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Purpose COVID-19 has severely affected urban and rural tourism in China, which has been extensively reported in the newspapers. This paper aims to discuss the impact of COVID-19 on urban and rural tourism at each critical juncture of the COVID-19 pandemic, as well as the studies, measures and attitudes of various stakeholders toward urban and rural tourism and their future development. Design/methodology/approach This paper collects Chinese news reports as the research object and uses ROST Content Mining 6.0 and Gephi software to analyze and visualize the data for high-frequency words and analyzed the discourse of stakeholders within the newspaper text. Findings This study found that prior to February 20, 2020, the Chinese Government mainly focused on pandemic prevention. From February 21 to April 3 (before The Qingming Festival), resumption of work and production became the theme. From April 4 to May 1 (before May Day), a time-sharing reservation system was widely implemented and the differentiation between urban and rural tourism became more prominent. After May 1, rural tourism attracted greater attention. Originality/value This paper uses discourse analysis to understand the impact of COVID-19 on urban and rural tourism from the perspective of time nodes and the relationship between various stakeholders and predicts the development trends within urban and rural tourism. This paper enriches and expands tourism management knowledge in the face of major crises, providing valuable advice for managers and investors and makes suggestions for the development of urban and rural tourism. COVID-19下中国的城乡旅游：恢复措施与旅游发展研究 目的 COVID-19已严重影响了中国的城市和乡村旅游业, 报纸上对此进行了广泛报道。本文旨在探讨在COVID-19大流行的每个关键时刻对城乡旅游的影响, 以及各种利益相关者对城乡旅游及其未来发展的观点, 措施和态度。 设计/方法 本文以中文新闻报道为研究对象, 利用ROST Content Mining 6.0和Gephi软件对高频词数据进行分析和可视化, 分析了报纸正文中利益相关者的话语。 结果 这项研究发现, 在2020年2月20日之前, 中国政府主要将重点放在疫情防控上。 2月21日至4月3日（清明节之前）, 复工复产成为主题。 从4月4日至5月1日（五一节之前）, 分时预约系统得到广泛实施, 城市和乡村旅游业的区别更加突出。 5月1日之后, 乡村旅游引起了更多关注。 创意/价值 本文通过话语分析从时间节点和各个利益相关者之间的关系的角度了解了COVID-19对城乡旅游业的影响, 并预测了城乡旅游业的发展趋势。 面对重大危机, 本文丰富和扩展了旅游管理知识, 为管理者和投资者提供了宝贵的建议, 并为城乡旅游业的发展提供了建议。 Turismo urbano y rural bajo COVID-19 en China Investigación sobre medidas de recuperación y desarrollo turístico Propósito COVID-19 ha afectado gravemente al turismo urbano y rural en China, que ha sido ampliamente informado en los periódicos. Este documento tiene como objetivo discutir el impacto de COVID-19 en el turismo urbano y rural en cada coyuntura crítica de la pandemia de COVID-19, así como los puntos de vista, las medidas y las actitudes de diversos interesados en relación con el turismo urbano y rural y su desarrollo futuro. Diseño/metodología/enfoque Este documento recopiló informes de noticias chinos como objeto de investigación y utilizó el software ROST Content Mining 6.0 y Gephi para analizar y visualizar los datos de palabras de alta frecuencia y analizó el discurso de los interesados dentro del texto del periódico. Conclusiones Este estudio encontró que antes del 20 de febrero de 2020, el gobierno chino se centró principalmente en la prevención de la pandemia en lugares pintorescos. Del 21 de febrero al 3 de abril (antes del Festival de Qingming), la reanudación del trabajo y la producción se convirtió en el tema. Desde el 4 de abril hasta el 1 de mayo (antes del Primero de Mayo), se implementó ampliamente un sistema de reserva de tiempo compartido y la diferenciación entre el turismo urbano y el rural se hizo más prominente. Después del 1 de mayo, el turismo rural atrajo mayor atención. Originalidad/valor En este documento, se utiliza el análisis del discurso para comprender el impacto de COVID-19 en el turismo urbano y rural desde la perspectiva de los nodos de tiempo y la relación entre los diversos interesados y se predice las tendencias de desarrollo dentro del turismo urbano y rural. Este trabajo enriquece y amplía el conocimiento de la gestión turística frente a las grandes crisis, brindando valiosos consejos a administradores e inversores, y hace sugerencias para el desarrollo del turismo urbano y rural.

Membrane fusion triggered by Ca 2+ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes 1 – 4 . For neurotransmitter release, the Ca 2+ sensitivity is introduced by interactions between the Ca 2+ sensor synaptotagmin and the SNARE complex 5 , and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion 6 . Disruption of Ca 2+ -triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis 7 – 11 . Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca 2+ -triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca 2+ -binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca 2+ -triggered fusion at physiological Ca 2+ concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca 2+ -triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways. Peptides that disrupt Ca 2+ -triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.

