Infection with wild-type SARS-CoV-2 elicits broadly neutralizing and protective antibodies against omicron subvariants.

The omicron variants of SARS-CoV-2 have substantial ability to escape infection- and vaccine-elicited antibody immunity. Here, we investigated the extent of such escape in nine convalescent patients infected with the wild-type SARS-CoV-2 during the first wave of the pandemic. Among the total of 476 monoclonal antibodies (mAbs) isolated from peripheral memory B cells, we identified seven mAbs with broad neutralizing activity to all variants tested, including various omicron subvariants. Biochemical and structural analysis indicated the majority of these mAbs bound to the receptor-binding domain, mimicked the receptor ACE2 and were able to accommodate or inadvertently improve recognition of omicron substitutions. Passive delivery of representative antibodies protected K18-hACE2 mice from infection with omicron and beta SARS-CoV-2. A deeper understanding of how the memory B cells that produce these antibodies could be selectively boosted or recalled can augment antibody immunity against SARS-CoV-2 variants.

Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge during the global pandemic and may facilitate escape from current antibody therapies and vaccine protection. Here we showed that the South African variant B.1.351 was the most resistant to current monoclonal antibodies and convalescent plasma from coronavirus disease 2019 (COVID-19)-infected individuals, followed by the Brazilian variant P.1 and the United Kingdom variant B.1.1.7. This resistance hierarchy corresponded with Y144del and 242-244del mutations in the N-terminal domain and K417N/T, E484K, and N501Y mutations in the receptor-binding domain (RBD) of SARS-CoV-2. Crystal structure analysis of the B.1.351 triple mutant (417N-484K-501Y) RBD complexed with the monoclonal antibody P2C-1F11 revealed the molecular basis for antibody neutralization and escape. B.1.351 and P.1 also acquired the ability to use mouse and mink ACE2 receptors for entry. Our results demonstrate major antigenic shifts and potential broadening of the host range for B.1.351 and P.1 variants, which poses serious challenges to current antibody therapies and vaccine protection.

Human neutralizing antibodies elicited by SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global health emergency that is in urgent need of intervention1-3. The entry of SARS-CoV-2 into its target cells depends on binding between the receptor-binding domain (RBD) of the viral spike protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2)2,4-6. Here we report the isolation and characterization of 206 RBD-specific monoclonal antibodies derived from single B cells from 8 individuals infected with SARS-CoV-2. We identified antibodies that potently neutralize SARS-CoV-2; this activity correlates with competition with ACE2 for binding to RBD. Unexpectedly, the anti-SARS-CoV-2 antibodies and the infected plasma did not cross-react with the RBDs of SARS-CoV or Middle East respiratory syndrome-related coronavirus (MERS-CoV), although there was substantial plasma cross-reactivity to their trimeric spike proteins. Analysis of the crystal structure of RBD-bound antibody revealed that steric hindrance inhibits viral engagement with ACE2, thereby blocking viral entry. These findings suggest that anti-RBD antibodies are largely viral-species-specific inhibitors. The antibodies identified here may be candidates for development of clinical interventions against SARS-CoV-2.

Key gp120 Glycans Pose Roadblocks to the Rapid Development of VRC01-Class Antibodies in an HIV-1-Infected Chinese Donor.

VRC01-class antibodies neutralize diverse HIV-1 strains by targeting the conserved CD4-binding site. Despite extensive investigations, crucial events in the early stage of VRC01 development remain elusive. We demonstrated how VRC01-class antibodies emerged in a Chinese donor by antigen-specific single B cell sorting, structural and functional studies, and longitudinal antibody and virus repertoire analyses. A monoclonal antibody DRVIA7 with modest neutralizing breadth was isolated that displayed a subset of VRC01 signatures. X-ray and EM structures revealed a VRC01-like angle of approach, but less favorable interactions between the DRVIA7 light-chain CDR1 and the N terminus with N276 and V5 glycans of gp120. Although the DRVIA7 lineage was unable to acquire broad neutralization, longitudinal analysis revealed a repertoire-encoded VRC01 light-chain CDR3 signature and VRC01-like neutralizing heavy-chain precursors that rapidly matured within 2 years. Thus, light chain accommodation of the glycan shield should be taken into account in vaccine design targeting this conserved site of vulnerability.

