Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion.

Recent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism.

The native structure of the assembled matrix protein 1 of influenza A virus.

Influenza A virus causes millions of severe cases of disease during annual epidemics. The most abundant protein in influenza virions is matrix protein 1 (M1), which mediates virus assembly by forming an endoskeleton beneath the virus membrane1. The structure of full-length M1, and how it oligomerizes to mediate the assembly of virions, is unknown. Here we determine the complete structure of assembled M1 within intact virus particles, as well as the structure of M1 oligomers reconstituted in vitro. We find that the C-terminal domain of M1 is disordered in solution but can fold and bind in trans to the N-terminal domain of another M1 monomer, thus polymerizing M1 into linear strands that coat the interior surface of the membrane of the assembling virion. In the M1 polymer, five histidine residues-contributed by three different monomers of M1-form a cluster that can serve as the pH-sensitive disassembly switch after entry into a target cell. These structures therefore reveal mechanisms of influenza virus assembly and disassembly.

Structures and distributions of SARS-CoV-2 spike proteins on intact virions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2-6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9-12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.

Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2.

SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus' evolution.

Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike.

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct "S-R/x3" to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

NiCo-LDH coupled with 2D ZIF-derived Co nitrogen doped carbon nanosheet arrays as a self-supporting electrocatalyst for detection of formaldehyde.

Formaldehyde is susceptible to illegal addition to foodstuffs to extend their shelf life due to its antimicrobial, preservative and bleaching properties. In this study, a self-supporting "nanosheet on nanosheet" arrays electrocatalyst with core-shell heterostructure was prepared in situ by coupling NiCo layer double hydroxide with 2D ZIF derived Co-nitrogen-doped porous carbon on carbon cloth (Co-N/C@NiCo-LDH NSAs/CC). Co-N/C nanosheet arrays act as a scaffold core with good electrical conductivity, providing more NiCo-LDH nucleation sites to avoid NiCo-LDH agglomeration, thus having fast mass/charge transfer performance. While the NiCo-LDH nanosheet arrays shell with high specific surface area provide more active sites for electrochemical reactions. As an electrocatalytic sensing electrode, Co-N/C@NiCo-LDH NSAs/CC has a wide linear range of 1 µM to 13 mM for formaldehyde detection, and the detection limit is 82 nM. Besides, the sensor has been applied to the detection of formaldehyde in food samples with satisfactory results.

A novel mutation in intron 1 of Wnt1 causes developmental loss of dopaminergic neurons in midbrain and ASD-like behaviors in rats.

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders with a strong genetic liability. Despite extensive studies, however, the underlying pathogenic mechanism still remains elusive. In the present study, we identified a homozygous mutation in the intron 1 of Wnt1 via large-scale screening of ASD risk/causative genes and verified that this mutation created a new splicing donor site in the intron 1, and consequently, a decrease of WNT1 expression. Interestingly, humanized rat models harboring this mutation exhibited robust ASD-like behaviors including impaired ultrasonic vocalization (USV), decreased social interactions, and restricted and repetitive behaviors. Moreover, in the substantia nigra compacta (SNpc) and the ventral tegmental area (VTA) of mutant rats, dopaminergic (DAergic) neurons were dramatically lost, together with a comparable decrease in striatal DAergic fibers. Furthermore, using single-cell RNA sequencing, we demonstrated that the decreased DAergic neurons in these midbrain areas might attribute to a shift of the boundary of the local pool of progenitor cells from the hypothalamic floor plate to the midbrain floor plate during the early embryonic stage. Moreover, treatments of mutant rats with levodopa could attenuate the impaired USV and social interactions almost completely, but not the restricted and repetitive behaviors. Our results for the first time documented that the developmental loss of DAergic neurons in the midbrain underlies the pathogenesis of ASD, and that the abnormal progenitor cell patterning is a cellular underpinning for this developmental DAergic neuronal loss. Importantly, the effective dopamine therapy suggests a translational significance in the treatment of ASD.

Construction of Co-ZIF-derived CoS2@Cu hollow heterogeneous nanotube array for the detection of hydrazine in environmental water samples.

