Synchronous Diagnosis of Respiratory Viruses Variants via Receptonics Based on Modeling Receptor-Ligand Dynamics.

The transmission and pathogenesis of highly contagious fatal respiratory viruses are increasing, and the need for an on-site diagnostic platform has arisen as an issue worldwide. Furthermore, as the spread of respiratory viruses continues, different variants have become the dominant circulating strains. To prevent virus transmission, the development of highly sensitive and accurate on-site diagnostic assays is urgently needed. Herein, a facile diagnostic device is presented for multi-detection based on the results of detailed receptor-ligand dynamics simulations for the screening of various viral strains. The novel bioreceptor-treated electronics (receptonics) device consists of a multichannel graphene transistor and cell-entry receptors conjugated to N-heterocyclic carbene (NHC). An ultrasensitive multi-detection performance is achieved without the need for sample pretreatment, which will enable rapid diagnosis and prevent the spread of pathogens. This platform can be applied for the diagnosis of variants of concern in clinical respiratory virus samples and primate models. This multi-screening platform can be used to enhance surveillance and discriminate emerging virus variants before they become a severe threat to public health.

Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV.

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Suramin binds and inhibits infection of SARS-CoV-2 through both spike protein-heparan sulfate and ACE2 receptor interactions.

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19.

Coordinated neurostimulation promotes circuit rewiring and unlocks recovery after spinal cord injury.

Functional recovery after incomplete spinal cord injury depends on the effective rewiring of neuronal circuits. Here, we show that selective chemogenetic activation of either corticospinal projection neurons or intraspinal relay neurons alone led to anatomically restricted plasticity and little functional recovery. In contrast, coordinated stimulation of both supraspinal centers and spinal relay stations resulted in marked and circuit-specific enhancement of neuronal rewiring, shortened EMG latencies, and improved locomotor recovery.

Monocytes as suitable carriers for dissemination of dengue viral infection.

Dengue viruses (DENVs) exploit monocytes and macrophages for tropism and replication, therefore, establishing a long-term reservoir. However, their roles in dengue pathogenesis remains unclear. Here, using the human monocytic cell line THP-1, human primary monocytes, and non-human primate models, we show that DENV-infected monocytes represent suitable carriers for circulatory viral dissemination. Monocyte-derived macrophages expressing M2 surface markers at the gene level efficiently replicated, while the productivity of monocyte replication was low. However, attachment of DENVs to the cellular surface of monocytes was similar to that of macrophages. Furthermore, after differentiation with type-2 cytokines, DENV-attached monocytes could replicate DENVs. Productive DENV infection was confirmed by intravenous injection of DENVs into nonhuman primate model, in which, DENV attachment to monocytes was positively correlated with viremia. These results provide insight into the role of circulating monocytes in DENV infection, suggesting that monocytes directly assist in DENV dissemination and replication during viremia and could be applied to design antiviral intervention.

The Chimeric Adenovirus (Ad5/35) Expressing Engineered Spike Protein Confers Immunity against SARS-CoV-2 in Mice and Non-Human Primates.

Several COVID-19 platforms have been licensed across the world thus far, but vaccine platform research that can lead to effective antigen delivery is still ongoing. Here, we constructed AdCLD-CoV19 that could modulate humoral immunity by harboring SARS-CoV-2 antigens onto a chimeric adenovirus 5/35 platform that was effective in cellular immunity. By replacing the S1/S2 furin cleavage sequence of the SARS-CoV-2 Spike (S) protein mounted on AdCLD-CoV19 with the linker sequence, high antigen expression was confirmed in various cell lines. The high levels of antigen expression contributed to antigen-specific antibody activity in mice and non-human primates (NHPs) with a single vaccination of AdCLD-CoV19. Furthermore, the adenovirus-induced Th1 immune response was specifically raised for the S protein, and these immune responses protected the NHP against live viruses. While AdCLD-CoV19 maintained neutralizing antibody activity against various SARS-CoV-2 variants, it was reduced to single vaccination for ß and ο variants, and the reduced neutralizing antibody activity was restored with booster shots. Hence, AdCLD-CoV19 can prevent SARS-CoV-2 with a single vaccination, and the new vaccine administration strategy that responds to various variants can maintain the efficacy of the vaccine.

Cynomolgus Macaque Model for COVID-19 Delta Variant.