MolProphet: A One-Stop, General Purpose, and AI-Based Platform for the Early Stages of Drug Discovery.

Artificial intelligence (AI) is an effective tool to accelerate drug discovery and cut costs in discovery processes. Many successful AI applications are reported in the early stages of small molecule drug discovery. However, most of those applications require a deep understanding of software and hardware, and focus on a single field that implies data normalization and transfer between those applications is still a challenge for normal users. It usually limits the application of AI in drug discovery. Here, based on a series of robust models, we formed a one-stop, general purpose, and AI-based drug discovery platform, MolProphet, to provide complete functionalities in the early stages of small molecule drug discovery, including AI-based target pocket prediction, hit discovery and lead optimization, and compound targeting, as well as abundant analyzing tools to check the results. MolProphet is an accessible and user-friendly web-based platform that is fully designed according to the practices in the drug discovery industry. The molecule screened, generated, or optimized by the MolProphet is purchasable and synthesizable at low cost but with good drug-likeness. More than 400 users from industry and academia have used MolProphet in their work. We hope this platform can provide a powerful solution to assist each normal researcher in drug design and related research areas. It is available for everyone at https://www.molprophet.com/.

Effect of polystyrene nanoplastics exposure on gene expression and pathogenesis of zoonotic pathogen, Edwardsiella piscicida.

A surge in the number of anthropogenic pollutants has been caused by increasing industrial activities. Nanoplastics are spotlighted as a new aquatic pollutant that are a threat to microbes and larger organisms. Our previous study showed that the subinhibitory concentrations of aquatic pollutants such as phenol and formalin act as signaling molecules and modulate global gene expression and metabolism. In this study, we aimed to investigate the impact of a new type of anthropogenic contaminant, polystyrene (PS) nanoplastics, on the expression of key virulence factors in zoonotic pathogen Edwardsiella piscicida and the assessment of potential changes in the susceptibility of zebrafish as a model host. The TEM data indicated a noticeable change in the cell membrane indicating that PS particles were possibly entering the bacterial cells. Transcriptome analyses performed to identify the differentially expressed genes upon PS exposure revealed that the genes involved in major virulence factor type VI secretion system (T6SS) were down-regulated. However, the expression of T6SS-related genes was recovered from the PS adapted E. piscicida when nanoplastics are free. This demonstrated the hypervirulence of pathogen in infection assays with both cell lines and in vivo zebrafish model. Therefore, this study provides experimental evidence elucidating the direct regulatory impact of nanoplastics influx into aquatic ecosystems on fish pathogenic bacteria, notably influencing the expression of virulence factors.

iDMer: an integrative and mechanism-driven response system for identifying compound interventions for sudden virus outbreak.

Emerging viral infections seriously threaten human health globally. Several challenges exist in identifying effective compounds against viral infections: (1) at the initial stage of a new virus outbreak, little information, except for its genome information, may be available; (2) although the identified compounds may be effective, they may be toxic in vivo and (3) cytokine release syndrome (CRS) triggered by viral infections is the primary cause of mortality. Currently, an integrative tool that takes all those aspects into consideration for identifying effective compounds to prevent viral infections is absent. In this study, we developed iDMer, as an integrative and mechanism-driven response system for addressing these challenges during the sudden virus outbreaks. iDMer comprises three mechanism-driven compound identification modules, that is, a virus-host interaction-oriented module, an autophagy-oriented module and a CRS-oriented module. As a one-stop integrative platform, iDMer incorporates compound toxicity evaluation and compound combination identification for virus treatment with clear mechanisms. iDMer was successfully tested on five viruses, including the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results indicated that, for all five tested viruses, compounds that were reported in the literature or experimentally validated for virus treatment were enriched at the top, demonstrating the generalized effectiveness of iDMer. Finally, we demonstrated that combinations of the individual modules successfully identified combinations of compounds effective for virus intervention with clear mechanisms.