As a neurotoxin, it is necessary to establish a low cost, stable and sensitive method for the quantitative detection of hydrazine. Using Co-ZIF (zeolite imidazole framework) nanorods as precursor, CoS2 hollow nanotube array heterogeneous structure loaded with Cu nanoparticles were prepared on carbon cloth (CC) by etching, calcination and plasma magnetron sputtering (CoS2@Cu HNTA/CC). As a self-supporting electrode, its hollow heterogeneous structure provides a large area of electron transfer channel for the oxidation of the food pollutant hydrazine. In addition, bimetallic synergies and in situ N doping regulated the electronic structure of CoS2@Cu HNTA/CC, and thus significantly improved the electrical conductivity and catalytic activity. As an efficient hydrazine sensor with a wide linear range of 1 µM L-1-10 mM (1 µM-1 mM and 1 mM-10 mM), its sensitivity and the limit of detection are 7996 µA mM-1 cm-2, 3772 µA mM-1 cm-2 and 0.276 µM (S/N = 3), respectively. This study provides a new strategy for the construction of MOFs (Metal Organic Framework)-derived bimetallic composites and their application in electrochemical sensing.

De novo mutations within metabolism networks of amino acid/protein/energy in Chinese autistic children with intellectual disability.

BACKGROUND: Autism spectrum disorder (ASD) is often accompanied by intellectual disability (ID). Despite extensive studies, however, the genetic basis for this comorbidity is still not clear. In this study, we tried to develop an analyzing pipeline for de novo mutations and possible pathways related to ID phenotype in ASD. Whole-exome sequencing (WES) was performed to screen de novo mutations and candidate genes in 79 ASD children together with their parents (trios). The de novo altering genes and relative pathways which were associated with ID phenotype were analyzed. The connection nodes (genes) of above pathways were selected, and the diagnostic value of these selected genes for ID phenotype in the study population was also evaluated. RESULTS: We identified 89 de novo mutant genes, of which 34 genes were previously reported to be associated with ASD, including double hits in the EGF repeats of NOTCH1 gene (p.V999M and p.S1027L). Interestingly, of these 34 genes, 22 may directly affect intelligence quotient (IQ). Further analyses revealed that these IQ-related genes were enriched in protein synthesis, energy metabolism, and amino acid metabolism, and at least 9 genes (CACNA1A, ALG9, PALM2, MGAT4A, PCK2, PLEKHA1, PSME3, ADI1, and TLE3) were involved in all these three pathways. Seven patients who harbored these gene mutations showed a high prevalence of a low IQ score (< 70), a non-verbal language, and an early diagnostic age (< 4 years). Furthermore, our panel of these 9 genes reached a 10.2% diagnostic rate (5/49) in early diagnostic patients with a low IQ score and also reached a 10% diagnostic yield in those with both a low IQ score and non-verbal language (4/40). CONCLUSION: We found some new genetic disposition for ASD accompanied with intellectual disability in this study. Our results may be helpful for etiologic research and early diagnoses of intellectual disability in ASD. Larger population studies and further mechanism studies are warranted.

NR1 Splicing Variant NR1a in Cerebellar Granule Neurons Constitutes a Better Motor Learning in the Mouse.

As an excitatory neuron in the cerebellum, the granule cells play a crucial role in motor learning. The assembly of NMDAR in these neurons varies in developmental stages, while the significance of this variety is still not clear. In this study, we found that motor training could specially upregulate the expression level of NR1a, a splicing form of NR1 subunit. Interestingly, overexpression of this splicing variant in a cerebellar granule cell-specific manner dramatically elevated the NMDAR binding activity. Furthermore, the NR1a transgenic mice did not only show an enhanced motor learning, but also exhibit a higher efficacy for motor training in motor learning. Our results suggested that as a "junior" receptor, NR1a facilitates NMDAR activity as well as motor skill learning.

Clinical characteristics and host immunity responses of SARS-CoV-2 Omicron variant BA.2 with deletion of ORF7a, ORF7b and ORF8.