With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, which are randomly mutated, the dominant strains in regions are changing globally. The development of preclinical animal models is imperative to validate vaccines and therapeutics against SARS-CoV-2 variants. The objective of this study was to develop a non-human primate (NHP) model for SARS-CoV-2 Delta variant infection. Cynomolgus macaques infected with Delta variants showed infectious viruses and viral RNA in the upper (nasal and throat) and lower respiratory (lung) tracts during the acute phase of infection. After 3 days of infection, lesions consistent with diffuse alveolar damage were observed in the lungs. For cellular immune responses, all macaques displayed transient lymphopenia and neutrophilia in the early stages of infection. SARS-CoV-2 Delta variant spike protein-specific IgM, IgG, and IgA levels were significantly increased in the plasma of these animals 14 days after infection. This new NHP Delta variant infection model can be used for comparative analysis of the difference in severity between SARS-CoV-2 variants of concern and may be useful in the efficacy evaluation of vaccines and universal therapeutic drugs for mutations.

Germinal Center-Induced Immunity Is Correlated With Protection Against SARS-CoV-2 Reinfection But Not Lung Damage.

Germinal centers (GCs) elicit protective humoral immunity through a combination of antibody-secreting cells and memory B cells, following pathogen invasion or vaccination. However, the possibility of a GC response inducing protective immunity against reinfection following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown. We found GC activity was consistent with seroconversion observed in recovered macaques and humans. Rechallenge with a different clade of virus resulted in significant reduction in replicating virus titers in respiratory tracts in macaques with high GC activity. However, diffuse alveolar damage and increased fibrotic tissue were observed in lungs of reinfected macaques. Our study highlights the importance of GCs developed during natural SARS-CoV-2 infection in managing viral loads in subsequent infections. However, their ability to alleviate lung damage remains to be determined. These results may improve understanding of SARS-CoV-2-induced immune responses, resulting in better coronavirus disease 2019 (COVID-19) diagnosis, treatment, and vaccine development.

Host- and Species-Dependent Quasispecies Divergence of Severe Acute Respiratory Syndrome Coronavirus-2 in Non-human Primate Models.

Recently, newly emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been continuously reported worldwide. However, the precise evaluation of SARS-CoV-2 microevolution in host is very limited because the exact genetic information of infected virus could not be acquired in human researches. In this report, we performed deep sequencing for seed virus and SARS-CoV-2 isolated in eight cynomolgus and rhesus macaques at 3 days postinoculation and evaluated single-nucleotide polymorphisms (SNPs) in SARS-CoV-2 by variant analysis. A total of 69 single-nucleotide variants (SNVs) were present in the 5'-untranslated region (UTR), 3'-UTR, ORF1ab, S, ORF3a, ORF8, and N genes of the seed virus passaged in VERO cells. Between those present on the seed virus and those on each SARS-CoV-2 isolated from the lungs of the macaques, a total of 29 variants was identified in 4 coding proteins (ORF1ab, S, ORF3a, and N) and non-coding regions (5'- and 3'-UTR). Variant number was significantly different according to individuals and ranged from 2 to 11. Moreover, the average major frequency variation was identified in six sites between the cynomolgus monkeys and rhesus macaques. As with diverse SNPs in SARS-CoV-2, the values of viral titers in lungs were significantly different according to individuals and species. Our study first revealed that the genomes of SARS-CoV-2 differ according to individuals and species despite infection of the identical virus in non-human primates (NHPs). These results are important for the interpretation of longitudinal studies evaluating the evolution of the SARS-CoV-2 in human beings and development of new diagnostics, vaccine, and therapeutics targeting SARS-CoV-2.

Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates.

Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), various vaccines are being developed, with most vaccine candidates focusing on the viral spike protein. Here, we developed a previously unknown subunit vaccine comprising the receptor binding domain (RBD) of the spike protein fused with the tetanus toxoid epitope P2 (RBD-P2) and tested its efficacy in rodents and nonhuman primates (NHPs). We also investigated whether the SARS-CoV-2 nucleocapsid protein (N) could increase vaccine efficacy. Immunization with N and RBD-P2 (RBDP2/N) + alum increased T cell responses in mice and neutralizing antibody levels in rats compared with those obtained using RBD-P2 + alum. Furthermore, in NHPs, RBD-P2/N + alum induced slightly faster SARS-CoV-2 clearance than that induced by RBD-P2 + alum, albeit without statistical significance. Our study supports further development of RBD-P2 as a vaccine candidate against SARS-CoV-2. Also, it provides insights regarding the use of N in protein-based vaccines against SARS-CoV-2.

Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates.

The unprecedented and rapid spread of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) has motivated the need for a rapidly producible and scalable vaccine. Here, we developed a synthetic soluble SARS-CoV-2 spike (S) DNA-based vaccine candidate, GX-19. In mice, immunization with GX-19 elicited not only S-specific systemic and pulmonary antibody responses but also Th1-biased T cell responses in a dose-dependent manner. GX-19-vaccinated nonhuman primates seroconverted rapidly and exhibited a detectable neutralizing antibody response as well as multifunctional CD4+ and CD8+ T cell responses. Notably, when the immunized nonhuman primates were challenged at 10 weeks after the last vaccination with GX-19, they had reduced viral loads in contrast to non-vaccinated primates as a control. These findings indicate that GX-19 vaccination provides a durable protective immune response and also support further development of GX-19 as a vaccine candidate for SARS-CoV-2.

Molecular evolution of dengue virus types 1 and 4 in Korean travelers.

Dengue virus (DV) is a mosquito-borne virus that is endemic to many tropical and subtropical areas. Recently, the annual incidence of DV infection has increased worldwide, including in Korea, due to global warming and increased global travel. We therefore sought to characterize the molecular and evolutionary features of DV-1 and DV-4 isolated from Korean overseas travelers. We used phylogenetic analysis based on the full coding region to classify isolates of DV-1 in Korea into genotype I (43251, KP406802), genotype IV (KP406803), and genotype V (KP406801). In addition, we found that strains of DV-4 belonged to genotype I (KP406806) and genotype II (43257). Evidence of positive selection in DV-1 strains was identified in the C, prM, NS2A, and NS5 proteins, whereas DV-4 showed positive selection only in the non-structural proteins NS2A, NS3, and NS5. The substitution rates per site per year were 5.58 × 10-4 and 6.72 × 10-4 for DV-1 and DV-4, respectively, and the time of the most recent common ancestor was determined using the Bayesian skyline coalescent method. In this study, the molecular, phylogenetic, and evolutionary characteristics of Korean DV-1 and DV-4 isolates were evaluated for the first time.

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

Comparison of high flow nasal oxygen and conventional nasal cannula during gastrointestinal endoscopic sedation in the prone position: a randomized trial.

PURPOSE: Deep sedation for endoscopic retrograde cholangiopancreatography (ERCP) can be challenging in elderly patients in the prone position. This study investigated the effect of a high flow nasal oxygen (HFNO) delivery system on oxygenation in this procedure compared with that of conventional nasal cannula oxygen administration. METHODS: A prospective randomized trial was conducted using HFNO and conventional nasal cannula in patients undergoing ERCP in the prone position. For each patient, the lowest oxygen saturation (SpO2), the incidence of hypoxemia defined as an SpO2 below 90%, and interruptions due to airway interventions were recorded during the procedure. RESULTS: The lowest mean (standard deviation) SpO2 recorded during the procedure was higher in the HFNO group than in the conventional control group [99.8 (0.6)% vs 95.1 (7.3)%; mean difference, 4.7%; 95% confidence interval, 2.3% to 7.1%; P Group x Time < 0.001]. While the lowest SpO2 during the procedure was lower than the baseline SpO2 in the control group, the lowest SpO2 during the procedure was higher than the baseline SpO2 in the HFNO group. Hypoxemia occurred only in the control group (n = 7; 19%; P = 0.01). Procedural interruptions, including discontinuation of sedation, patient stimulation, and jaw thrusting, occurred only in the control group (n = 9 [25%], n = 10 [28%], and n = 10 [28%] cases, respectively; P = 0.001 for each). CONCLUSION: In contrast to conventional nasal cannula, high flow nasal oxygen provided adequate oxygenation without causing procedural interruptions during ERCP, suggesting that HFNO may be used as a standard oxygen delivery method during these procedures. TRIAL REGISTRATION: www.ClinicalTrials.gov (NCT03872674); registered 11 March 2019.