Retinoic Acid Prevents the Neuronal Damage Through the Regulation of Parvalbumin in an Ischemic Stroke Model.

Ischemic stroke is a neurological disease that causes brain damage by increasing oxidative stress and ion imbalance. Retinoic acid is a major metabolite of vitamin A and regulates oxidative stress, calcium homeostasis, and cell death. Intracellular calcium is involved in neuronal growth and synaptic plasticity. Parvalbumin is a calcium-binding protein that is mainly expressed in brain. In this study, we investigated whether retinoic acid has neuroprotective effects by controlling intracellular calcium concentration and parvalbumin expression in ischemic brain damage. Middle cerebral artery occlusion (MCAO) was performed to induce cerebral ischemia. Retinoic acid (5 mg/kg) or vehicle was injected into the abdominal cavity for four days before surgery and cerebral cortices were collected 24 h after MCAO for further studies. MCAO damage induced neurological deficits and histopathological changes and decreased parvalbumin expression. However, retinoic acid treatment alleviated these changes. In cultured neurons, glutamate (5 mM) exposure induced neuronal cell death, increased intracellular calcium concentration, and decreased parvalbumin expression. Retinoic acid treatment attenuated these changes against glutamate toxicity in a dose-dependent manner. It also regulates glutamate induced change in bcl-2 and bax expression. The mitigation effects of retinoic acid were greater under non-transfection conditions than under parvalbumin siRNA transfection conditions. Our findings showed that retinoic acid modulates intracellular calcium concentration and parvalbumin expression and prevents apoptosis in ischemic brain injury. In conclusion, retinoic acid contributes to the preservation of neurons from ischemic stroke by controlling parvalbumin expression and apoptosis-related proteins.

Efficacy and safety of Kumpe access catheter for pre-percutaneous nephrolithotomy renal access in modified supine percutaneous nephrolithotomy.

INTRODUCTION: Traditionally, a pigtail catheter (PCN) is placed for preoperative renal access before performing percutaneous nephrolithotomy (PCNL). However, PCN can hamper the passage of the guidewire to the ureter, due to which, access tract can be lost. Therefore, Kumpe Access Catheter (KMP) has been proposed for preoperative renal access before PCNL. In this study, we analyzed the efficacy and safety of KMP for surgical outcomes in modified supine PCNL compared to those in PCN. MATERIALS AND METHODS: From July 2017 to December 2020, 232 patients underwent modified supine PCNL at a single tertiary center, of which 151 patients were enrolled in this study after excluding patients who underwent bilateral surgery, multiple punctures, or combined operations. Enrolled patients were divided into two groups according to the type of pre-PCNL nephrostomy catheter used: PCN versus KMP. A pre-PCNL nephrostomy catheter was selected based on the radiologist's preference. A single surgeon performed all PCNL procedures. Patient characteristics and surgical outcomes, including stone-free rate, operation time, radiation exposure time (RET), and complications, were compared between the two groups. RESULTS: Of the 151 patients, 53 underwent PCN placement, and 98 underwent KMP placement for pre-PCNL nephrostomy. Patient baseline characteristics were comparable between the two groups, except for the renal stone type and multiplicity. The operation time, stone-free rate, and complication rate were not significantly different between the two groups; however, RET was significantly shorter in the KMP group. CONCLUSION: The surgical outcomes of KMP placement were comparable to those of PCN and showed shorter RET during modified supine PCNL. Based on our results, we recommend KMP placement for pre-PCNL nephrostomy, particularly for reducing RET during supine PCNL.

A fourth dose of Omicron RBD vaccine enhances broad neutralization against SARS-CoV-2 variants including BA.1 and BA.2 in vaccinated mice.