BACKGROUND: The pathogenicity and virulence of the Omicron strain have weakened significantly pathogenesis of Omicron variants. Accumulating data indicated accessory proteins play crucial roles in host immune evasion and virus pathogenesis of SARS-CoV-2. Therefore, the impact of simultaneous deletion of accessory protein ORF7a, ORF7b and ORF8 on the clinical characteristics and specific immunity in Omicron breakthrough infected patients (BIPs) need to be verified. METHODS: Herein, plasma cytokines were identified using a commercial Multi-cytokine detection kit. Enzyme-linked immunosorbent assay and pseudovirus neutralization assays were utilized to determine the titers of SARS-CoV-2 specific binding antibodies and neutralizing antibodies, respectively. In addition, an enzyme-linked immunospot assay was used to quantify SARS-CoV-2 specific T cells and memory B cells. RESULTS: A local COVID-19 outbreak was caused by the Omicron BA.2 variant, which featured a deletion of 871 base pairs (∆871 BA.2), resulting in the removal of ORF7a, ORF7b, and ORF8. We found that hospitalized patients with ∆871 BA.2 had significantly shorter hospital stays than those with wild-type (WT) BA.2. Plasma cytokine levels in both ∆871 BA.2 and WT BA.2 patients were within the normal range of reference, and there was no notable difference in the titers of SARS-CoV-2 ancestor or Omicron-specific binding IgG antibodies, neutralizing antibody titers, effector T cells, and memory B cells frequencies between ∆871 BA.2 and WT BA.2 infected adult patients. However, antibody titers in ∆871 BA.2 infected adolescents were higher than in adults. CONCLUSIONS: The simultaneous deletion of ORF7a, ORF7b, and ORF8 facilitates the rapid clearance of the BA.2 variant, without impacting cytokine levels or affecting SARS-CoV-2 specific humoral and cellular immunity in Omicron-infected individuals.

SARS-CoV-2 Spike Protein Stabilized in the Closed State Induces Potent Neutralizing Responses.

The majority of SARS-CoV-2 vaccines in use or advanced development are based on the viral spike protein (S) as their immunogen. S is present on virions as prefusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described against both open and closed conformations. The long-term success of vaccination strategies depends upon inducing antibodies that provide long-lasting broad immunity against evolving SARS-CoV-2 strains. Here, we have assessed the results of immunization in a mouse model using an S protein trimer stabilized in the closed state to prevent full exposure of the receptor binding site and therefore interaction with the receptor. We compared this with other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induced a T cell response and long-lived, strongly neutralizing antibody responses against 2019 SARS-CoV-2 and variants of concern P.1 and B.1.351. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralizing responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, immune responses than open spikes and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. We suggest that closed spikes, together with their improved stability and storage properties, may be a valuable component of refined, next-generation vaccines. IMPORTANCE Vaccines in use against SARS-CoV-2 induce immune responses against the spike protein. There is intense interest in whether the antibody response induced by vaccines will be robust against new variants, as well as in next-generation vaccines for use in previously infected or immunized individuals. We assessed the use as an immunogen of a spike protein engineered to be conformationally stabilized in the closed state where the receptor binding site is occluded. Despite occlusion of the receptor binding site, the spike induces potently neutralizing sera against multiple SARS-CoV-2 variants. Antibodies are raised against a different pattern of epitopes to those induced by other spike constructs, preferring conformational epitopes present in the closed conformation. Closed spikes, or mRNA vaccines based on their sequence, can be a valuable component of next-generation vaccines.

Alteration of the allergenicity of cow's milk proteins using different food processing modifications.

Milk is an essential source of protein for infants and young children. At the same time, cow's milk is also one of the most common allergenic foods causing food allergies in children. Recently, cow's milk allergy (CMA) has become a common public health issue worldwide. Modern food processing technologies have been developed to reduce the allergenicity of milk proteins and improve the quality of life of patients with CMA. In this review, we summarize the main allergens in cow's milk, and introduce the recent findings on CMA responses. Moreover, the reduced effects and underlying mechanisms of different food processing techniques (such as heating, high pressure, γ-ray irradiation, ultrasound irradiation, hydrolysis, glycosylation, etc.) on the allergenicity of cow's milk proteins, and the application of processed cow's milk in clinical studies, are discussed. In addition, we describe the changes of nutritional value in cow's milk treated by different food processing technologies. This review provides an in-depth understanding of the allergenicity reduction of cow's milk proteins by various food processing techniques.