RéSUMé: OBJECTIF: La sédation profonde pour cholangiopancréatographie rétrograde endoscopique (CPRE) peut être difficile à réaliser chez des patients âgés en position ventrale. Cette étude a exploré l'effet d'un système d'oxygénothérapie nasale à haut débit (ONHD) sur l'oxygénation pendant cette intervention par rapport à l'administration conventionnelle d'oxygène via une lunette nasale. MéTHODE: Une étude randomisée prospective a été réalisée en utilisant une ONHD ou une lunette nasale conventionnelle chez des patients subissant une CPRE en position ventrale. Pour chaque patient, la saturation en oxygène (SpO2) la plus basse, l'incidence d'hypoxémie définie en tant qu'une SpO2 inférieure à 90 %, et les interruptions provoquées par des interventions au niveau des voies aériennes ont été enregistrées au cours de l'intervention. RéSULTATS: La SpO2 moyenne (écart type) la plus basse enregistrée pendant l'intervention était plus élevée dans le groupe ONHD que dans le groupe témoin conventionnel [99,8 (0,6) % vs 95,1 (7,3) %; différence moyenne, 4,7%; intervalle de confiance 95 %, 2,3 % à 7,1 %; P Groupe x Temps < 0,001]. Alors que la SpO2 la plus basse pendant l'intervention était plus basse que la SpO2 de base dans le groupe témoin, la SpO2 la plus basse pendant l'intervention était plus élevée que la SpO2 de base dans le groupe ONHD. L'hypoxémie n'est survenue que dans le groupe témoin (n = 7; 19 %; P = 0,01). Il n'y a eu d'interruptions de l'intervention, y compris la cessation de la sédation, la stimulation du patient et le déplacement de la mâchoire inférieure vers l'avant, que dans le groupe témoin (n = 9 [25 %], n = 10 [28 %], et n = 10 [28 %] cas, respectivement; P = 0,001 pour chaque intervention). CONCLUSION: Comparativement à une lunette nasale conventionnelle, l'oxygénothérapie nasale à haut débit a procuré une oxygénation adéquate sans provoquer d'interruptions de l'intervention pendant une CPRE, suggérant que cette modalité pourrait être utilisée comme méthode standard d'oxygénothérapie pendant de telles interventions. ENREGISTREMENT DE L'éTUDE: www.ClinicalTrials.gov (NCT03872674); enregistrée le 11 mars 2019.

Bile duct ligation of C57BL/6 mice as a model of hepatic encephalopathy.

BACKGROUND: Bile duct ligation (BDL) has been used for experimental research on hepatic encephalopathy (HE) caused by chronic liver disease. However, little research has been done on a BDL model in C57BL/6 mouse. Therefore, we evaluated the suitability of a BDL model in C57BL/6 mouse for the study of HE and determined which behavioral tests are appropriate for the identification of HE in this model. METHODS: Twelve to fourteen-week-old male C57BL/6 mice were randomly assigned to either sham group or BDL group. Histological changes in liver were confirmed by hematoxylin/eosin and Masson's trichrome staining. Liver function alterations were detected by alanine aminotransferase (ALT) and ammonia levels. To identify behavioral changes, open field, elevated plus maze, novel object recognition, and passive avoidance tests were performed. RESULTS: Inflammatory liver injury and fibrosis were observed 14 days after BDL. ALT and ammonia levels were significantly higher in BDL group than in sham group. There were no differences in general locomotor activity or anxiety between the groups. No difference was observed between these two groups in the novel object recognition test, but BDL group showed significant learning/memory impairment in the passive avoidance test compared to sham group. CONCLUSIONS: Fourteen days of BDL in 12-14-week-old male C57BL/6 mice is a clinically relevant model for HE, as these mice have liver fibrosis with impaired liver function, hyperammonemia, and learning/memory impairment. Passive avoidance can be used as the major behavioral test in this model of HE.

Sleep disturbance and delirium in patients with acromegaly in the early postoperative period after transsphenoidal pituitary surgery.

