Neutralizing monoclonal antibodies against the Gc fusion loop region of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus, prevalent in more than 30 countries worldwide. Human infection by this virus leads to severe illness, with an average case fatality of 40%. There is currently no approved vaccine or drug to treat the disease. Neutralizing antibodies are a promising approach to treat virus infectious diseases. This study generated 37 mouse-derived specific monoclonal antibodies against CCHFV Gc subunit. Neutralization assays using pseudotyped virus and authentic CCHFV identified Gc8, Gc13, and Gc35 as neutralizing antibodies. Among them, Gc13 had the highest neutralizing activity and binding affinity with CCHFV Gc. Consistently, Gc13, but not Gc8 or Gc35, showed in vivo protective efficacy (62.5% survival rate) against CCHFV infection in a lethal mouse infection model. Further characterization studies suggested that Gc8 and Gc13 may recognize a similar, linear epitope in domain II of CCHFV Gc, while Gc35 may recognize a different epitope in Gc. Cryo-electron microscopy of Gc-Fab complexes indicated that both Gc8 and Gc13 bind to the conserved fusion loop region and Gc13 had stronger interactions with sGc-trimers. This was supported by the ability of Gc13 to block CCHFV GP-mediated membrane fusion. Overall, this study provides new therapeutic strategies to treat CCHF and new insights into the interaction between antibodies with CCHFV Gc proteins.

AcMNPV P74 is cleaved at R325 and R334 by proteinases of both OB and BBMV to expose a potential fusion peptide for oral infection.

Baculoviruses enter insect midgut epithelial cells via a set of occlusion-derived virion (ODV) envelope proteins called per os infectivity factors (PIFs). P74 of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which was the first identified PIF, is cleaved by an endogenous proteinase embedded within the occlusion body during per os infection, but the target site(s) and function of the cleavage have not yet been ascertained. Here, based on bioinformatics analyses, we report that cleavage was predicted at an arginine and lysine-rich region in the middle of P74. A series of recombinant viruses with site-directed mutants in this region of P74 were generated. R325 or R334 was identified as primary cleavage site. In addition, we showed that P74 is also cleaved by brush border membrane vesicles (BBMV) of the host insect at R325 or R334, instead of R195, R196, and R199, as previously reported. Simultaneous mutations in R195, R196, and R199 lead to instability of P74 during ODV release. Bioassays showed that mutations at both R325 and R334 significantly affected oral infectivity. Taken together, our data show that both R325 and R334 of AcMNPV P74 are the primary cleavage site for both occlusion body endogenous proteinase and BBMV proteinase during ODV release and are critical for oral infection. IMPORTANCE: Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a per os infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus per os infection.

Empirical estimates of the mutation rate for an alphabaculovirus.

Mutation rates are of key importance for understanding evolutionary processes and predicting their outcomes. Empirical mutation rate estimates are available for a number of RNA viruses, but few are available for DNA viruses, which tend to have larger genomes. Whilst some viruses have very high mutation rates, lower mutation rates are expected for viruses with large genomes to ensure genome integrity. Alphabaculoviruses are insect viruses with large genomes and often have high levels of polymorphism, suggesting high mutation rates despite evidence of proofreading activity by the replication machinery. Here, we report an empirical estimate of the mutation rate per base per strand copying (s/n/r) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). To avoid biases due to selection, we analyzed mutations that occurred in a stable, non-functional genomic insert after five serial passages in Spodoptera exigua larvae. Our results highlight that viral demography and the stringency of mutation calling affect mutation rate estimates, and that using a population genetic simulation model to make inferences can mitigate the impact of these processes on estimates of mutation rate. We estimated a mutation rate of µ = 1×10-7 s/n/r when applying the most stringent criteria for mutation calling, and estimates of up to µ = 5×10-7 s/n/r when relaxing these criteria. The rates at which different classes of mutations accumulate provide good evidence for neutrality of mutations occurring within the inserted region. We therefore present a robust approach for mutation rate estimation for viruses with stable genomes, and strong evidence of a much lower alphabaculovirus mutation rate than supposed based on the high levels of polymorphism observed.

