Trimerized S expressed by modified vaccinia virus Ankara (MVA) confers superior protection against lethal SARS-CoV-2 challenge in mice.

The reoccurrence of successive waves of SARS-CoV-2 variants suggests the exploration of more vaccine alternatives is imperative. Modified vaccinia virus Ankara (MVA) is a virus vector exhibiting excellent safety as well as efficacy for vaccine development. Here, a series of recombinant MVAs (rMVAs) expressing monomerized or trimerized S proteins from different SARS-CoV-2 variants are engineered. Trimerized S expressed from rMVAs is found predominantly as trimers on the surface of infected cells. Remarkably, immunization of mice with rMVAs demonstrates that S expressed in trimer elicits higher levels of binding IgG and IgA, as well as neutralizing antibodies for matched and mismatched S proteins than S in the monomer. In addition, trimerized S expressed by rMVA induces enhanced cytotoxic T-cell responses than S in the monomer. Importantly, the rMVA vaccines expressing trimerized S exhibit superior protection against a lethal SARS-CoV-2 challenge as the immunized animals all survive without displaying any pathological conditions. This study suggests that opting for trimerized S may represent a more effective approach and highlights that the MVA platform serves as an ideal foundation to continuously advance SARS-CoV-2 vaccine development. IMPORTANCE: MVA is a promising vaccine vector and has been approved as a vaccine for smallpox and mpox. Our analyses suggested that recombinant MVA expressing S in trimer (rMVA-ST) elicited robust cellular and humoral immunity and was more effective than MVA-S-monomer. Importantly, the rMVA-ST vaccine was able to stimulate decent cross-reactive neutralization against pseudoviruses packaged using S from different sublineages, including Wuhan, Delta, and Omicron. Remarkably, mice immunized with rMVA-ST were completely protected from a lethal challenge of SARS-CoV-2 without displaying any pathological conditions. Our results demonstrated that an MVA vectored vaccine expressing trimerized S is a promising vaccine candidate for SARS-CoV-2 and the strategy might be adapted for future vaccine development for coronaviruses.

Genomic analysis of lumpy skin disease virus asian variants and evaluation of its cellular tropism.

Lumpy skin disease virus (LSDV) is a poxvirus that mainly affects cattle and can lead to symptoms such as severe reduction in milk production as well as infertility and mortality, which has resulted in dramatic economic loss in affected countries in Africa, Europe, and Asia. In this study, we successfully isolated two strains of LSDV from different geographical regions in China. Comparative genomic analyses were performed by incorporating additional LSDV whole genome sequences reported in other areas of Asia. Our analyses revealed that LSDV exhibited an 'open' pan-genome. Phylogenetic analysis unveiled distinct branches of LSDV evolution, signifying the prevalence of multiple lineages of LSDV across various regions in Asia. In addition, a reporter LSDV expressing eGFP directed by a synthetic poxvirus promoter was generated and used to evaluate the cell tropism of LSDV in various mammalian and avian cell lines. Our results demonstrated that LSDV replicated efficiently in several mammalian cell lines, including human A549 cells. In conclusion, our results underscore the necessity for strengthening LSD outbreak control measures and continuous epidemiological surveillance.

Human transferrin receptor can mediate SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been detected in almost all organs of coronavirus disease-19 patients, although some organs do not express angiotensin-converting enzyme-2 (ACE2), a known receptor of SARS-CoV-2, implying the presence of alternative receptors and/or co-receptors. Here, we show that the ubiquitously distributed human transferrin receptor (TfR), which binds to diferric transferrin to traffic between membrane and endosome for the iron delivery cycle, can ACE2-independently mediate SARS-CoV-2 infection. Human, not mouse TfR, interacts with Spike protein with a high affinity (KD ~2.95 nM) to mediate SARS-CoV-2 endocytosis. TfR knock-down (TfR-deficiency is lethal) and overexpression inhibit and promote SARS-CoV-2 infection, respectively. Humanized TfR expression enables SARS-CoV-2 infection in baby hamster kidney cells and C57 mice, which are known to be insusceptible to the virus infection. Soluble TfR, Tf, designed peptides blocking TfR-Spike interaction and anti-TfR antibody show significant anti-COVID-19 effects in cell and monkey models. Collectively, this report indicates that TfR is a receptor/co-receptor of SARS-CoV-2 mediating SARS-CoV-2 entry and infectivity by likely using the TfR trafficking pathway.

