Large-Scale Comparative Analyses of Tick Genomes Elucidate Their Genetic Diversity and Vector Capacities.

Among arthropod vectors, ticks transmit the most diverse human and animal pathogens, leading to an increasing number of new challenges worldwide. Here we sequenced and assembled high-quality genomes of six ixodid tick species and further resequenced 678 tick specimens to understand three key aspects of ticks: genetic diversity, population structure, and pathogen distribution. We explored the genetic basis common to ticks, including heme and hemoglobin digestion, iron metabolism, and reactive oxygen species, and unveiled for the first time that genetic structure and pathogen composition in different tick species are mainly shaped by ecological and geographic factors. We further identified species-specific determinants associated with different host ranges, life cycles, and distributions. The findings of this study are an invaluable resource for research and control of ticks and tick-borne diseases.

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

The I7L protein of African swine fever virus is involved in viral pathogenicity by antagonizing the IFN-γ-triggered JAK-STAT signaling pathway through inhibiting the phosphorylation of STAT1.

Cell-passage-adapted strains of African swine fever virus (ASFV) typically exhibit substantial genomic alterations and attenuated virulence in pigs. We have indicated that the human embryonic kidney (HEK293T) cells-adapted ASFV strain underwent genetic alterations and the I7L gene in the right variable region was deleted compared with the ASFV HLJ/2018 strain (ASFV-WT). A recent study has revealed that the deletion of the I7L-I11L genes results in attenuation of virulent ASFV in vivo, but the underlying mechanism remains largely unknown. Therefore, we hypothesized that the deletion of the I7L gene may be related to the pathogenicity of ASFV in pigs. We generated the I7L gene-deleted ASFV mutant (ASFV-ΔI7L) and found that the I7L gene deletion does not influence the replication of ASFV in primary porcine alveolar macrophages (PAMs). Using transcriptome sequencing analysis, we identified that the differentially expressed genes in the PAMs infected with ASFV-ΔI7L were mainly involved in antiviral immune responses induced by interferon gamma (IFN-γ) compared with those in the ASFV-WT-infected PAMs. Meanwhile, we further confirmed that the I7L protein (pI7L) suppressed the IFN-γ-triggered JAK-STAT signaling pathway. Mechanistically, pI7L interacts with STAT1 and inhibits its phosphorylation and homodimerization, which depends on the tyrosine at position 98 (Y98) of pI7L, thereby preventing the nuclear translocation of STAT1 and leading to the decreased production of IFN-γ-stimulated genes. Importantly, ASFV-ΔI7L exhibited reduced replication and virulence compared with ASFV-WT in pigs, likely due to the increased production of IFN-γ-stimulated genes, indicating that pI7L is involved in the virulence of ASFV. Taken together, our findings demonstrate that pI7L is associated with pathogenicity and antagonizes the IFN-γ-triggered JAK-STAT signaling pathway via inhibiting the phosphorylation and homodimerization of STAT1 depending on the Y98 residue of pI7L and the Src homology 2 domain of STAT1, which provides more information for understanding the immunoevasion strategies and designing the live attenuated vaccines against ASFV infection.

The roles of nuclear orphan receptor NR2F6 in anti-viral innate immunity.

Proper transcription regulation by key transcription factors, such as IRF3, is critical for anti-viral defense. Dynamics of enhancer activity play important roles in many biological processes, and epigenomic analysis is used to determine the involved enhancers and transcription factors. To determine new transcription factors in anti-DNA-virus response, we have performed H3K27ac ChIP-Seq and identified three transcription factors, NR2F6, MEF2D and MAFF, in promoting HSV-1 replication. NR2F6 promotes HSV-1 replication and gene expression in vitro and in vivo, but not dependent on cGAS/STING pathway. NR2F6 binds to the promoter of MAP3K5 and activates AP-1/c-Jun pathway, which is critical for DNA virus replication. On the other hand, NR2F6 is transcriptionally repressed by c-Jun and forms a negative feedback loop. Meanwhile, cGAS/STING innate immunity signaling represses NR2F6 through STAT3. Taken together, we have identified new transcription factors and revealed the underlying mechanisms involved in the network between DNA viruses and host cells.

Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins.

The ongoing outbreak of viral pneumonia in China and across the world is associated with a new coronavirus, SARS-CoV-21. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection2. Although bats are probable reservoir hosts for SARS-CoV-2, the identity of any intermediate host that may have facilitated transfer to humans is unknown. Here we report the identification of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity in the receptor-binding domain to SARS-CoV-2. The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of new coronaviruses and should be removed from wet markets to prevent zoonotic transmission.

Structural basis for difunctional mechanism of m-AMSA against African swine fever virus pP1192R.

The African swine fever virus (ASFV) type II topoisomerase (Topo II), pP1192R, is the only known Topo II expressed by mammalian viruses and is essential for ASFV replication in the host cytoplasm. Herein, we report the structures of pP1192R in various enzymatic stages using both X-ray crystallography and single-particle cryo-electron microscopy. Our data structurally define the pP1192R-modulated DNA topology changes. By presenting the A2+-like metal ion at the pre-cleavage site, the pP1192R-DNA-m-AMSA complex structure provides support for the classical two-metal mechanism in Topo II-mediated DNA cleavage and a better explanation for nucleophile formation. The unique inhibitor selectivity of pP1192R and the difunctional mechanism of pP1192R inhibition by m-AMSA highlight the specificity of viral Topo II in the poison binding site. Altogether, this study provides the information applicable to the development of a pP1192R-targeting anti-ASFV strategy.

The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKKα and IKKß through the recruitment of TOLLIP.

The multigene family genes (MGFs) in the left variable region (LVR) of the African swine fever virus (ASFV) genome have been reported to be involved in viral replication in primary porcine alveolar macrophages (PAMs) and virulence in pigs. However, the exact functions of key MGFs in the LVR that regulate the replication and virulence of ASFV remain unclear. In this study, we identified the MGF300-2R gene to be critical for viral replication in PAMs by deleting different sets of MGFs in the LVR from the highly virulent strain ASFV HLJ/18 (ASFV-WT). The ASFV mutant lacking the MGF300-2R gene (Del2R) showed a 1-log reduction in viral titer, and induced higher IL-1ß and TNF-α production in PAMs than did ASFV-WT. Mechanistically, the MGF300-2R protein was found to interact with and degrade IKKα and IKKß via the selective autophagy pathway. Furthermore, we showed that MGF300-2R promoted the K27-linked polyubiquitination of IKKα and IKKß, which subsequently served as a recognition signal for the cargo receptor TOLLIP-mediated selective autophagic degradation. Importantly, Del2R exhibited a significant reduction in both replication and virulence compared with ASFV-WT in pigs, likely due to the increased IL-1ß and TNF-α, indicating that MGF300-2R is a virulence determinant. These findings reveal that MGF300-2R suppresses host innate immune responses by mediating the degradation of IKKα and IKKß, which provides clues to paving the way for the rational design of live attenuated vaccines to control ASF.

N4-acetylcytidine modifies primary microRNAs for processing in cancer cells.

N4 acetylcytidine (ac4C) modification mainly occurs on tRNA, rRNA, and mRNA, playing an important role in the expression of genetic information. However, it is still unclear whether microRNAs have undergone ac4C modification and their potential physiological and pathological functions. In this study, we identified that NAT10/THUMPD1 acetylates primary microRNAs (pri-miRNAs) with ac4C modification. Knockdown of NAT10 suppresses and augments the expression levels of mature miRNAs and pri-miRNAs, respectively. Molecular mechanism studies found that pri-miRNA ac4C promotes the processing of pri-miRNA into precursor miRNA (pre-miRNA) by enhancing the interaction of pri-miRNA and DGCR8, thereby increasing the biogenesis of mature miRNA. Knockdown of NAT10 attenuates the oncogenic characters of lung cancer cells by regulating miRNA production in cancers. Moreover, NAT10 is highly expressed in various clinical cancers and negatively correlated with poor prognosis. Thus, our results reveal that NAT10 plays a crucial role in cancer initiation and progression by modulating pri-miRNA ac4C to affect miRNA production, which would provide an attractive therapeutic strategy for cancers.