Structural basis for SARS-CoV-2 neutralizing antibodies with novel binding epitopes.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.

Exposure to high dose of polystyrene nanoplastics causes trophoblast cell apoptosis and induces miscarriage.

BACKGROUND: With rapid increase in the global use of various plastics, microplastics (MPs) and nanoplastics (NPs) pollution and their adverse health effects have attracted global attention. MPs have been detected out in human body and both MPs and NPs showed female reproductive toxicological effects in animal models. Miscarriage (abnormal early embryo loss), accounting for 15-25% pregnant women worldwide, greatly harms human reproduction. However, the adverse effects of NPs on miscarriage have never been explored. RESULTS: In this study, we identified that polystyrene (PS) plastics particles were present in women villous tissues. Their levels were higher in villous tissues of unexplained recurrent miscarriage (RM) patients vs. healthy control (HC) group. Furthermore, mouse assays further confirmed that exposure to polystyrene nanoplastics (PS-NPs, 50 nm in diameter, 50 or 100 mg/kg) indeed induced miscarriage. In mechanism, PS-NPs exposure (50, 100, 150, or 200 µg/mL) increased oxidative stress, decreased mitochondrial membrane potential, and increased apoptosis in human trophoblast cells by activating Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3 signaling through mitochondrial pathway. The alteration in this signaling was consistent in placental tissues of PS-NPs-exposed mouse model and in villous tissues of unexplained RM patients. Supplement with Bcl-2 could efficiently suppress apoptosis in PS-NPs-exposed trophoblast cells and reduce apoptosis and alleviate miscarriage in PS-NPs-exposed pregnant mouse model. CONCLUSIONS: Exposure to PS-NPs activated Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3, leading to excessive apoptosis in human trophoblast cells and in mice placental tissues, further inducing miscarriage.

Structural transitions during the cooperative assembly of baculovirus single-stranded DNA-binding protein on ssDNA.

Single-stranded DNA-binding proteins (SSBs) interact with single-stranded DNA (ssDNA) to form filamentous structures with various degrees of cooperativity, as a result of intermolecular interactions between neighboring SSB subunits on ssDNA. However, it is still challenging to perform structural studies on SSB-ssDNA filaments at high resolution using the most studied SSB models, largely due to the intrinsic flexibility of these nucleoprotein complexes. In this study, HaLEF-3, an SSB protein from Helicoverpa armigera nucleopolyhedrovirus, was used for in vitro assembly of SSB-ssDNA filaments, which were structurally studied at atomic resolution using cryo-electron microscopy. Combined with the crystal structure of ssDNA-free HaLEF-3 octamers, our results revealed that the three-dimensional rearrangement of HaLEF-3 induced by an internal hinge-bending movement is essential for the formation of helical SSB-ssDNA complexes, while the contacting interface between adjacent HaLEF-3 subunits remains basically intact. We proposed a local cooperative SSB-ssDNA binding model, in which, triggered by exposure to oligonucleotides, HaLEF-3 molecules undergo ring-to-helix transition to initiate continuous SSB-SSB interactions along ssDNA. Unique structural features revealed by the assembly of HaLEF-3 on ssDNA suggest that HaLEF-3 may represent a new class of SSB.

Environmental BaP/BPDE suppressed trophoblast cell invasion/migration and induced miscarriage by down-regulating lnc-HZ01/MEST/VIM axis.

