Type-I-interferon-responsive microglia shape cortical development and behavior.

Microglia are brain-resident macrophages that shape neural circuit development and are implicated in neurodevelopmental diseases. Multiple microglial transcriptional states have been defined, but their functional significance is unclear. Here, we identify a type I interferon (IFN-I)-responsive microglial state in the developing somatosensory cortex (postnatal day 5) that is actively engulfing whole neurons. This population expands during cortical remodeling induced by partial whisker deprivation. Global or microglial-specific loss of the IFN-I receptor resulted in microglia with phagolysosomal dysfunction and an accumulation of neurons with nuclear DNA damage. IFN-I gain of function increased neuronal engulfment by microglia in both mouse and zebrafish and restricted the accumulation of DNA-damaged neurons. Finally, IFN-I deficiency resulted in excess cortical excitatory neurons and tactile hypersensitivity. These data define a role for neuron-engulfing microglia during a critical window of brain development and reveal homeostatic functions of a canonical antiviral signaling pathway in the brain.

SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming.

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

A defective viral genome strategy elicits broad protective immunity against respiratory viruses.

RNA viruses generate defective viral genomes (DVGs) that can interfere with replication of the parental wild-type virus. To examine their therapeutic potential, we created a DVG by deleting the capsid-coding region of poliovirus. Strikingly, intraperitoneal or intranasal administration of this genome, which we termed eTIP1, elicits an antiviral response, inhibits replication, and protects mice from several RNA viruses, including enteroviruses, influenza, and SARS-CoV-2. While eTIP1 replication following intranasal administration is limited to the nasal cavity, its antiviral action extends non-cell-autonomously to the lungs. eTIP1 broad-spectrum antiviral effects are mediated by both local and distal type I interferon responses. Importantly, while a single eTIP1 dose protects animals from SARS-CoV-2 infection, it also stimulates production of SARS-CoV-2 neutralizing antibodies that afford long-lasting protection from SARS-CoV-2 reinfection. Thus, eTIP1 is a safe and effective broad-spectrum antiviral generating short- and long-term protection against SARS-CoV-2 and other respiratory infections in animal models.

Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant.

We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.

SARS-CoV-2 Nsp1 cooperates with initiation factors EIF1 and 1A to selectively enhance translation of viral RNA.

A better mechanistic understanding of virus-host dependencies can help reveal vulnerabilities and identify opportunities for therapeutic intervention. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in protein synthesis dynamics during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones that are used by CoV2 to promote the synthesis and maturation of its own proteins. These can be targeted to inhibit infection, without major toxicity to the host. We also find that CoV2 non-structural protein 1 (Nsp1) cooperates with initiation factors EIF1 and 1A to selectively enhance translation of viral RNA. When EIF1/1A are depleted, more ribosomes initiate translation from a conserved upstream CUG start codon found in all genomic and subgenomic viral RNAs. This results in higher translation of an upstream open reading frame (uORF1) and lower translation of the main ORF, altering the stoichiometry of viral proteins and attenuating infection. Replacing the upstream CUG with AUG strongly inhibits translation of the main ORF independently of Nsp1, EIF1, or EIF1A. Taken together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and proposes a model for dosage control of viral proteins through Nsp1-mediated control of translation start site selection.

High-resolution mapping reveals the mechanism and contribution of genome insertions and deletions to RNA virus evolution.