Phase-separated DropCRISPRa platform for efficient gene activation in mammalian cells and mice.

Liquid-liquid phase separation (LLPS) plays a critical role in regulating gene transcription via the formation of transcriptional condensates. However, LLPS has not been reported to be engineered as a tool to activate endogenous gene expression in mammalian cells or in vivo. Here, we developed a droplet-forming CRISPR (clustered regularly interspaced short palindromic repeats) gene activation system (DropCRISPRa) to activate transcription with high efficiency via combining the CRISPR-SunTag system with FETIDR-AD fusion proteins, which contain an N-terminal intrinsically disordered region (IDR) of a FET protein (FUS or TAF15) and a transcription activation domain (AD, VP64/P65/VPR). In this system, the FETIDR-AD fusion protein formed phase separation condensates at the target sites, which could recruit endogenous BRD4 and RNA polymerase II with an S2 phosphorylated C-terminal domain (CTD) to enhance transcription elongation. IDR-FUS9Y>S and IDR-FUSG156E, two mutants with deficient and aberrant phase separation respectively, confirmed that appropriate phase separation was required for efficient gene activation. Further, the DropCRISPRa system was compatible with a broad set of CRISPR-associated (Cas) proteins and ADs, including dLbCas12a, dAsCas12a, dSpCas9 and the miniature dUnCas12f1, and VP64, P65 and VPR. Finally, the DropCRISPRa system could activate target genes in mice. Therefore, this study provides a robust tool to activate gene expression for foundational research and potential therapeutics.

Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility.

DNA double-strand breaks (DSBs) are functionally linked to genomic instability in spermatocytes and to male infertility. The heavy metal cadmium (Cd) is known to induce DNA damage in spermatocytes by unknown mechanisms. Here, we showed that Cd ions impaired the canonical non-homologous end-joining (NHEJ) repair pathway, but not the homologous recombination (HR) repair pathway, through stimulation of Ser2056 and Thr2609 phosphorylation of DNA-PKcs at DSB sites. Hyper-phosphorylation of DNA-PKcs led to its premature dissociation from DNA ends and the Ku complex, preventing recruitment of processing enzymes and further ligation of DNA ends. Specifically, this cascade was initiated by the loss of PP5 phosphatase activity, which results from the dissociation of PP5 from its activating ions (Mn), that is antagonized by Cd ions through a competitive mechanism. In accordance, in a mouse model Cd-induced genomic instability and consequential male reproductive dysfunction were effectively reversed by a high dosage of Mn ions. Together, our findings corroborate a protein phosphorylation-mediated genomic instability pathway in spermatocytes that is triggered by exchange of heavy metal ions.

Room-Temperature Ferromagnetism in Epitaxial Bilayer FeSb/SrTiO3(001) Terminated with a Kagome Lattice.

Two-dimensional (2D) magnets exhibit unique physical properties for potential applications in spintronics. To date, most 2D ferromagnets are obtained by mechanical exfoliation of bulk materials with van der Waals interlayer interactions, and the synthesis of single- or few-layer 2D ferromagnets with strong interlayer coupling remains experimentally challenging. Here, we report the epitaxial growth of 2D non-van der Waals ferromagnetic bilayer FeSb on SrTiO3(001) substrates stabilized by strong coupling to the substrate, which exhibits in-plane magnetic anisotropy and a Curie temperature above 390 K. In situ low-temperature scanning tunneling microscopy/spectroscopy and density-functional theory calculations further reveal that an Fe Kagome layer terminates the bilayer FeSb. Our results open a new avenue for further exploring emergent quantum phenomena from the interplay of ferromagnetism and topology for application in spintronics.