Transferrin Is Up-Regulated by Microbes and Acts as a Negative Regulator of Immunity to Induce Intestinal Immunotolerance.

Cross-talks (e.g., host-driven iron withdrawal and microbial iron uptake between host gastrointestinal tract and commensal microbes) regulate immunotolerance and intestinal homeostasis. However, underlying mechanisms that regulate the cross-talks remain poorly understood. Here, we show that bacterial products up-regulate iron-transporter transferrin and transferrin acts as an immunosuppressor by interacting with cluster of differentiation 14 (CD14) to inhibit pattern recognition receptor (PRR) signaling and induce host immunotolerance. Decreased intestinal transferrin is found in germ-free mice and human patients with ulcerative colitis, which are characterized by impaired intestinal immunotolerance. Intestinal transferrin and host immunotolerance are returned to normal when germ-free mice get normal microbial commensalism, suggesting an association between microbial commensalism, transferrin, and host immunotolerance. Mouse colitis models show that transferrin shortage impairs host's tolerogenic responses, while its supplementation promotes immunotolerance. Designed peptide blocking transferrin-CD14 interaction inhibits immunosuppressive effects of transferrin. In monkeys with idiopathic chronic diarrhea, transferrin shows comparable or even better therapeutic effects than hydrocortisone. Our findings reveal that by up-regulating host transferrin to silence PRR signaling, commensal bacteria counteract immune activation induced by themselves to shape host immunity and contribute for intestinal tolerance.

A practical key recovery attack on the lightweight WG-5 stream cipher.

WG-5 is a lightweight stream cipher proposed for usage in the resource-constrained devices, e.g., passive RFID tags, industrial controllers, contactless smart cards and sensors. In this paper, a weakness called slide property of WG-5 which has not been discovered in previous works is for the first time explored and analyzed. The result shows that the probability that two related key-IV pairs of WG-5 generate the shifted keystreams can be up to 2-20, which is significantly high compared with an ideal stream cipher that generates the random keystreams. The correctness and accuracy of this theoretical probability is confirmed experimentally. Based on the slide property of WG-5, some key recovery attacks on WG-5 in the related key setting are proposed. The cryptanalytic result shows that the 80-bit secret key of WG-5 can be recovered with a time complexity of 225.615, requiring 6 related keys and 80 keystream bits for each of 224.585 chosen IVs. The experimental result validates our attack and shows that WG-5 can be broken within about 92.054 seconds on a common PC in the related key setting. These results imply that the design of WG-5 is far from optimal and needs to be strengthened to provide enough security for the lightweight constrained applications.

Poliovirus receptor-based chimeric antigen receptor T cells combined with NK-92 cells exert potent activity against glioblastoma.

BACKGROUND: Poliovirus receptor interacts with 3 receptors: T-cell immunoglobulin immunoreceptor tyrosine-based inhibitory motif, CD96, and DNAX accessory molecule 1, which are predominantly expressed on T cells and natural killer (NK) cells. Many solid tumors, including IDH wild-type glioblastoma, have been reported to overexpress poliovirus receptor, and this overexpression is associated with poor prognosis. However, there are no preclinical or clinical trials investigating the use of cell-based immunotherapies targeting poliovirus receptor in IDH wild-type glioblastoma. METHODS: We analyzed poliovirus receptor expression in transcriptome sequencing databases and specimens from IDH wild-type glioblastoma patients. We developed poliovirus receptor targeting chimeric antigen receptor T cells using lentivirus. The antitumor activity of chimeric antigen receptor T cells was demonstrated in patient-derived glioma stem cells, intracranial and subcutaneous mouse xenograft models. RESULTS: We verified poliovirus receptor expression in primary glioma stem cells, surgical specimens from IDH wild-type glioblastoma patients, and organoids. Accordingly, we developed poliovirus receptor-based second-generation chimeric antigen receptor T cells. The antitumor activity of chimeric antigen receptor T cells was demonstrated in glioma stem cells and xenograft models. Tumor recurrence occurred in intracranial xenograft models because of antigen loss. The combinational therapy of tyrosine-based inhibitory motif extracellular domain-based chimeric antigen receptor T cells and NK-92 cells markedly suppressed tumor recurrence and prolonged survival. CONCLUSIONS: Poliovirus receptor-based chimeric antigen receptor T cells were capable of killing glioma stem cells and suppressing tumor recurrence when combined with NK-92 cells.

Blood-brain barrier injury and neuroinflammation induced by SARS-CoV-2 in a lung-brain microphysiological system.