The SARS-CoV-2 vaccines have been widely used to build an immunologic barrier in the population against the COVID-19 pandemic. However, a newly emerging Omicron variant, including BA.1, BA.1.1, BA.2, and BA.3 sublineages, largely escaped the neutralization of existing neutralizing antibodies (nAbs), even those elicited by three doses of vaccines. Here, we used the Omicron BA.1 RBD as a fourth dose of vaccine to induce potent Omicron-specific nAbs and evaluated the broadly neutralizing activities against SARS-CoV-2 variants. The BA.1-based vaccine was indeed prone to induce a strain-specific antibody response substantially cross-reactive with BA.2 sublineage, and yet triggered broad neutralization against SARS-CoV-2 variants when it was used in the sequential immunization with WT and other variant vaccines. These results demonstrated that the booster of Omicron RBD vaccine could be a rational strategy to enhance the broadly nAb response.

A Δ42PD1 fusion-expressing DNA vaccine elicits enhanced adaptive immune response to HIV-1: the key role of TLR4.

Since its discovery in the 1990s, the DNA vaccine has been of great interest because of its ability to elicit both humoral and cellular immune responses while showing relative advantages regarding producibility, stability and storage. However, when applied to human subjects, inadequate immunogenicity remains as the greatest challenge for the practical use of DNA vaccines. In this study, we generated a DNA vaccine Δ42PD1-P24 encoding a fusion protein comprised of the HIV-1 Gag p24 antigen and the extracellular domain of murine Δ42PD1, a novel endogenous Toll-like receptor 4 (TLR4) agonist. Using a mouse model, we found that Δ42PD1-P24 DNA vaccine elicited a higher antibody response and an increased number of IFN-γ-producing CD4 and CD8 T cells. Moreover, mice with Δ42PD1-P24 DNA vaccination were protected from a subcutaneous challenge with murine mesothelioma cells expressing the HIV-1 p24 antigen. Importantly, the Δ42PD1-mediated enhancement of immune responses was not observed in TLR4 knockout mice. Collectively, these data demonstrate that the immunogenicity and efficacy of DNA vaccines could be improved by the fusion of the extracellular domain of Δ42PD1 to target the immunogen to dendritic cells.

Identification and application of a pair of noncompeting monoclonal antibodies broadly binding to the nucleocapsid proteins of SARS-CoV-2 variants including Omicron.

The SARS-CoV-2 nucleocapsid protein (NP) is an important indicator for the virus infection, highlighting the crucial role of NP-specific monoclonal antibodies (mAbs) used in multiple biochemical assays and clinical diagnosis for detecting the NP antigen. Here, we reported a pair of noncompeting human NP-specific mAbs, named P301-F7 and P301-H5, targeting two distinct linear epitopes on SARS-CoV-2 or SARS-CoV. We evaluated the application of P301-F7 in the analysis of enzyme linked immunosorbent assay, western blot, flow cytometry, immunofluorescence, and focus reduction neutralization test. We for the first time report a broad mAb effectively recognizing various live viruses of SARS-CoV-2 variants including Alpha, Beta, Delta, and Omicron, indicating a wide range of application prospects.

Sequential immunization with SARS-CoV-2 RBD vaccine induces potent and broad neutralization against variants in mice.

The current COVID-19 pandemic caused by constantly emerging SARS-CoV-2 variants still poses a threat to public health worldwide. Effective next-generation vaccines and optimized booster vaccination strategies are urgently needed. Here, we sequentially immunized mice with a SARS-CoV-2 wild-type inactivated vaccine and a heterologous mutant RBD vaccine, and then evaluated their neutralizing antibody responses against variants including Beta, Delta, Alpha, Iota, Kappa, and A.23.1. These data showed that a third booster dose of heterologous RBD vaccine especially after two doses of inactivated vaccines significantly enhanced the GMTs of nAbs against all SARS-CoV-2 variants we tested. In addition, the WT and variants all displayed good cross-immunogenicity and might be applied in the design of booster vaccines to induce broadly neutralizing antibodies.

Characterization of Edwardsiella piscicida CK108 flagellin genes and evaluation of their potential as vaccine targets in the zebrafish model.