The mutations on the envelope glycoprotein D contribute to the enhanced neurotropism of the pseudorabies virus variant.

The pseudorabies virus (PRV) TJ strain, a variant of PRV, induces more severe neurological symptoms and higher mortality in piglets and mice than the PRV SC strain isolated in 1980. However, the mechanism underlying responsible for the discrepancy in virulence between these strains remains unclear. Our study investigated the differences in neurotropism between PRV TJ and PRV SC using both in vitro and in vivo models. We discovered that PRV TJ enters neural cells more efficiently than PRV SC. Furthermore, we found that PRV TJ has indistinguishable genomic DNA replication capability and axonal retrograde transport dynamics compared to the PRV SC. To gain deeper insights into the mechanisms underlying these differences, we constructed gene-interchanged chimeric virus constructs and assessed the affinity between envelope glycoprotein B, C, and D (gD) and corresponding receptors. Our findings confirmed that mutations in these envelope proteins, particularly gD, significantly contributed to the heightened attachment and penetration capabilities of PRV TJ. Our study revealed the critical importance of the gDΔR278/P279 and gDV338A in facilitating viral invasion. Furthermore, our observations indicated that mutations in envelope proteins have a more significant impact on viral invasion than on virulence in the mouse model. Our findings provide valuable insights into the roles of natural mutations on the PRV envelope glycoproteins in cell tropism, which sheds light on the relationship between cell tropism and clinical symptoms and offers clues about viral evolution.

Enantioselective Construction of Quaternary Stereocenters via Cooperative Photoredox/Fe/Chiral Primary Amine Triple Catalysis.

The catalytic and enantioselective construction of quaternary (all-carbon substituents) stereocenters poses a formidable challenge in organic synthesis due to the hindrance caused by steric factors. One conceptually viable and potentially versatile approach is the coupling of a C-C bond through an outer-sphere mechanism, accompanied by the realization of enantiocontrol through cooperative catalysis; however, examples of such processes are yet to be identified. Herein, we present such a method for creating different compounds with quaternary stereocenters by photoredox/Fe/chiral primary amine triple catalysis. This approach facilitates the connection of an unactivated alkyl source with a tertiary alkyl moiety, which is also rare. The scalable process exhibits mild conditions, does not necessitate the use of a base, and possesses a good functional-group tolerance. Preliminary investigations into the underlying mechanisms have provided valuable insights into the reaction pathway.

Streptococcus pneumoniae endopeptidase O induces trained immunity and confers protection against various pathogenic infections.

Antibiotic resistance and the surge of infectious diseases during the pandemic present significant threats to human health. Trained immunity emerges as a promising and innovative approach to address these infections. Synthetic or natural fungal, parasitic and viral components have been reported to induce trained immunity. However, it is not clear whether bacterial virulence proteins can induce protective trained immunity. Our research demonstrates Streptococcus pneumoniae virulence protein PepO, is a highly potent trained immunity inducer for combating broad-spectrum infection. Our findings showcase that rPepO training confers robust protection to mice against various pathogenic infections by enhancing macrophage functionality. rPepO effectively re-programs macrophages, re-configures their epigenetic modifications and bolsters their immunological responses, which is independent of T or B lymphocytes. In vivo and in vitro experiments confirm that trained macrophage-secreted complement C3 activates peritoneal B lymphocyte and enhances its bactericidal capacity. In addition, we provide the first evidence that granulocyte colony-stimulating factor (G-CSF) derived from trained macrophages plays a pivotal role in shaping central-trained immunity. In summation, our research demonstrates the capability of rPepO to induce both peripheral and central trained immunity in mice, underscoring its potential application in broad-spectrum anti-infection therapy. Our research provides a new molecule and some new target options for infectious disease prevention.

Effects of different karst fissures and rainfall distribution on the biomass, mineral nutrient elements, antioxidant substances, and photosynthesis of two coniferous seedlings.