In some patients, COVID-19 can trigger neurological symptoms with unclear pathogenesis. Here we describe a microphysiological system integrating alveolus and blood-brain barrier (BBB) tissue chips that recapitulates neuropathogenesis associated with infection by SARS-CoV-2. Direct exposure of the BBB chip to SARS-CoV-2 caused mild changes to the BBB, and infusion of medium from the infected alveolus chip led to more severe injuries on the BBB chip, including endothelial dysfunction, pericyte detachment and neuroinflammation. Transcriptomic analyses indicated downregulated expression of the actin cytoskeleton in brain endothelium and upregulated expression of inflammatory genes in glial cells. We also observed early cerebral microvascular damage following lung infection with a low viral load in the brains of transgenic mice expressing human angiotensin-converting enzyme 2. Our findings suggest that systemic inflammation is probably contributing to neuropathogenesis following SARS-CoV-2 infection, and that direct viral neural invasion might not be a prerequisite for this neuropathogenesis. Lung-brain microphysiological systems should aid the further understanding of the systemic effects and neurological complications of viral infection.

Lipopolysaccharide-binding protein expression is increased by stress and inhibits monoamine synthesis to promote depressive symptoms.

Monoamine insufficiency is suggested to be associated with depressive features such as sadness, anhedonia, insomnia, and cognitive dysfunction, but the mechanisms that cause it are unclear. We found that the acute-phase protein lipopolysaccharide-binding protein (LBP) inhibits monoamine biosynthesis by acting as an endogenous inhibitor of dopamine-ß-hydroxylase (DBH) and aromatic-L-amino-acid-decarboxylase (DDC). LBP expression was increased in individuals with depression and by diverse stress challenges in mice. LBP antibodies and LBP knockdown inhibited monoamine insufficiency and depression-like features in mice, which worsened with LBP overexpression or administration. Monoamine insufficiency and depression-like symptoms were not induced by stressful stimuli in LBP-deficient mice, further highlighting a role for LBP in stress-induced depression, and a peptide we designed that blocks LBP-DBH and LBP-DDC interactions showed anti-depression effects in mice. This study reveals an important role for LBP in regulating monoamine biosynthesis and suggests that targeting LBP may have potential as a treatment for some individuals with depression.

Hypoxia and low temperature upregulate transferrin to induce hypercoagulability at high altitude.

Studies have shown significantly increased thromboembolic events at high altitude. We recently reported that transferrin could potentiate blood coagulation, but the underlying mechanism for high altitude-related thromboembolism is still poorly understood. Here, we examined the activity and concentration of plasma coagulation factors and transferrin in plasma collected from long-term human residents and short-stay mice exposed to varying altitudes. We found that the activities of thrombin and factor XIIa (FXIIa) along with the concentrations of transferrin were significantly increased in the plasma of humans and mice at high altitudes. Furthermore, both hypoxia (6% O2) and low temperature (0°C), 2 critical high-altitude factors, enhanced hypoxia-inducible factor 1α (HIF-1α) levels to promote the expression of the transferrin gene, whose enhancer region contains HIF-1α binding site, and consequently, to induce hypercoagulability by potentiating thrombin and FXIIa. Importantly, thromboembolic disorders and pathological insults in mouse models induced by both hypoxia and low temperature were ameliorated by transferrin interferences, including transferrin antibody treatment, transferrin downregulation, and the administration of our designed peptides that inhibit the potentiation of transferrin on thrombin and FXIIa. Thus, low temperature and hypoxia upregulated transferrin expression-promoted hypercoagulability. Our data suggest that targeting the transferrin-coagulation pathway is a novel and potentially powerful strategy against thromboembolic events caused by harmful environmental factors under high-altitude conditions.

An inhibitor of leukotriene-A4 hydrolase from bat salivary glands facilitates virus infection.

SignificanceAn immunosuppressant protein (MTX), which facilitates virus infection by inhibiting leukotriene A4 hydrolase (LTA4H) to produce the lipid chemoattractant leukotriene B4 (LTB4), was identified and characterized from the submandibular salivary glands of the bat Myotis pilosus. To the best of our knowledge, this is a report of an endogenous LTA4H inhibitor in animals. MTX was highly concentrated in the bat salivary glands, suggesting a mechanism for the generation of immunological privilege and immune tolerance and providing evidence of viral shedding through oral secretions. Moreover, given that the immunosuppressant MTX selectively inhibited the proinflammatory activity of LTA4H, without affecting its antiinflammatory activity, MTX might be a potential candidate for the development of antiinflammatory drugs by targeting the LTA4-LTA4H-LTB4 inflammatory axis.