Environmental benzo(a)pyrene (BaP) and itsmetabolite benzo(a)pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE), classic endocrine disrupting chemical and persistent organic pollutant, could cause miscarriage. However, the detailed mechanisms are still largely unclear and should be further explored. In this study, we discovered that exposure of trophoblast cells with BPDE could suppressed cell invasion/migration by inhibiting MEST/VIM (Vimentin) pathway. Moreover, BPDE exposure also increased lnc-HZ01 expression level, which further inhibited MEST/VIM pathway and then suppressed invasion/migration. Knockdown of lnc-HZ01 or overexpression of MEST could efficiently rescue invasion/migration of BPDE-exposed Swan 71 cells. Furthermore, lnc-HZ01 was highly expressed and MEST/VIM were lowly expressed in recurrent miscarriage (RM) villous tissues compared with healthy control (HC) group. Finally, we also found that BaP exposure inhibited murine Mest/Vim pathway in placental tissues and induced miscarriage in BaP-exposed mice. Therefore, the regulatory mechanisms were similar in BPDE-exposed human trophoblast cells, RM villous tissues, and placental tissues of BaP-exposed mice with miscarriage, building a bridge to connect BaP/BPDE exposure, invasion/migration, and miscarriage. This study provided novel insights in the toxicological effects and molecular mechanisms of BaP/BPDE-induced miscarriage, which is helpful for better elucidating the toxicological risks of BaP/BPDE on female reproduction.

Enhanced Stability and Solidification of Volatile Eugenol by Cyclodextrin-Metal Organic Framework for Nasal Powder Delivery.

Eugenol (Eug) holds potential as a treatment for bacterial rhinosinusitis by nasal powder drug delivery. To stabilization and solidification of volatile Eug, herein, nasal inhalable γ-cyclodextrin metal-organic framework (γ-CD-MOF) was investigated as a carrier by gas-solid adsorption method. The results showed that the particle size of Eug loaded by γ-CD-MOF (Eug@γ-CD-MOF) distributed in the range of 10-150 µm well. In comparison to γ-CD and ß-CD-MOF, γ-CD-MOF has higher thermal stability to Eug. And the intermolecular interactions between Eug and the carriers were verified by characterizations and molecular docking. Based on the bionic human nasal cavity model, Eug@γ-CD-MOF had a high deposition distribution (90.07 ± 1.58%). Compared with free Eug, the retention time Eug@γ-CD-MOF in the nasal cavity was prolonged from 5 min to 60 min. In addition, the cell viability showed that Eug@γ-CD-MOF (Eug content range 3.125-200 µg/mL) was non-cytotoxic. And the encapsulation of γ-CD-MOF could not reduce the bacteriostatic effect of Eug. Therefore, the biocompatible γ-CD-MOF could be a potential and valuable carrier for nasal drug delivery to realize solidification and nasal therapeutic effects of volatile oils.

Integrative analysis of metabolomics and proteomics unravels purine metabolism dysregulation in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with progressive paralysis of limbs and bulb in patients, the cause of which remains unclear. Accumulating studies suggest that motor neuron degeneration is associated with systemic metabolic impairment in ALS. However, the metabolic reprogramming and underlying mechanism in the longitudinal progression of the disease remain poorly understood. In this study, we aimed to investigate the molecular changes at both metabolic and proteomic levels during disease progression to identify the most critical metabolic pathways and underlying mechanisms involved in ALS pathophysiological changes. Utilizing liquid chromatography-mass spectrometry-based metabolomics, we analyzed the metabolites' levels of plasma, lumbar spinal cord, and motor cortex from SOD1G93A mice and wildtype (WT) littermates at different stages. To elucidate the regulatory network underlying metabolic changes, we further analyzed the proteomics profile in the spinal cords of SOD1G93A and WT mice. A group of metabolites implicated in purine metabolism, methionine cycle, and glycolysis were found differentially expressed in ALS mice, and abnormal expressions of enzymes involved in these metabolic pathways were also confirmed. Notably, we first demonstrated that dysregulation of purine metabolism might contribute to the pathogenesis and disease progression of ALS. Furthermore, we discovered that fatty acid metabolism, TCA cycle, arginine and proline metabolism, and folate-mediated one­carbon metabolism were also significantly altered in this disease. The identified differential metabolites and proteins in our study could complement existing data on metabolic reprogramming in ALS, which might provide new insight into the pathological mechanisms and novel therapeutic targets of ALS.