RNA viruses rapidly adapt to selective conditions due to the high intrinsic mutation rates of their RNA-dependent RNA polymerases (RdRps). Insertions and deletions (indels) in viral genomes are major contributors to both deleterious mutational load and evolutionary novelty, but remain understudied. To characterize the mechanistic details of their formation and evolutionary dynamics during infection, we developed a hybrid experimental-bioinformatic approach. This approach, called MultiMatch, extracts insertions and deletions from ultradeep sequencing experiments, including those occurring at extremely low frequencies, allowing us to map their genomic distribution and quantify the rates at which they occur. Mapping indel mutations in adapting poliovirus and dengue virus populations, we determine the rates of indel generation and identify mechanistic and functional constraints shaping indel diversity. Using poliovirus RdRp variants of distinct fidelity and genome recombination rates, we demonstrate tradeoffs between fidelity and Indel generation. Additionally, we show that maintaining translation frame and viral RNA structures constrain the Indel landscape and that, due to these significant fitness effects, Indels exert a significant deleterious load on adapting viral populations. Conversely, we uncover positively selected Indels that modulate RNA structure, generate protein variants, and produce defective interfering genomes in viral populations. Together, our analyses establish the kinetic and mechanistic tradeoffs between misincorporation, recombination, and Indel rates and reveal functional principles defining the central role of Indels in virus evolution, emergence, and the regulation of viral infection.

Effects of perceived stress on turnover intention of female healthcare staff: a serial multiple mediation model.

BACKGROUND: Healthcare staff in China, especially females, work in a high-pressure, high-load, and high-risk environment, which affects the physical and mental health, the efficiency and quality of work, and increases turnover intention. The present study investigated the relationship between perceived stress and turnover intention in female healthcare staff, and the effects of future-oriented coping and work-family balance on this relationship. METHODS: Four hundred thirty-five female medical workers were recruited to perform a perceived stress scale, future-oriented coping inventory, work-family balance scale and turnover intention scale. Meanwhile, serial multiple mediation analysis was performed using PROCESS. RESULTS: 1) Perceived stress positively predicted the level of turnover intention in female healthcare staff; 2) Preventive coping and proactive coping showed mediation effects on the relationship between perceived stress and turnover intention, and preventive coping positively related to proactive coping; 3) The work-family balance also showed mediation effects on the relationship between perceived stress and turnover intention; 4) Preventive coping, proactive coping and work-family balance showed a serial multiple mediation on the relationship between perceived stress and turnover intention in female healthcare workers. CONCLUSIONS: Perceived stress affects the level of turnover intention in female healthcare staff through preventive coping, proactive coping, and work-family balance. In addition, the sequential model of future-oriented coping was validated among female healthcare staff.

Experimental and mathematical insights on the interactions between poliovirus and a defective interfering genome.

During replication, RNA viruses accumulate genome alterations, such as mutations and deletions. The interactions between individual variants can determine the fitness of the virus population and, thus, the outcome of infection. To investigate the effects of defective interfering genomes (DI) on wild-type (WT) poliovirus replication, we developed an ordinary differential equation model, which enables exploring the parameter space of the WT and DI competition. We also experimentally examined virus and DI replication kinetics during co-infection, and used these data to infer model parameters. Our model identifies, and our experimental measurements confirm, that the efficiencies of DI genome replication and encapsidation are two most critical parameters determining the outcome of WT replication. However, an equilibrium can be established which enables WT to replicate, albeit to reduced levels.

Ethacridine inhibits SARS-CoV-2 by inactivating viral particles.

The respiratory disease COVID-19 is caused by the coronavirus SARS-CoV-2. Here we report the discovery of ethacridine as a potent drug against SARS-CoV-2 (EC50 ~ 0.08 µM). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescence assay. Plaque assays, RT-PCR and immunofluorescence imaging at various stages of viral infection demonstrate that the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Consistently, ethacridine is effective in various cell types, including primary human nasal epithelial cells that are cultured in an air-liquid interface. Taken together, our work identifies a promising, potent, and new use of the old drug via a distinct mode of action for inhibiting SARS-CoV-2.

RNA virus population diversity, an optimum for maximal fitness and virulence.