The control of bacterial pathogens, including Edwardsiella piscicida, in the aquaculture industry has high economic importance. This study aimed to identify a potential live vaccine candidate against E. piscicida infection to minimize the side effects and elicit immunity in the host. This study evaluated the virulence factors of E. piscicida CK108, with a special focus on the flagella. E. piscicida has two important homologous flagellin genes, namely flagellin-associated protein (fap) and flagellin domain-containing protein (fdp). CK226 (Δfap), CK247 (Δfdp) and CK248 (Δfap, fdp) mutant strains were constructed. Both CK226 and CK247 displayed decreased length and thickness of flagellar filaments, resulting in reduced bacterial swimming motility, while CK248 was non-motile as it lacked flagella. The loss of flagella and decreased motility was expected to decrease the pathogenicity of CK248. However, the median lethal dose (LD50 ) of CK248 against zebrafish was lower than those of the wild-type, CK226 and CK247 strains. The protective immunity and cytokine gene expression levels in the CK248-infected zebrafish were lower than those in the wild type-infected zebrafish. In conclusion, Fap and Fdp are essential for flagella formation and motility, and for stimulating fish immune response, which can be utilized as a potential adjuvants for E. piscicida vaccination.

Mandibular incisors with two canals are associated with the presence of the distolingual root in mandibular first molars: a cone-beam computed tomographic study.

BACKGROUND: This study aimed to evaluate the root canal configurations and the association between mandibular first molars and distolingual root (DLR) and mandibular incisors using cone-beam computed tomography (CBCT). METHODS: Mandibular first molars and incisors were evaluated using CBCT images of 150 patients. Mandibular first molars were classified according to the presence or absence of DLR. The curvature of the DLR was evaluated using Schneider's method in buccolingual and mesiodistal orientations. The angle of the mesiolingual-distolingual-distobuccal canal orifices (ML-DL-DB) was evaluated at the pulpal floor level. The mandibular central and lateral incisors were classified as either single canal or two canals. The association between the root canal system of the mandibular first molar and mandibular incisors was evaluated. Data were analyzed using the Chi-square test, Student's t-test, and odds ratios from binary logistic regression. The significance level was set at 5%. RESULTS: The frequency of mandibular first molars with DLR was 27.0% and that of mandibular incisors with two canals was 25.8%. The curvature of the DLR was 37.1° in the buccolingual orientation and 10.4° in the mesiodistal orientation. The angle of ML-DL-DB was 79.0°. The presence of two canal systems in mandibular incisors was associated with the presence of DLR in the mandibular first molar (left central incisors: p = 0.001, odds ratio = 4.25; left lateral incisors: p < 0.001, odds ratio = 3.8; right central incisors: p = 0.003, odds ratio = 3.86; right lateral incisors: p = 0.001, odds ratio = 3.44) but not with the curvature of the DLR or angle of the ML-DL-DB orifice (p > 0.05). CONCLUSIONS: The mandibular first molar showed a high incidence of separate DLRs. It was confirmed that DLR in the mandibular first molar is highly associated with the presence of two-canal system in the mandibular incisors. This association can aid clinicians in successful endodontic treatments.

Impact of the N501Y substitution of SARS-CoV-2 Spike on neutralizing monoclonal antibodies targeting diverse epitopes.

The emergence and rapid spread of the B.1.1.7 lineage (VOC-202012/01) SARS-CoV-2 variant has aroused global concern. The N501Y substitution is the only mutation in the interface between the RBD of B.1.1.7 and ACE2, raising concerns that its recognition by neutralizing antibodies may be affected. Here, we assessed the neutralizing activity and binding affinity of a panel of 12 monoclonal antibodies against the wild type and N501Y mutant SARS-CoV-2 pseudovirus and RBD protein, respectively. We found that the neutralization activity and binding affinity of most detected antibodies (10 out of 12) were unaffected, although the N501Y substitution decreased the neutralizing and binding activities of CB6 and increased that of BD-23. These findings could be of value in the development of therapeutic antibodies.