Shrew's venom quickly causes circulation disorder, analgesia and hypokinesia.

Multiple representatives of eulipotyphlan mammals such as shrews have oral venom systems. Venom facilitates shrews to hunt and/or hoard preys. However, little is known about their venom composition, and especially the mechanism to hoard prey in comatose states for meeting their extremely high metabolic rates. A toxin (BQTX) was identified from venomous submaxillary glands of the shrew Blarinella quadraticauda. BQTX is specifically distributed and highly concentrated (~ 1% total protein) in the organs. BQTX shares structural and functional similarities to toxins from snakes, wasps and snails, suggesting an evolutional relevancy of venoms from mammalians and non-mammalians. By potentiating thrombin and factor-XIIa and inhibiting plasmin, BQTX induces acute hypertension, blood coagulation and hypokinesia. It also shows strong analgesic function by inhibiting elastase. Notably, the toxin keeps high plasma stability with a 16-h half-life in-vivo, which likely extends intoxication to paralyze or immobilize prey hoarded fresh for later consumption and maximize foraging profit.

Hypermethylation of EFEMP1 in the Hippocampus May Be Related to the Deficit in Spatial Memory of Rat Neonates Triggered by Repeated Administration of Propofol.

It has been confirmed that repeated application of propofol, as an intravenous and short-fast-acting anesthetic, in neonatal animals or humans may produce long-term deficits in cognitive functions. With the aim of explaining the neurotoxic effects of repeated administration of propofol on neonatal rat pups from P7 to P9 especially from an epigenetic perspective, the present study used the Morris water maze to detect cognitive deficits in spatial learning and memory, Sequenom methylation on the CpG island located in the promoter region of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) to assess the methylation level of this region, and Western blot to measure the expression of EFEMP1, TIMP-3, and MMP-9. As the results have shown, repeated propofol administration on neonatal rats caused significant systemic growth retardation, impairment of spatial learning and memory, and hypermethylation of the CpG sites in the promoter region of EFEMP1 accompanied by lower expression of EFEMP1 and TIMP-3 and enhanced expression of MMP-9. These data suggest that repeated propofol administration in neonatal rats may generate hypermethylation in the promoter region of EFEMP1 which results in downregulation of the expression of EFEMP1 and tissue inhibitor of metalloproteinase-3 (TIMP-3) but upregulation of the expression of matrix metalloproteinase-9 (MMP-9), which together may affect the stability of ECM to hamper the development of the central nervous system and therefore lead to deficits in cognitive functions.

Knockdown of MCM10 Gene Impairs Glioblastoma Cell Proliferation, Migration and Invasion and the Implications for the Regulation of Tumorigenesis.

Minichromosome maintenance 10 (MCM10) plays an important role in DNA replication and is expressed in a variety of tumors, including glioma. However, its role and mechanism in glioma remain elusive. The purpose of this study was to examine the molecular function of MCM10 in glioblastoma cell lines in vitro and to further investigate the molecular mechanisms in the network mediated by MCM10. Cell proliferation, invasion, and migration were investigated in the absence of MCM10 mediated by RNA interference (RNAi) in U87 and U251 cell lines. Microarray data were obtained from U87 cells infected with a lentivirus expressing a small interfering RNA (siRNA) targeting MCM10, and ingenuity pathway analysis (IPA) was performed. Molecular signaling pathways, gene functions, and upstream and downstream regulatory genes and networks were analyzed. MCM10 was positively stained in human glioblastoma multiforme (GBM) samples according to immunohistochemistry. Silencing MCM10 in U87 and U251 cells significantly reduced cell proliferation, migration, and invasion. In U87 cells transfected with MCM10, 274 genes were significantly upregulated, while 313 genes were downregulated. IPA revealed that MCM10 is involved in the IGF-1 signaling pathway, and calcitriol appears to be a significant upstream regulator of MCM10. Other factors, such as TWIST1 and Stat3, also interact within the MCM10-mediated network. Our data indicate that MCM10 is involved in the regulation of GBM in vitro and may provide more evidence for understanding the molecular mechanisms of this fatal disease.

Prognostic value of H3K27me3 in children with ependymoma.