BACKGROUND: Studying the physiological growth status of Pinus yunnanensis Franch and Pinus elliottii Engelm. seedlings under different karst fissure thicknesses and rainfall distributions is of great significance for the management, vegetation restoration, and tree species selection in karst rocky desertification areas. In this study, we used a two-factor block experiment and set different rainfall durations, namely reduced rainfall duration (I3d), natural rainfall duration (I6d), and extended rainfall duration (I9d); Different karst small habitats, i.e., stone-free soil (S0), less stone and more soil (S1/4), and half stone and half soil (S1/2), are simulated at these three levels. Analyze the changes in physiological growth and photosynthetic characteristics in two coniferous seedlings under different treatments with different karst thicknesses. RESULTS: The results showed that with the increase of karst thickness, the growth volumes of height and diameter of P. yunnanensis seedlings, the biomass of various organs, and the accumulation of K+, Ca2+, Na+, and Mg2+ showed a significant change pattern of first increasing and then decreasing (P < 0.05); P. elliottii seedlings show a gradually decreasing trend (except for Ca2+). The biomass accumulation of each organ in two coniferous seedlings showed that leaves > stems > roots. The K+, Ca2+, and Mg2+ content in various organs of P. yunnanensis seedlings showed that leaves > roots > stems, while Na+ shows the order of roots > leaves > stems. The accumulation of mineral elements in various organs of P. elliottii seedlings is manifested as roots > stems > leaves and the accumulation of mineral elements in both coniferous seedlings is manifested as Ca2+ > Mg2+ > K+ > Na+. Root length, root volume, root surface area, root diameter, SOD, POD, SP, photosynthetic pigment content, fluorescence parameters, and gas exchange parameters of P. yunnanensis seedlings gradually increase with the increase of karst thickness (except for the 9-day rainfall duration), while those of P. elliottii seedlings gradually decrease. The light saturation point of P. yunnanensis seedlings is highest under the I6dS1/2 treatment, while that of P. elliottii is highest under the I3dS0 treatment. CONCLUSIONS: In summary, prolonging rainfall duration has an inhibitory effect on the growth of two types of coniferous seedlings. Increasing karst thickness inhibits the growth of P. elliottii seedlings, and to some extent, promotes the growth and development of P. yunnanensis seedlings. I6dS1/4 and I3dS0 treatments have the best growth effects on P. yunnanensis and P. elliottii seedlings. Therefore, we give priority to P. yunnanensis as the tree species for vegetation restoration or rocky desertification management in karst areas. Our study reveals the role of limestone-filled different karst fissures in mitigating the effects of drought as "containers" for plant growth. These findings help us understand the response of plants to drought stress and provide valuable insights for vegetation restoration in karst environments affected by global climate change. Therefore, further experiments with various karst fissure sizes are necessary to test the universality of the reactions of various plants under different karst fissures.

A Less-is-More Strategy for Mitochondria-Targeted Photodynamic Therapy of Rheumatoid Arthritis.

Conventional photodynamic therapy (PDT) of rheumatoid arthritis (RA) faces a dilemma: low-power is insufficient to kill pro-inflammatory cells while high-power exacerbates inflammation. Herein, mitochondrial targeting is introduced in PDT of RA to implement a "less-is-more" strategy, where higher apoptosis in pro-inflammatory cells are achieved with lower laser power. In arthritic rats, chlorine 6-loaded and mitochondria-targeting liposomes (Ce6@M-Lip) passively accumulated in inflamed joints, entered pro-inflammatory macrophages, and actively localized to mitochondria, leading to enhanced mitochondrial dysfunction under laser irradiation. By effectively disrupting mitochondria, pro-inflammatory macrophages are more susceptible to PDT, resulting in increased apoptosis initiation. Additionally, it identifies that high-power irradiation caused cell rupture and release of endogenous danger signals that recruited and activated additional macrophages. In contrast, under low-power irradiation, mitochondria-targeting Ce6@M-Lip not only prevented inflammation but also reduced pro-inflammatory macrophage infiltration and pro-inflammatory cytokine secretion. Overall, targeting mitochondria reconciled therapeutic efficacy and inflammation, thus enabling efficacious yet inflammation-sparing PDT for RA. This highlights the promise of mitochondrial targeting to resolve the dilemma between anti-inflammatory efficacy and inflammatory exacerbation in PDT by implementing a "less-is-more" strategy.

