead a model in which each segment contains specific incorporation signals that enable the RNPs to be recruited and packaged as a complete set. A selective mechanism of RNP incorporation into virions and the unique organization of the eight RNP segments may be crucial to maintaining the integrity of the viral genome during repeated cycles of replication.

The influenza A virus genome consists of eight single-stranded negative-sense RNA (vRNA) segments. Although genome segmentation provides advantages such as genetic reassortment, which contributes to the emergence of novel strains with pandemic potential, it complicates the genome packaging of progeny virions. Here we elucidate, using electron tomography, the three-dimensional structure of ribonucleoprotein complexes (RNPs) within progeny virions. Each virion is packed with eight well-organized RNPs that possess rod-like structures of different lengths. Multiple interactions are found among the RNPs. The position of the eight RNPs is not consistent among virions, but a pattern suggests the existence of a specific mechanism for assembly of these RNPs. Analyses of budding progeny virions suggest two independent roles for the viral spike proteins: RNP association on the plasma membrane and the subsequent formation of the virion shell. Our data provide further insights into the mechanisms responsible for segmented-genome packaging into virions., The influenza A virus genome consists of eight RNA segments, which permits genetic reassortment and contributes to the emergence of novel strains with pandemic potential. Here, electron tomography is used to study the three-dimensional structure of ribonucleoprotein complexes within progeny virions.

The mechanism that causes high-temperature superconductivity in copper oxide materials (cuprates) is still unknown, more than 15 years after it was discovered1. As the charge carriers (electrons or holes) are introduced into the parent antiferromagnetic insulator, a process called doping, the material evolves from an insulator to a superconductor, and eventually to a normal metal. This marked change of physical properties with doping2,3,4,5,6,7 indicates that doping dependence (non-universality) might be a general feature of these materials, but we find that, on the contrary, the low-energy Fermi velocity of electrons is in fact universal, even among different superconductor families.