OBJECTIVE: To investigate the expression of H3K27me3 in different anatomical sites and analyze its prognostic value in children with ependymoma. METHODS: A total of 188 children diagnosed with ependymoma were admitted to the Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, between 2012 and 2017, and regular follow-up was conducted. Expression of H3K27me3 was analyzed by immunohistochemistry and scored semiquantitatively. The prognostic correlation was analyzed by Kaplan-Meier and Cox regression survival analyses. RESULTS: Of the 188 children with ependymoma, 61.7% were male, and the median and average age was five years (0-17 years) and 6.26 years, respectively. There were 65 cases of supratentorial ependymoma, 115 cases of infratentorial ependymoma, and 8 cases of spinal cord ependymoma. The median follow-up time was 39.95 months (0.3-90.19 months). Five-year progression-free survival (PFS) and overall survival (OS) were 48.5% and 61.4%, respectively. Kaplan-Meier univariate survival analysis showed that H3K27me3 expression had significant effects on PFS (P = 0.0003) and OS (P < 0.0001) in infratentorial ependymoma, but only affected OS (P = 0.03) in supratentorial ependymoma. CONCLUSION: In Chinese children, infratentorial ependymoma with incomplete resection and no adjuvant radiotherapy is associated with poor OS. On the other hand, low expression of H3K27me3 indicates poor prognosis of infratentorial ependymoma, but it has no significant prognostic value for supratentorial ependymoma. In addition, high expression of H3K27me3 in spinal ependymoma may indicate a better prognosis.

Establishing reproducible predictors of cerebellar mutism syndrome based on pre-operative imaging.

PURPOSE: To establish some explicit, feasible, and reproducible predictors for CMS. MATERIALS AND METHODS: This study was a retrospective case study. Data were obtained from 82 patients with medulloblastoma at a single center, Beijing Tiantan Hospital. Based on medical records, we created two independent samples: the CMS group comprising 23 patients and the non-CMS group comprising 23 patients. Pre-operative imaging was studied by performing quantitative assessments of specific indicators. RESULTS: The CMS group showed greater differences in pre-operative imaging data with the non-CMS group. The Aaxi/daxi ratio in pre-operative MR imaging captured in the axial plane was used to quantify the compression of the cerebellum and brainstem, and significant differences were observed between the CMS group and non-CMS group (p = 0.0002). In the sagittal plane, Dsag*dsag was used to quantify the area of the tumor that invaded the brainstem, and significant differences were observed between the two groups (p = 0.0003). In the coronal plane, Acor/dcor was used to quantify the compression of the upper functional brain region, and significant differences were noted between the two groups (p = 0.0219). Additionally, Evans' index was introduced to quantify the degree of hydrocephalus. The CMS group tended to show an increased Evans' index (p = 0.0027). CONCLUSION: Based on pre-operative imaging data, some reproducible predictors, such as Aaxi/daxi, Dsag*dsag, Acor/dcor, and Evans' index, were established.

A20 rescues hepatocytes from apoptosis through the NF-κB signaling pathway in rats with acute liver failure.

Background: Acute liver failure (ALF) is a disease of acute derangements in the hepatic synthetic function with defects involving innate immune responses, which was reported to be negatively regulated by tumor necrosis factor α-induced protein 3 (A20). Herein, the present study was conducted to investigate the effects the A20 protein on the proliferation and apoptosis of hepatocytes through the nuclear factor (NF)-κB signaling pathway in the rat models simulating ALF.Methods: Male Wistar rats were used to simulate ALF in the model rats. Next, the positive expression of A20 and Caspase-3 proteins was measured in liver tissues. Rat hepatocytes were separated and subjected to pyrrolidine dithiocarbamate (PDTC, inhibitor of NF-κB pathway) or A20 siRNA. Additionally, both mRNA and protein levels of A20, NF-κB, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and receptor-interacting protein 1 (RIP1) were determined. Finally, we detected the hepatocyte proliferation, cell cycle entry, and apoptosis.Results: ALF rats displayed a lower positive expression of A20 protein and a higher expression of Caspase-3 protein. Furthermore, A20 was down-regulated, while NF-κB, TRAF6, and RIP1 were all up-regulated in ALF rats. Notably, A20 inhibited activation of NF-κB signaling pathway. The blockade of NF-κB signaling pathway enhanced proliferation and cell cycle progression of hepatocytes, whereas inhibited apoptosis of hepatocytes. On the contrary, A20 siRNA reversed the above situation.Conclusion: A20 inhibits apoptosis of hepatocytes and promotes the proliferation through the NF-κB signaling pathway in ALF rats, potentially providing new insight into the treatment of ALF.