N-(4-Aminobutyl)-N-ethylisopropanol and Co2+ Dual-Functionalized Core-Shell Fe3O4@Au/Ag Magnetic Nanomaterials with Strong and Stable Chemiluminescence for a Label-Free Exosome Immunosensor.

Recently, magnetic beads (MBs) are moving toward chemiluminescence (CL) functional magnetic nanomaterials with a great potential for constructing label-free immunosensors. However, most of the CL-functionalized MBs suffer from scarce binding sites, easy aggregation, and leakage of CL reagents, which will ultimately affect the analytical performance of immunosensors. Herein, by using core-shell Fe3O4@Au/Ag magnetic nanomaterials as a nanoplatform, a novel N-(4-aminobutyl)-N-ethylisopropanol (ABEI) and Co2+ dual-functionalized magnetic nanomaterial, namely, Fe3O4@Au/Ag/ABEI/Co2+, with strong and stable CL emission was successfully synthesized. Its CL intensity was 36 and 3.5 times higher than that of MB@ABEI-Au/Co2+ and ABEI and Co2+ dual-functionalized chemiluminescent MBs previously reported by our group, respectively. It was found that the excellent CL performance of Fe3O4@Au/Ag/ABEI/Co2+ could be attributed to the enrichment effect of the Au/Ag shell and the synergistic enhance effect of the Au/Ag shell and Co2+. A related CL mechanism has been proposed. Afterward, based on the intense and stable CL emission of Fe3O4@Au/Ag/ABEI/Co2+, a sensitive and effective label-free CL immunosensor for exosome detection was established. It exhibited excellent analytical performance with a wide detection range of 3.1 × 103 to 3.1 × 108 particles/mL and a low detection limit of 2.1 × 103 particles/mL, which were better than the vast majority of the reported CL immunosensors. Moreover, the proposed label-free CL immunosensor was successfully used to detect exosomes in human serum samples and enabled us to distinguish healthy persons and lung cancer patients. It has the potential to be a powerful tool for exosome study and early cancer diagnosis.

AC81 Is a Putative Disulfide Isomerase Involved in Baculoviral Disulfide Bond Formation.

The correct formation of native disulfide bonds is critical for the proper structure and function of many proteins. Cellular disulfide bond formation pathways commonly consist of two parts: sulfhydryl oxidase-mediated oxidation and disulfide isomerase-mediated isomerization. Some large DNA viruses, such as baculoviruses, encode sulfhydryl oxidases, but viral disulfide isomerases have not yet been identified, although G4L in poxvirus has been suggested to serve such a function. Here, we report that the baculovirus core gene ac81 encodes a putative disulfide isomerase. ac81 is conserved in baculoviruses, nudiviruses, and hytrosaviruses. We found that AC81 homologs contain a typical thioredoxin fold conserved in disulfide isomerases. To determine the role of AC81, a series of Autographa californica nucleopolyhedrovirus (AcMNPV) bacmids containing ac81 knockout or point mutations was generated, and the results showed that AC81 is essential for budded virus production, multinucleocapsid occlusion-derived virus (ODV) formation, and ODV embedding in occlusion bodies. Nonreducing Western blot analysis indicated that disulfide bond formation in per os infectivity factor 5 (PIF5), a substrate of the baculoviral sulfhydryl oxidase P33, was abnormal when ac81 was knocked out or mutated. Pulldown assays showed that AC81 interacted with PIF5 and P33 in infected cells. In addition, two critical regions that harbor key amino acids for function were identified in AC81. Taken together, our results suggest that AC81 is a key component involved in the baculovirus disulfide bond formation pathway and likely functions as a disulfide isomerase. IMPORTANCE Many large DNA viruses, such as poxvirus, asfarvirus, and baculovirus, encode their own sulfhydryl oxidase to facilitate the disulfide bond formation of viral proteins. Here, we show that AC81 functions as a putative disulfide isomerase and is involved in multiple functions of the baculovirus life cycle. Interestingly, AC81 and P33 (sulfhydryl oxidase) are conserved in baculoviruses, nudiviruses, and hytrosaviruses, which are all insect-specific large DNA viruses replicating in the nucleus, suggesting that viral disulfide bond formation is an ancient mechanism shared by these viruses.