Sleep disturbance is a common comorbidity among patients with acromegaly [patients with growth hormone (GH)-secreting tumor] due to somatotropic axis change and sleep apnea. However, no previous studies exist concerning sleep disturbance and delirium in the early postoperative period in patients with acromegaly undergoing transsphenoidal tumor surgery. Herein, we aimed to compare the incidence of postoperative sleep disturbance and delirium in the early postoperative period between patients with GH-secreting and nonfunctioning pituitary tumors.We retrospectively reviewed the medical records of 1286 patients (969 with nonfunctioning and 317 with GH-secreting tumors) without history of psychological disease and sedative or antipsychotic use. We examined the use of antipsychotics/sedatives and findings of psychology consultation within the first postoperative week. Only patients with sleep disturbance noted in medical records were considered to have postoperative sleep disturbance. Patients with an Intensive Care Delirium Screening Checklist score of 4 or more were considered to have postoperative delirium.The incidence of postoperative sleep disturbance was higher in the GH-secreting group than in the nonfunctioning tumor group (2/969 [0.2%] vs 6/317 [1.9%]; P = .004; odds ratio = 9.328 [95% confidence interval, 1.873-46.452]). Univariable regression analysis showed that only diagnosis (GH-secreting tumor or nonfunctioning tumor) was a risk factor for sleep disturbance, and not sex, age, body mass index, American Society of Anesthesiologists physical status score, surgery duration, anesthesia duration, anesthesia type, tumor size, cavernous sinus invasion, or bleeding. The incidence of postoperative delirium was comparable between the 2 groups (6/969 [0.6%] vs 0/317 [0%]; P = .346).Patients with acromegaly showed increased incidence of sleep disturbance than those with nonfunctioning tumors in the early postoperative period after transsphenoidal tumor surgery. A prospective study evaluating sleep quality in patients with GH-secreting tumors in the early postoperative period could be conducted based on our findings.

Comparison of Biocompatibility of Calcium Silicate-Based Sealers and Epoxy Resin-Based Sealer on Human Periodontal Ligament Stem Cells.

The aim of this study was to evaluate the biocompatibility of calcium silicate-based sealers (CeraSeal and EndoSeal TCS) and epoxy resin-based sealer (AH-Plus) in terms of cell viability, inflammatory response, expression of mesenchymal phenotype, osteogenic potential, cell attachment, and morphology, of human periodontal ligament stem cells (hPDLSCs). hPDLSCs were acquired from the premolars (n = 4) of four subjects, whose ages extended from 16 to 24 years of age. Flow cytometry analysis showed stemness of hPDLSCs was maintained in all materials. In cell viability test, AH-Plus showed the lowest cell viability, and CeraSeal showed significantly higher cell viability than others. In ELISA test, AH-Plus showed higher expression of IL-6 and IL-8 than calcium silicate-based sealers. In an osteogenic potential test, AH-Plus showed a lower expression level than other material; however, EndoSeal TCS showed a better expression level than others. All experiments were repeated at least three times per cell line. Scanning electronic microscopy studies showed low degree of cell proliferation on AH-Plus, and high degree of cell proliferation on calcium silicate-based sealers. In this study, calcium silicate-based sealers appear to be more biocompatible and less cytotoxic than epoxy-resin based sealers.

Transient Lymphopenia and Interstitial Pneumonia With Endotheliitis in SARS-CoV-2-Infected Macaques.

Using a reliable primate model is critical for developing therapeutic advances to treat humans infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we exposed macaques to high titers of SARS-CoV-2 via combined transmission routes. We observed acute interstitial pneumonia with endotheliitis in the lungs of all infected macaques. All macaques had a significant loss of total lymphocytes during infection, which were restored over time. These data show that SARS-CoV-2 causes a coronavirus disease 2019 (COVID-19)-like disease in macaques. This new model could investigate the interaction between SARS-CoV-2 and the immune system to test therapeutic strategies.

Immunohistochemical analysis of periostin in the hearts of Lewis rats with experimental autoimmune myocarditis.

Periostin plays a critical role in tissue regeneration and homeostasis. The aim of this study was to evaluate the changes in periostin levels in the hearts of rats with experimental autoimmune myocarditis (EAM). Western blot analysis revealed that the expression levels of periostin and alpha-smooth muscle actin were significantly increased at day 14 post-immunization. Immunohistochemical analysis indicated that periostin was expressed in macrophages and fibroblasts in the hearts of EAM-induced rats. In conclusion, these results suggest that increased periostin expression in macrophages and fibroblasts promotes cardiac fibrosis in EAM-induced rats, potentially by enhancing immune cell infiltration. Therefore, periostin should be further investigated as a candidate therapeutic target for myocarditis.