The ability of an RNA virus to exist as a population of genetically distinct variants permits the virus to overcome events during infections that would otherwise limit virus multiplication or drive the population to extinction. Viral genetic diversity is created by the ribonucleotide misincorporation frequency of the viral RNA-dependent RNA polymerase (RdRp). We have identified a poliovirus (PV) RdRp derivative (H273R) possessing a mutator phenotype. GMP misincorporation efficiency for H273R RdRp in vitro was increased by 2-3-fold that manifested in a 2-3-fold increase in the diversity of the H273R PV population in cells. Circular sequencing analysis indicated that some mutations were RdRp-independent. Consistent with the population genetics theory, H273R PV was driven to extinction more easily than WT in cell culture. Furthermore, we observed a substantial reduction in H273R PV virulence, measured as the ability to cause paralysis in the cPVR mouse model. Reduced virulence correlated with the inability of H273R PV to sustain replication in tissues/organs in which WT persists. Despite the attenuated phenotype, H273R PV was capable of replicating in mice to levels sufficient to induce a protective immune response, even when the infecting dose used was insufficient to elicit any visual signs of infection. We conclude that optimal RdRp fidelity is a virulence determinant that can be targeted for viral attenuation or antiviral therapies, and we suggest that the RdRp may not be the only source of mutations in a RNA virus genome.

GO-COO-HP-ß-CD nanosphere: a complex construction and its drug-loading properties.

A novel nanosphere based on carboxylated GO (GO-COOH) and hydroxypropyl-beta-CD (HP-ß-CD) was synthesized to construct a complex of GO-COO-HP-ß-CD. The complex formation process was studied using spectral characterization and transmission electron microscopy (TEM). X-ray diffraction and energy dispersive spectroscopy patterns show that HP-ß-CD molecules either cover or intercalate into GO-COOH interlayers in the complex. Fourier transform infrared spectroscopy results indicate that GO-COOH and HP-ß-CD are linked with covalent bonds formed via esterification. When employed as nanohybrid drug carriers for dexamethasone, the inclusion displays good dispersibility validated by dynamic light scattering (DLS). Cytotoxicity assays and hemolysis testing demonstrate that the nanospheres possess good biological compatibility. The loading capacity of dexamethasone is as high as 32.33%, with loading efficiency 64.66%.

SARS-CoV-2 Nsp1 regulates translation start site fidelity to promote infection.

A better mechanistic understanding of virus-host interactions can help reveal vulnerabilities and identify opportunities for therapeutic interventions. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in polysome composition and protein synthesis during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones whose inhibition impairs infectious particle production without major toxicity to the host. We find that CoV2 non-structural protein Nsp1 selectively enhances virus translation through functional interactions with initiation factor EIF1A. When EIF1A is depleted, more ribosomes initiate translation from an upstream CUG start codon, inhibiting translation of non-structural genes and reducing viral titers. Together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and identifies druggable targets for potential antiviral development.

Type I interferon responsive microglia shape cortical development and behavior.

Microglia are brain resident phagocytes that can engulf synaptic components and extracellular matrix as well as whole neurons. However, whether there are unique molecular mechanisms that regulate these distinct phagocytic states is unknown. Here we define a molecularly distinct microglial subset whose function is to engulf neurons in the developing brain. We transcriptomically identified a cluster of Type I interferon (IFN-I) responsive microglia that expanded 20-fold in the postnatal day 5 somatosensory cortex after partial whisker deprivation, a stressor that accelerates neural circuit remodeling. In situ, IFN-I responsive microglia were highly phagocytic and actively engulfed whole neurons. Conditional deletion of IFN-I signaling (Ifnar1fl/fl) in microglia but not neurons resulted in dysmorphic microglia with stalled phagocytosis and an accumulation of neurons with double strand DNA breaks, a marker of cell stress. Conversely, exogenous IFN-I was sufficient to drive neuronal engulfment by microglia and restrict the accumulation of damaged neurons. IFN-I deficient mice had excess excitatory neurons in the developing somatosensory cortex as well as tactile hypersensitivity to whisker stimulation. These data define a molecular mechanism through which microglia engulf neurons during a critical window of brain development. More broadly, they reveal key homeostatic roles of a canonical antiviral signaling pathway in brain development.

Fluorogenic reporter enables identification of compounds that inhibit SARS-CoV-2.