Insights into Two-Metal-Ion Catalytic Mechanism of Cap-Snatching Endonuclease of Ebinur Lake Virus in Bunyavirales.

The cap-snatching endonuclease (EN) of segmented negative-strand RNA viruses (sNSVs) produces short capped primers for viral transcription by cleaving the host mRNAs. EN requires divalent metals as cofactors for nucleic acid substrates cleavage; however, the detailed mechanism of metal ion-dependent catalysis of ENs remains obscure. In this work, we reported the EN crystal structure of the Ebinur Lake virus (EBIV), an emerging mosquito-borne orthobunyavirus, and investigated its enzymatic properties and metal ion-based catalytic mechanism. In vitro biochemical data showed that EBIV EN is a specific RNA nuclease and prefers to cleave unstructured uridine-rich ssRNA. Structural comparison indicated that the overall structural architecture of EBIV EN is similar to that of other sNSV ENs, while the detailed active site configuration including the binding state of metal ions and the conformation of the LA/LB loop pair is different. Based on sequence conservation analysis, nine active site mutants were constructed, and seven crystal structures of them were determined. Mutations of active site residues associated with the two metal ions (Mn1 and Mn2) coordination abolished EN activity. Crystallographic analyses further revealed that none of these mutants bound two metal ions simultaneously in the active site. Importantly, we found that the perturbation of Mn1-coordination (metal site 1), resulted in the enhancement or elimination of Mn2-coordination (metal site 2). Taken together, our data provide structural evidence to support the two-metal-ion catalytic mechanism of EBIV EN and the correlation of metal binding at the two binding sites, which may be commonly shared by bunyaviruses or other sNSVs. IMPORTANCE The viral endonucleases (ENs) encoded by bunyaviruses and orthomyxoviruses play an essential role in initiating transcription by "snatching" capped primers from the host mRNAs. These ENs are metal-ion-dependent nucleases; however, the details of their catalytic mechanism remain elusive. Here, we reported high-resolution crystal structures of the wild-type and mutant ENs of a novel bunyavirus, the Ebinur Lake virus (EBIV), and revealed the structure and function relationship of EN. The EBIV EN exhibited differences in the details of active site structure compared to its homologues. Our data provided structural evidence to support a two-metal-ion catalytic mechanism of EBIV EN, and found the correlation of metal binding at both binding sites, which might reflect the dynamic structural properties that correlate to EN catalytic function. Taken together, our results revealed the structural characteristics of EBIV EN and made important implications for understanding the catalytic mechanism of cap-snatching ENs.

Functional Peroral Infectivity Complex of White Spot Syndrome Virus of Shrimp.

White spot syndrome virus (WSSV) is a major cause of disease in shrimp cultures worldwide. The infection process of this large circular double-stranded DNA virus has been well studied, but its entry mechanism remains controversial. The major virion envelope protein VP28 has been implicated in oral and systemic viral infection in shrimp. However, genetic analysis of viral DNA has shown the presence of a few genes related to proteins of per os infectivity factor (PIF) complex in baculoviruses. This complex is essential for the entry of baculoviruses, large terrestrial circular DNA viruses, into the midgut epithelial cells of insect larvae. In this study, we aimed to determine whether a PIF complex exists in WSSV, the components of this complex, whether it functions as an oral infectivity complex in shrimp, and the biochemical properties that contribute to its function in a marine environment. The results revealed a WSSV PIF complex (~720 kDa) comprising at least eight proteins, four of which were not identified as PIF homologs: WSV134, VP124 (WSV216), WSSV021, and WSV136. WSV134 is suggested to be a PIF4 homolog due to predicted structural similarity and amino acid sequence identity. The WSSV PIF complex is resistant to alkali, proteolysis, and high salt, properties that are important for maintaining infectivity in aquatic environments. Oral infection can be neutralized by PIF-specific antibodies but not by VP28-specific antibodies. These results indicate that the WSSV PIF complex is critical for WSSV entry into shrimp; the complex's evolutionary significance is also discussed. IMPORTANCE White spot disease, caused by the white spot syndrome virus (WSSV), is a major scourge in cultured shrimp production facilities worldwide. This disease is only effectively controlled by sanitation. Intervention strategies are urgently needed but are limited by a lack of appropriate targets. Our identification of a per os infectivity factor (PIF) complex, which is pivotal for the entry of WSSV into shrimp, could provide new targets for antibody- or dsRNA-based intervention strategies. In addition, the presence of a PIF complex with at least eight components in WSSV, which is ancestrally related to the PIF complex of invertebrate baculoviruses, suggests that this complex is structurally and functionally conserved in disparate virus taxa.