Single-stranded RNA bacteriophages (ssRNA phages) infect Gram-negative bacteria via a single maturation protein (Mat), which attaches to a retractile pilus of the host. Here we present structures of the ssRNA phage MS2 in complex with the Escherichia coli F-pilus, showing a network of hydrophobic and electrostatic interactions at the Mat-pilus interface. Moreover, binding of the pilus induces slight orientational variations of the Mat relative to the rest of the phage capsid, priming the Mat-connected genomic RNA (gRNA) for its release from the virions. The exposed tip of the attached Mat points opposite to the direction of the pilus retraction, which may facilitate the translocation of the gRNA from the capsid into the host cytosol. In addition, our structures determine the orientation of the assembled F-pilin subunits relative to the cell envelope, providing insights into the F-like type IV secretion systems. Single-stranded RNA bacteriophages use a single maturation protein (Mat) to attach to a retractile pilus of the bacterial host. Here, the authors report the structures of the MS2 phage bound to the host receptor F-pili and define the orientations of Mat relative to the cell and emanating F-pili, providing new insights into the F-like type IV secretion systems.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies due to structural and dynamic changes of the viral spike glycoprotein (S). The heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains of S drive virus–host membrane fusion by assembly into a six-helix bundle, resulting in delivery of viral RNA into the host cell. We surveyed mutations of currently reported SARS-CoV-2 variants and selected eight mutations, including Q954H, N969K, and L981F from the Omicron variant, in the postfusion HR1HR2 bundle for functional and structural studies. We designed a molecular scaffold to determine cryogenic electron microscopy (cryo-EM) structures of HR1HR2 at 2.2–3.8 Å resolution by linking the trimeric N termini of four HR1 fragments to four trimeric C termini of the Dps4 dodecamer from Nostoc punctiforme. This molecular scaffold enables efficient sample preparation and structure determination of the HR1HR2 bundle and its mutants by single-particle cryo-EM. Our structure of the wild-type HR1HR2 bundle resolves uncertainties in previously determined structures. The mutant structures reveal side-chain positions of the mutations and their primarily local effects on the interactions between HR1 and HR2. These mutations do not alter the global architecture of the postfusion HR1HR2 bundle, suggesting that the interfaces between HR1 and HR2 are good targets for developing antiviral inhibitors that should be efficacious against all known variants of SARS-CoV-2 to date. We also note that this work paves the way for similar studies in more distantly related viruses.

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX’s mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site.We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition 1 , 2 , yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains 3 . The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the proton gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters 4 , 5 . Synaptophysin 6 and its paralogues synaptoporin 7 and synaptogyrin 8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin-knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin-knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin. Using cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles, a V-ATPase–synaptophysin interface was found to regulate synaptic vesicle biogenesis and alter seizure susceptibility.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.

Telomeres are the essential nucleoprotein structures that provide a physical cap for the ends of linear chromosomes. The highly conserved CST (CTC1/STN1/TEN1) protein complex facilitates telomeric DNA replication and promotes telomere stability. Here we report three unexpected properties of Arabidopsis thaliana TEN1 that indicate it possesses functions distinct from other previously characterized telomere proteins. First, we show that telomeres in ten1 mutants are highly sensitive to thermal stress. Heat shock causes abrupt and dramatic loss of telomeric DNA in ten1 plants, likely via deletional recombination. Second, we show that AtTEN1 has the properties of a heat-shock induced molecular chaperone. At elevated temperature, AtTEN1 rapidly assembles into high molecular weight homo-oligomeric complexes that efficiently suppress heat-induced aggregation of model protein substrates in vitro. Finally, we report that AtTEN1 specifically protects CTC1 from heat-induced aggregation in vitro, and from heat-induced protein degradation and loss of telomere association in vivo. Collectively, these observations define Arabidopsis TEN1 as a highly dynamic protein that works in concert with CTC1 to preserve telomere integrity in response to environmental stress.

Gene expression in the human pathogen, Mycobacterium tuberculosis (M. tuberculosis), may possess species-specific structural features that can be targeted by drugs. Previous studies have identified, for example, several ribosomal RNA (rRNA) expansion segments and ribosomal proteins (rProtein) encoded by paralogous genes in the M. tuberculosis ribosome. To better understand the functions of these features and facilitate drug design, high-resolution structures are essential. Here we have used cryo-electron microscopy (cryo-EM) to solve the structures of RNA polymerase, the ribosome, and their higher order complexes.The RNA polymerase structure we have solved is a core complex consisting of all five subunits. The structure indicates a new conformation of a bridging α-helix, termed “B2”, on the β subunit. In this new conformation, the B2 helix can potentially interact with rifampicin, a firstline drug against tuberculosis, suggesting exciting potential approach to design rifampicin derivatives. For the M. tuberculosis ribosome, we have solved multiple structures. These structures reveal that, upon joining of the large and small ribosomal subunits, a 100-nucleotide long expansion segment of the M. tuberculosis 23S rRNA, named H54a or the “handle”, switches from interacting with rRNA helix H68 and rProtein uL2 to interact with rProtein bS6, forming a new intersubunit bridge “B9”. In M. tuberculosis 70S, bridge B9 is mostly maintained, leading to correlated motions among the handle, the L1 stalk and the small subunit in the rotated and non-rotated states. Two new protein densities were discovered near the decoding center and the peptidyl transferase center, respectively. We have also characterized the high order complexes involved in gene expression, including ribosome dimers and expressome.The biochemical and structural characterizations in this work provided a glimpse of the key components in the mycobacterial gene expression system. Most, if not all, unique features were unveiled, which should be not only further complemented with functional studies, but also taken into account for anti-tuberculosis drug development.