The coronavirus SARS-CoV-2 causes the severe disease COVID-19. SARS-CoV-2 infection is initiated by interaction of the viral spike protein and host receptor angiotensin-converting enzyme 2 (ACE2). We report an improved bright and reversible fluorogenic reporter, named SURF (split UnaG-based reversible and fluorogenic protein-protein interaction reporter), that we apply to monitor real-time interactions between spike and ACE2 in living cells. SURF has a large dynamic range with a dark-to-bright fluorescence signal that requires no exogenous cofactors. Utilizing this reporter, we carried out a high-throughput screening of small-molecule libraries. We identified three natural compounds that block replication of SARS-CoV-2 in both Vero cells and human primary nasal and bronchial epithelial cells. Cell biological and biochemical experiments validated all three compounds and showed that they block the early stages of viral infection. Two of the inhibitors, bruceine A and gamabufotalin, were also found to block replication of the Delta and Omicron variants of SARS-CoV-2. Both bruceine A and gamabufotalin exhibited potent antiviral activity in K18-hACE2 and wild-type C57BL6/J mice, as evidenced by reduced viral titres in the lung and brain, and protection from alveolar and peribronchial inflammation in the lung, thereby limiting disease progression. We propose that our fluorescent assay can be applied to identify antiviral compounds with potential as therapeutic treatment for COVID-19 and other respiratory diseases.

Nursing Practice Based on Evidence-Based Concepts to Prevent Enteral Nutrition Complications for Critically Ill Neurosurgical Patients.

Purpose: To explore the practical value of enteral nutrition care guided by evidence-based concepts in preventing enteral nutritional complications in critically ill neurosurgical patients. Methods: Three hundred critically ill patients from March 2020 to October 2021 from our neurosurgery department were included in the study. Patients were divided into a control group (March to December 2020, n = 150) and a study group (January to October 2021, n = 150) according to the order of their admission. The control group received conventional enteral nutrition care, and the study group received enteral nutrition care based on evidence-based concept guidance. The levels of serum nutritional indicators [hemoglobin (Hb), albumin (ALB), and total protein (TP)], feeding compliance rate, the incidence of complications (gastric retention, bloating, diarrhea, reflux, vomiting, aspiration, stress ulcers, etc.), and prognosis during the observation period were compared between the two groups. The scores of the questionnaire of knowledge, attitude, and practice on nutrition among neurosurgical nurses before and after the implementation of evidence-based care were compared among nursing staff in the study group. Results: At 1 and 2 weeks after enrollment, Hb, ALB, and TP levels were lower in both groups than before enrollment in the same group (P < 0.05). At 2 weeks after enrollment, Hb, ALB, and TP levels were higher in both groups than at 1 week after enrollment in the same group (P < 0.05). At 1 and 2 weeks after enrollment, Hb, ALB, and TP levels were higher in the study group than in the control group (P < 0.05). At 7 days after feeding, the feeding compliance rate was higher in the study group (94.67%) than in the control group (70.00%) (P < 0.05). The total complication rate was lower in the study group (8.00%) than in the control group (16.00%) (P < 0.05). The percentage of good prognosis was higher in the study group (34.00%) than in the control group (23.33%) (P < 0.05). After the implementation of evidence-based care, caregivers in the study group scored higher on nutrition knowledge, nutrition attitudes, and nutrition practices than those before the implementation (P < 0.05). Conclusion: The implementation of evidence-based nursing interventions in critically ill neurosurgical patients based on evidence-based concepts is of great clinical value in correcting their nutritional status, preventing enteral nutritional complications, improving prognosis, and enhancing the nutritional knowledge, attitudes, and practices of nursing staff.

SARS-CoV-2 infects human pancreatic ß cells and elicits ß cell impairment.

Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic ß cells can be infected by SARS-CoV-2 and cause ß cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in ß cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic ß cells in patients who succumbed to COVID-19 and selectively infects human islet ß cells in vitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces ß cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic ß cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce ß cell killing.

Determinants of SARS-CoV-2 entry and replication in airway mucosal tissue and susceptibility in smokers.