Structural Characterization of Per Os Infectivity Factor 5 (PIF5) Reveals the Essential Role of Intramolecular Interactions in Baculoviral Oral Infectivity.

Baculoviruses initiate oral infection in the highly alkaline midgut of insects via a group of envelope proteins called per os infectivity factors (PIFs). To date, no high-resolution structural information has been reported for any PIF. Here, we present the crystal structure of the PIF5 ectodomain (PIF5e) from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) at a 2.2-Å resolution. It revealed an open cavity between the N-terminal E1 domain and the C-terminal E2 domain and a cysteine-rich region with three pairs of disulfide bonds in the E2 domain. Multiple conserved intramolecular interactions within PIF5 are essential for maintaining its tertiary structure. Two conserved arginines (Arg8 and Arg74) play critical roles in E1-E2 interactions, and mutagenesis analysis supported their crucial role in oral infection. Importantly, the reduction in the oral infectivity of the Arg8, Arg74, or cysteine mutant viruses was related to the proteolytic cleavage of PIF5 by the endogenous protease embedded in occlusion bodies during alkaline treatment. This suggested that the structural stability of PIF5 under physiological conditions in the insect midgut is critical for baculoviral oral infectivity. IMPORTANCEPer os infection mediated by PIFs is the highly complex mechanism by which baculoviruses initiate infection in insects. Previous studies revealed that multiple PIF proteins form a large PIF complex on the envelope of virions, while PIF5 functions independently of the PIF complex. Here, we report the crystal structure of AcMNPV PIF5e, which, to our knowledge, is the first atomic structure reported for a PIF protein. The structure revealed the precise locations of three previously proposed disulfide bonds and other conserved intramolecular interactions, which are important for the structural stability of PIF5 and are also essential for oral infectivity. These findings advance our understanding of the molecular mechanism of baculovirus oral infection under alkaline conditions.

Structural and Biochemical Basis for Development of Diketo Acid Inhibitors Targeting the Cap-Snatching Endonuclease of the Ebinur Lake Virus (Order: Bunyavirales).