Epigallocatechin Gallate Alleviates Down-Regulation of Thioredoxin in Ischemic Brain Damage and Glutamate-Exposed Neuron.

Epigallocatechin gallate (EGCG) is one of polyphenol that is abundant in green tea. It has anti-oxidative activity and exerts neuroprotective effects in ischemic brain damage. Ischemic conditions induce oxidative stress and result in cell death. Thioredoxin is a small redox protein that plays an important role in the regulation of oxidation and reduction. This study was designed to investigate the regulation of thioredoxin by EGCG in ischemic brain damage. Middle cerebral artery occlusion (MCAO) was performed to induce focal cerebral ischemia in male Sprague-Dawley rats. The EGCG (50 mg/kg) or was administered before MCAO surgical operation. Neurological behavior test, reactive oxygen species (ROS), and lipid peroxidation (LPO) measurement were performed 24 h after MCAO. The cerebral cortex was isolated for further experiments. EGCG alleviated MCAO-induced neurological deficits and increases in ROS and LPO levels. EGCG also ameliorated the decrease in thioredoxin expression by MCAO. This finding was confirmed using various techniques such as Western blot analysis, reverse transcription PCR, and immunofluorescence staining. Results of immunoprecipitation showed that MCAO decreases the interaction between apoptosis signal-regulating kinase 1 (ASK1) and thioredoxin, while EGCG treatment attenuates this decrease. EGCG also attenuated decrease of cell viability and thioredoxin expression in glutamate-exposed neuron in a dose-dependent manner. It alleviated the increase of caspase-3 by glutamate exposure. However, this effect of EGCG on caspase-3 change was weakened in thioredoxin siRNA-transfected neurons. These findings suggest that EGCG exerts a neuroprotective effect by regulating thioredoxin expression and modulating ASK1 and thioredoxin binding in ischemic brain damage.

Endoplasmic Reticulum (ER) Stress and Unfolded Protein Response (UPR) in Mammalian Oocyte Maturation and Preimplantation Embryo Development.

Mammalian oocytes and early embryos derived from in vitro production are highly susceptible to a variety of cellular stresses. During oocyte maturation and preimplantation embryo development, functional proteins must be folded properly in the endoplasmic reticulum (ER) to maintain oocyte and embryo development. However, some adverse factors negatively impact ER functions and protein synthesis, resulting in the activation of ER stress and unfolded protein response (UPR) signaling pathways. ER stress and UPR signaling have been identified in mammalian oocytes and embryos produced in vitro, suggesting that modulation of ER stress and UPR signaling play very important roles in oocyte maturation and the development of preimplantation embryos. In this review, we briefly describe the current state of knowledge regarding ER stress, UPR signaling pathways, and their roles and mechanisms in mammalian (excluding human) oocyte maturation and preimplantation embryo development.

A Method for Detecting Dynamic Mutation of Complex Systems Using Improved Information Entropy.

In this study, a nonlinear analysis method called improved information entropy (IIE) is proposed on the basis of constructing a special probability mass function for the normalized analysis of Shannon entropy for a time series. The definition is directly applied to several typical time series, and the characteristic of IIE is analyzed. This method can distinguish different kinds of signals and reflects the complexity of one-dimensional time series of high sensitivity to the changes in signal. Thus, the method is applied to the fault diagnosis of a rolling bearing. Experimental results show that the method can effectively extract the sensitive characteristics of the bearing running state and has fast operation time and minimal parameter requirements.

A Feature Extraction Method Using Improved Multi-Scale Entropy for Rolling Bearing Fault Diagnosis.

A feature extraction method named improved multi-scale entropy (IMSE) is proposed for rolling bearing fault diagnosis. This method could overcome information leakage in calculating the similarity of machinery systems, which is based on Pythagorean Theorem and similarity criterion. Features extracted from bearings under different conditions using IMSE are identified by the support vector machine (SVM) classifier. Experimental results show that the proposed method can extract the status information of the bearing. Compared with the multi-scale entropy (MSE) and sample entropy (SE) methods, the identification accuracy of the features extracted by IMSE is improved as well.