The African swine fever virus I10L protein inhibits the NF-κB signaling pathway by targeting IKKß.

Proinflammatory factors play important roles in the pathogenesis of African swine fever virus (ASFV), which is the causative agent of African swine fever (ASF), a highly contagious and severe hemorrhagic disease. Efforts in the prevention and treatment of ASF have been severely hindered by knowledge gaps in viral proteins responsible for modulating host antiviral responses. In this study, we identified the I10L protein (pI10L) of ASFV as a potential inhibitor of the TNF-α- and IL-1ß-triggered NF-κB signaling pathway, the most canonical and important part of host inflammatory responses. The ectopically expressed pI10L remarkably suppressed the activation of NF-κB signaling in HEK293T and PK-15 cells. The ASFV mutant lacking the I10L gene (ASFVΔI10L) induced higher levels of proinflammatory cytokines production in primary porcine alveolar macrophages (PAMs) compared with its parental ASFV HLJ/2018 strain (ASFVWT). Mechanistic studies suggest that pI10L inhibits IKKß phosphorylation by reducing the K63-linked ubiquitination of NEMO, which is necessary for the activation of IKKß. Morever, pI10L interacts with the kinase domain of IKKß through its N-terminus, and consequently blocks the association of IKKß with its substrates IκBα and p65, leading to reduced phosphorylation. In addition, the nuclear translocation efficiency of p65 was also altered by pI10L. Further biochemical evidence supported that the amino acids 1-102 on pI10L were essential for the pI10L-mediated suppression of the NF-κB signaling pathway. The present study clarifies the immunosuppressive activity of pI10L, and provides novel insights into the understanding of ASFV pathobiology and the development of vaccines against ASF. IMPORTANCE African swine fever (ASF), caused by the African swine fever virus (ASFV), is now widespread in many countries and severely affects the commercial rearing of swine. To date, few safe and effective vaccines or antiviral strategies have been marketed due to large gaps in knowledge regarding ASFV pathobiology and immune evasion mechanisms. In this study, we deciphered the important role of the ASFV-encoded I10L protein in the TNF-α-/IL-1ß-triggered NF-κB signaling pathway. This study provides novel insights into the pathogenesis of ASFV and thus contributes to the development of vaccines against ASF.

The D129L protein of African swine fever virus interferes with the binding of transcriptional coactivator p300 and IRF3 to prevent beta interferon induction.

IMPORTANCE: African swine fever (ASF) is an acute, hemorrhagic, and severe porcine infectious disease caused by African swine fever virus (ASFV). ASF outbreaks severely threaten the global pig industries and result in serious economic losses. No safe and efficacious commercial vaccine is currently available except in Vietnam. To date, large gaps in the knowledge concerning viral biological characteristics and immunoevasion strategies have hindered the ASF vaccine design. In this study, we demonstrate that pD129L negatively regulates the type I interferon (IFN) signaling pathway by interfering with the interaction of the transcriptional coactivator p300 and IRF3, thereby inhibiting the induction of type I IFNs. This study reveals a novel immunoevasion strategy employed by ASFV, shedding new light on the intricate mechanisms for ASFV to evade the host immune responses.

Generation of Human Embryonic Stem Cell-Derived Lung Organoids for Modeling Infection and Replication Differences between Human Adenovirus Types 3 and 55 and Evaluating Potential Antiviral Drugs.