The Bunyavirales contain many important human pathogens that lack an antiviral therapy. The cap-snatching endonuclease (EN) of segmented negative-strand RNA viruses is an attractive target for broad-spectrum antivirals due to its essential role in initiating viral transcription. L-742,001, a previously reported diketo acid inhibitor against influenza virus EN, demonstrated potent EN inhibition and antiviral activity on various bunyaviruses. However, the precise inhibitory mechanism of the compound is still poorly understood. We recently characterized a highly active EN from Ebinur Lake virus (EBIV), a newly identified member of the Orthobunyavirus genus, and obtained its high-resolution structures, paving the way for structure-guided inhibitor development. Here, nine L-742,001 derivatives were designed and synthesized de novo, and their structure-activity relationship with EBIV EN was studied. In vitro biochemical data showed that the compounds inhibited the EBIV EN activity with different levels and could be divided into three categories. Five representative compounds were selected for further cell-based antiviral assay, and the results largely agreed with those of the EN assays. Furthermore, the precise binding modes of L-742,001 and its derivatives in EN were revealed by determining the high-resolution crystal structures of EN-inhibitor complexes, which suggested that the p-chlorobenzene is essential for the inhibitory activity and the flexible phenyl has the greatest exploration potential. This study provides an important basis for the structure-based design and optimization of inhibitors targeting EN of segmented negative-strand RNA viruses. IMPORTANCE The Bunyavirales contain many important human pathogens such as Crimean-Congo hemorrhagic fever virus and Lassa virus that pose serious threats to public health; however, currently there are no specific antiviral drugs against these viruses. The diketo acid inhibitor L-742,001 is a potential drug as it inactivates the cap-snatching endonuclease (EN) encoded by bunyaviruses. Here, we designed and synthesized nine L-742,001 derivatives and assessed the structure-activity relationship using EN of the newly identified Ebinur Lake virus (EBIV) as a research model. Our results revealed that the p-chlorobenzene of this broad-spectrum EN inhibitor is crucial for the inhibitory activity and the flexible phenyl "arm" has the best potential for further optimization. As cap-snatching ENs are present not only in bunyaviruses but also in influenza viruses, our data provide important guidelines for the development of novel and more potent diketo acid-based antiviral drugs against those viruses.

FAP-α-Instructed Coumarin Excimer Formation for High Contrast Fluorescence Imaging of Tumor.

Emissive excimers, which are formed by planar polycyclic aromatic fluorophores (e.g., coumarin), enable high contrast tumor imaging. However, it is still challenging to "turn on" excimer fluorescence in physiological dilute solutions. The biocompatible CBT-Cys click condensation reaction enables both intra- and intermolecular aggregations of the as-loaded fluorophores on the probe molecules, which may promote the generation of emissive excimers in a synergistic manner. As a proof-of-concept, we herein design a fluorescence probe Cbz-Gly-Pro-Cys(StBu)-Lys(coumarin)-CBT (Cbz-GPC(StBu)K(Cou)-CBT), which can be activated by FAP-α under tumor-inherent reduction conditions, undergo a CBT-Cys click reaction, and self-assemble into coumarin nanoparticle Cou-CBT-NP to "turn on" the excimer fluorescence. In vitro and in vivo studies validate that this "smart" probe realizes efficient excimer fluorescence imaging of FAP-α-overexpressed tumor cells with high contrast and enhanced accumulation, respectively. We anticipate that this probe can be applied for diagnosis of FAP-α-related diseases in the clinic in near future.

The electrophysiological and neuropathological profiles of cerebellum in APPswe /PS1ΔE9 mice: A hypothesis on the role of cerebellum in Alzheimer's disease.

We propose the hypothesis that the cerebellar electrophysiology and sleep-wake cycles may be altered at the early stage of Alzheimer's disease (AD), proceeding the amyloid-ß neuropathological hallmarks. The electrophysiologic characteristics of cerebellum thereby might be served as a biomarker in the prepathological detection of AD. Sleep disturbances are common in preclinical AD patients, and the cerebellum has been implicated in sleep-wake regulation by several pioneer studies. Additionally, recent studies suggest that the structure and function of the cerebellum may be altered at the early stages of AD, indicating that the cerebellum may be involved in the disease's progression. We used APPswe /PS1ΔE9 mice as a model of AD, monitored and analyzed electroencephalogram data, and assessed neuropathological profiles in the cerebellum of AD mice. Our hypothesis may establish a linkage between the cerebellum and AD, thereby potentially providing new perspectives on the pathogenesis of the disease.

Residual status and source analysis of phthalate esters in Ulungur Lake, China.