Microplasma-assisted synthesis of a mixed-valence Ce-MOF with enhanced oxidase-like activity for colorimetric sensing of dopamine.

Due to their high catalytic activity, stability and low cost, nanozymes with oxidase-like activity have attracted widespread interest in the fields of analytical detection and colorimetric sensing. To further promote the catalytic activity and the sensitivity of dopamine (DA) sensing, herein, a mixed valence Ce-MOF (MVCM) with enhanced oxidase-like activity was synthesized by the dielectric barrier discharge (DBD) microplasma method. Compared with hydrothermal synthesis, the prepared MVCM synthesized using a microplasma showed a higher catalytic activity, which benefits from a low Ce3+/Ce4+ ratio. Due to the spontaneous redox properties of Ce3+/Ce4+ in a MVCM, the MVCM-based colorimetric sensing of dopamine was established and showed a limit of detection of 0.74 µM over a linear range of 5-100 µM with high selectivity and stability and has been applied for the detection of dopamine in sweat. The proposed study provides an effective synthesis method for nanozymes with enhanced activity and shows great promise in widespread analytical and sensing applications.

Vulnerability and resilience in children during the COVID-19 pandemic.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is having a profound impact on the health and development of children worldwide. There is limited evidence on the impact of COVID-19 and its related school closures and disease-containment measures on the psychosocial wellbeing of children; little research has been done on the characteristics of vulnerable groups and factors that promote resilience. METHODS: We conducted a large-scale cross-sectional population study of Hong Kong families with children aged 2-12 years. Parents completed an online survey on family demographics, child psychosocial wellbeing, functioning and lifestyle habits, parent-child interactions, and parental stress during school closures due to COVID-19. We used simple and multiple linear regression analyses to explore factors associated with child psychosocial problems and parental stress during the pandemic. RESULTS: The study included 29,202 individual families; of which 12,163 had children aged 2-5 years and 17,029 had children aged 6-12 years. The risk of child psychosocial problems was higher in children with special educational needs, and/or acute or chronic disease, mothers with mental illness, single-parent families, and low-income families. Delayed bedtime and/or inadequate sleep or exercise duration, extended use of electronic devices were associated with significantly higher parental stress and more psychosocial problems among pre-schoolers. CONCLUSIONS: This study identifies vulnerable groups of children and highlights the importance of strengthening family coherence, adequate sleep and exercise, and responsible use of electronic devices in promoting psychosocial wellbeing during the COVID-19 pandemic.

An assessment of the mental health of front-line medical staff involved in the COVID-19 outbreak in Chongqing, China.

In response to the coronavirus disease 2019 (COVID-19) outbreak in December 2019 in China, medical staff went to work across the country to combat widespread infection. When health workers are suddenly faced with such a serious event, it is important to assess their mental health in order to determine whether they can meet the challenge effectively. Herein, Symptom Checklist-90 (SCL-90) was used to assess the psychological problems of 382 front-line medical staff in Chongqing. The average SCL-90 score was low, and no specific mental health problems were found. With the exception of the phobic-anxiety factor, the scores were close to normal values. A single-factor analysis of variance showed that the SCL-90 scores of male and older staff were higher than those of female and younger staff, implying that they were at greater psychological risk. We found that both gender and age have a significant impact on mental health, and our findings suggest that more attention should be given to the mental health of male and older front-line medical staff.

Enhancing Neuraminidase Immunogenicity of Influenza A Viruses by Rewiring RNA Packaging Signals.