Human adenoviruses type 3 (HAdV-3) and type 55 (HAdV-55) are frequently encountered, highly contagious respiratory pathogens with high morbidity rate. In contrast to HAdV-3, one of the most predominant types in children, HAdV-55 is a reemergent pathogen associated with more severe community-acquired pneumonia (CAP) in adults, especially in military camps. However, the infectivity and pathogenicity differences between these viruses remain unknown as in vivo models are not available. Here, we report a novel system utilizing human embryonic stem cells-derived 3-dimensional airway organoids (hAWOs) and alveolar organoids (hALOs) to investigate these two viruses. Firstly, HAdV-55 replicated more robustly than HAdV-3. Secondly, cell tropism analysis in hAWOs and hALOs by immunofluorescence staining revealed that HAdV-55 infected more airway and alveolar stem cells (basal and AT2 cells) than HAdV-3, which may lead to impairment of self-renewal functions post-injury and the loss of cell differentiation in lungs. Additionally, the viral life cycles of HAdV-3 and -55 in organoids were also observed using Transmission Electron Microscopy. This study presents a useful pair of lung organoids for modeling infection and replication differences between respiratory pathogens, illustrating that HAdV-55 has relatively higher replication efficiency and more specific cell tropism in human lung organoids than HAdV-3, which may result in relatively higher pathogenicity and virulence of HAdV-55 in human lungs. The model system is also suitable for evaluating potential antiviral drugs, as demonstrated with cidofovir. IMPORTANCE Human adenovirus (HAdV) infections are a major threat worldwide. HAdV-3 is one of the most predominant respiratory pathogen types found in children. Many clinical studies have reported that HAdV-3 causes less severe disease. In contrast, HAdV-55, a reemergent acute respiratory disease pathogen, is associated with severe community-acquired pneumonia in adults. Currently, no ideal in vivo models are available for studying HAdVs. Therefore, the mechanism of infectivity and pathogenicity differences between human adenoviruses remain unknown. In this study, a useful pair of 3-dimensional (3D) airway organoids (hAWOs) and alveolar organoids (hALOs) were developed to serve as a model. The life cycles of HAdV-3 and HAdV-55 in these human lung organoids were documented for the first time. These 3D organoids harbor different cell types, which are similar to the ones found in humans. This allows for the study of the natural target cells for infection. The finding of differences in replication efficiency and cell tropism between HAdV-55 and -3 may provide insights into the mechanism of clinical pathogenicity differences between these two important HAdV types. Additionally, this study provides a viable and effective in vitro tool for evaluating potential anti-adenoviral treatments.

Velocity of cerebrospinal fluid in the aqueduct measured by phase-contrast MRI in rat.

Cerebrospinal fluid (CSF) circulation plays a key role in cerebral waste clearance via the glymphatic system. Although CSF flow velocity is an essential component of CSF dynamics, it has not been sufficiently characterized, and particularly, in studies of the glymphatic system in rat. To investigate the relationship between the flow velocity of CSF in the brain aqueduct and the glymphatic waste clearance rate, using phase-contrast MRI we performed the first measurements of CSF velocity in rats. Phase-contrast MRI was performed using a 7 T system to map mean velocity of CSF flow in the aqueduct in rat brain. The effects of age (3 months old versus 18 months old), gender, strain (Wistar, RNU, Dark Agouti), anesthetic agents (isoflurane versus dexmedetomidine), and neurodegenerative disorder (Alzheimer' disease in Fischer TgF344-AD rats, males and females) on CSF velocity were investigated in eight independent groups of rats (12 rats per group). Our results demonstrated that quantitative velocities of CSF flow in the aqueduct averaged 5.16 ± 0.86 mm/s in healthy young adult male Wistar rats. CSF flow velocity in the aqueduct was not altered by rat gender, strain, and the employed anesthetic agents in all rats, also age in the female rats. However, aged (18 months) Wistar male rats exhibited significantly reduced the CSF flow velocity in the aqueduct (4.31 ± 1.08 mm/s). In addition, Alzheimer's disease further reduced the CSF flow velocity in the aqueduct of male and female rats.