Understanding viral tropism is an essential step toward reducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, decreasing mortality from coronavirus disease 2019 (COVID-19) and limiting opportunities for mutant strains to arise. Currently, little is known about the extent to which distinct tissue sites in the human head and neck region and proximal respiratory tract selectively permit SARS-CoV-2 infection and replication. In this translational study, we discover key variabilities in expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), essential SARS-CoV-2 entry factors, among the mucosal tissues of the human proximal airways. We show that SARS-CoV-2 infection is present in all examined head and neck tissues, with a notable tropism for the nasal cavity and tracheal mucosa. Finally, we uncover an association between smoking and higher SARS-CoV-2 viral infection in the human proximal airway, which may explain the increased susceptibility of smokers to developing severe COVID-19. This is at least partially explained by differences in interferon (IFN)-ß1 levels between smokers and non-smokers.

Transmission, infectivity, and antibody neutralization of an emerging SARS-CoV-2 variant in California carrying a L452R spike protein mutation.

We identified a novel SARS-CoV-2 variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California. Named B.1.427/B.1.429 to denote its 2 lineages, the variant emerged around May 2020 and increased from 0% to >50% of sequenced cases from September 1, 2020 to January 29, 2021, exhibiting an 18.6-24% increase in transmissibility relative to wild-type circulating strains. The variant carries 3 mutations in the spike protein, including an L452R substitution. Our analyses revealed 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation found in the B.1.1.7, B.1.351, and P.1 variants. Antibody neutralization assays showed 4.0 to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California associated with decreased antibody neutralization warrants further investigation.

Increased female fertility in aquaporin 8-deficient mice.

Aquaporin-8 (AQP8) is a water channel expressed extensively in male and female reproductive systems. But its physiological functions are largely unknown. In the present study, we first found significantly increased number of offspring delivered by AQP8(-/-) mothers compared with wild-type mothers in cross-mating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8(-/-) ovaries released more oocytes (9.5 ± 1.9 vs. 7.1 ± 2.1 in normal ovulation and 37.8 ± 6.7 vs. 27.9 ± 5.7 in superovulation). Histological analysis showed increased number of corpus luteums in mature AQP8(-/-) ovaries, suggesting increased maturation and ovulation of follicles. By RT-PCR, western blot and immunohistochemistry analyses, we determined the expression of AQP8 in mouse ovarian granulosa cells. Granulosa cells isolated from AQP8(-/-) mice showed 45% of decreased membrane water permeability than wild-type mice. As the atresia of ovarian follicles is primarily due to apoptosis of granulosa cells, we analyzed the apoptosis of isolated granulosa cells from wild-type and AQP8(-/-) mice. The results indicated significantly lower apoptosis rate in AQP8(-/-) granulosa cells (21.3 ± 3.6% vs. 32.6 ± 4.3% in AQP8(+/+) granulosa cells). Taken together, we conclude that AQP8 deficiency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. This discovery may offer new insight of improving female fertility by reducing granulosa cell apoptosis through AQP8 inhibition.

Preparation and characterization of Keratin-PEG conjugate-based micelles as a tumor microenvironment-responsive drug delivery system.

Keratin-based drug carriers have attracted great interest due to their intrinsic biocompatibility and tumor micro-environmental responsiveness. In the study, keratin was first extracted from human hair with reduction method. The reduced keratin was successively conjugated with poly(ethylene glycol) (PEG) via thiol Michael addition reaction and iodoacetic acid (IAA) via substitution reaction to impart both physical stability and acidity responsiveness. Subsequently, the conjugated keratin was fabricated into micelles and loaded with doxorubicin (DOX) by self-assembly. The micelles exhibited pH, glutathione (GSH) and enzyme (trypsin) triple-responsiveness as well as charge reversibility under the simulated tumor microenvironment. These drug-loaded micelles exhibited high toxicity against A549 cells with low side effect on normal cells. Furthermore, anticancer efficacy in vivo revealed DOX-loaded micelles presented higher therapeutic efficiency than free DOX. Moreover, these micelles were stable under physiological conditions, and could be internalized through endocytosis without hemolysis. Based on the results, the drug-loaded micelles were satisfactory candidates for drug carriers.