Humoral immune protection against influenza virus infection is mediated largely by antibodies against hemagglutinin (HA) and neuraminidase (NA), the two major glycoproteins on the virus surface. While influenza virus vaccination efforts have focused mainly on HA, NA-based immunity has been shown to reduce disease severity and provide heterologous protection. Current seasonal vaccines do not elicit strong anti-NA responses-in part due to the immunodominance of the HA protein. Here, we demonstrate that by swapping the 5' and 3' terminal packaging signals of the HA and NA genomic segments, which contain the RNA promoters, we are able to rescue influenza viruses that express more NA and less HA. Vaccination with formalin-inactivated "rewired" viruses significantly enhances the anti-NA antibody response compared to vaccination with unmodified viruses. Passive transfer of sera from mice immunized with rewired virus vaccines shows better protection against influenza virus challenge. Our results provide evidence that the immunodominance of HA stems in part from its abundance on the viral surface, and that rewiring viral packaging signals-thereby increasing the NA content on viral particles-is a viable strategy for improving the immunogenicity of NA in an influenza virus vaccine.IMPORTANCE Influenza virus infections are a major source of morbidity and mortality worldwide. Increasing evidence highlights neuraminidase as a potential vaccination target. This report demonstrates the efficacy of rewiring influenza virus packaging signals for creating vaccines with more neuraminidase content which provide better neuraminidase (NA)-based protection.

A Comparison Between Chinese Children Infected with Coronavirus Disease-2019 and with Severe Acute Respiratory Syndrome 2003.

OBJECTIVES: To compare the clinical and laboratory features of severe acute respiratory syndrome 2003 (SARS) and coronavirus disease 2019 (COVID-19) in 2 Chinese pediatric cohorts, given that the causative pathogens and are biologically similar. STUDY DESIGN: This is a cross-sectional study reviewing pediatric patients with SARS (n = 43) and COVID-19 (n = 244) who were admitted to the Princess Margaret Hospital in Hong Kong and Wuhan Children's Hospital in Wuhan, respectively. Demographics, hospital length of stay, and clinical and laboratory features were compared. RESULTS: Overall, 97.7% of patients with SARS and 85.2% of patients with COVID-19 had epidemiologic associations with known cases. Significantly more patients with SARS developed fever, chills, myalgia, malaise, coryza, sore throat, sputum production, nausea, headache, and dizziness than patients with COVID-19. No patients with SARS were asymptomatic at the time of admission, whereas 29.1% and 20.9% of patients with COVID-19 were asymptomatic on admission and throughout their hospital stay, respectively. More patients with SARS required oxygen supplementation than patients with COVID-19 (18.6 vs 4.7%; P = .004). Only 1.6% of patients with COVID-19 and 2.3% of patients with SARS required mechanical ventilation. Leukopenia (37.2% vs 18.6%; P = .008), lymphopenia (95.4% vs 32.6%; P < .01), and thrombocytopenia (41.9% vs 3.8%; P < .001) were significantly more common in patients with SARS than in patients with COVID-19. The duration between positive and negative nasopharyngeal aspirate and the length in hospital stay were similar in patients with COVID-19, regardless of whether they were asymptomatic or symptomatic, suggesting a similar duration of viral shedding. CONCLUSIONS: Children with COVID-19 were less symptomatic and had more favorable hematologic findings than children with SARS.

Receptor binding by H10 influenza viruses.

H10N8 follows H7N9 and H5N1 as the latest in a line of avian influenza viruses that cause serious disease in humans and have become a threat to public health. Since December 2013, three human cases of H10N8 infection have been reported, two of whom are known to have died. To gather evidence relating to the epidemic potential of H10 we have determined the structure of the haemagglutinin of a previously isolated avian H10 virus and we present here results relating especially to its receptor-binding properties, as these are likely to be major determinants of virus transmissibility. Our results show, first, that the H10 virus possesses high avidity for human receptors and second, from the crystal structure of the complex formed by avian H10 haemagglutinin with human receptor, it is clear that the conformation of the bound receptor has characteristics of both the 1918 H1N1 pandemic virus and the human H7 viruses isolated from patients in 2013 (ref. 3). We conclude that avian H10N8 virus has sufficient avidity for human receptors to account for its infection of humans but that its preference for avian receptors should make avian-receptor-rich human airway mucins an effective block to widespread infection. In terms of surveillance, particular attention will be paid to the detection of mutations in the receptor-binding site of the H10 haemagglutinin that decrease its avidity for avian receptor, and could enable it to be more readily transmitted between humans.