Ulungur Lake is the largest lake in northern Xinjiang and undertakes important aquatic tasks. It is the No. 1 fishing ground in northern Xinjiang, and the problem of persistent organic pollution in the water has received much attention. However, there are few studies on phthalate esters (PAEs) in the water of Ulungur Lake. Understanding the pollution levels, distribution characteristics and sources of PAEs is of great significance for the protection and prevention of water. Fifteen sampling sites are established in Ulungur Lake to collect water samples during flood and dry periods, then seventeen PAEs are extracted from the water samples and purified by liquid-liquid extraction-solid-phase purification. Gas chromatography-mass spectrometry is used to detect the pollution levels and distribution characteristics of the 17 PAEs and analyse their sources. Results show that the concentrations of PAEs in the dry and flood periods are 0.451-9.97 µg/L and 0.0490-6.38 µg/L, respectively. The concentration of ∑PAEs with time is characterised by the dry period > the flood period. The change in flow is the main reason for the diverse concentration distributions of PAEs in different periods. The concentration of ΣPAEs in the dry period is much lower on the side near the lake entrance of the Ulungur River and Irtysh River. In the dry period, PAEs mainly come from chemical production and the use of cosmetics and personal care products; in the flood period, they mainly come from chemical production. River input and atmospheric sedimentation are the main sources of PAEs in the lake.

Solvent-Free Loading of Vitamin A Palmitate into ß-Cyclodextrin Metal-Organic Frameworks for Stability Enhancement.

Cyclodextrin metal-organic frameworks (CD-MOFs) exhibit a high structural diversity, which contributes to their functional properties. In this study, we have successfully synthesized a novel type of ß-cyclodextrin metal-organic framework (ß-CD-POF(I)) that exhibits excellent drug adsorption capacity and enhances stability. Single-crystal X-ray diffraction analysis revealed that ß-CD-POF(I) possessed the dicyclodextrin channel moieties and long-parallel tubular cavities. Compared with the reported ß-CD-MOFs, the ß-CD-POF(I) has a more promising drug encapsulation capability. Here, the stability of vitamin A palmitate (VAP) was effectively improved by the solvent-free method. Molecular modeling and other characterization techniques like synchrotron radiation Fourier transform infrared spectroscopy (SR-FTIR), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), and nitrogen adsorption isotherm were applied to confirm that the VAP was successfully encapsulated into the channel formed by the dicyclodextrin pairs. Furthermore, the mechanism of stability enhancement for VAP was determined to be due to the constraint and separation effects of ß-CD pairs on VAP. Therefore, ß-CD-POF(I) is capable of trapping and stabilizing certain unstable drug molecules, offering benefits and application possibilities. One kind of cyclodextrin particle with characteristic shapes of dicyclodextrin channel moieties and parallel tubular cavities, which was synthesized by a facile process. Subsequently, the spatial structure and characteristics of the ß-CD-POF(I) were primarily confirmed. The structure of ß-CD-POF(I) was then compared to that of KOH-ß-CD-MOF, and a better material for vitamin A palmitate (VAP) encapsulation was determined. VAP was successfully loaded into the particles by solvent-free method. The arrangement of spatial structure made cyclodextrin molecular cavity encapsulation in ß-CD-POF(I) more stable for VAP capture than that of KOH-ß-CD-MOF.

Covalently Engineered Protein Minibinders with Enhanced Neutralization Efficacy against Escaping SARS-CoV-2 Variants.

The rapid emergence and spread of escaping mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly challenged our efforts in fighting against the COVID-19 pandemic. A broadly neutralizing reagent against these concerning variants is thus highly desirable for the prophylactic and therapeutic treatments of SARS-CoV-2 infection. We herein report a covalent engineering strategy on protein minibinders for potent neutralization of the escaping variants such as B.1.617.2 (Delta), B.1.617.1 (Kappa), and B.1.1.529 (Omicron) through in situ cross-linking with the spike receptor binding domain (RBD). The resulting covalent minibinder (GlueBinder) exhibited enhanced blockage of RBD-human angiotensin-converting enzyme 2 (huACE2) interaction and more potent neutralization effect against the Delta variant than its noncovalent counterpart as demonstrated on authentic virus. By leveraging the covalent chemistry against escaping mutations, our strategy may be generally applicable for restoring and enhancing the potency of neutralizing antibodies to SARS-CoV-2 and other rapidly evolving viral targets.