Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

Receptor-binding domain of SARS-Cov spike protein: Soluble expression in E.coli, purification and functional characterization

AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

A Vaccine Based on the Receptor-Binding Domain of the Spike Protein Expressed in Glycoengineered Pichia pastoris Targeting SARS-CoV-2 Stimulates Neutralizing and Protective Antibody Responses

In 2020 and 2021,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),a novel coronavirus,caused a global pandemic.Vaccines are expected to reduce the pressure of prevention and control,and have become the most effective strategy to solve the pandemic crisis.SARS-CoV-2 infects the host by binding to the cellular receptor angiotensin converting enzyme 2(ACE2)via the receptor-binding domain(RBD)of the surface spike(S)glycoprotein.In this study,a candidate vaccine based on a RBD recombinant subunit was prepared by means of a novel glycoengineered yeast Pichia pastoris expression system with characteristics of glycosylation modification similar to those of mammalian cells.The candidate vaccine effectively stimulated mice to produce high-titer anti-RBD specific antibody.Furthermore,the specific antibody titer and virus-neutralizing antibody(NAb)titer induced by the vaccine were increased significantly by the combination of the double adjuvants Al(OH)_(3) and CpG.Our results showed that the virus-NAb lasted for more than six months in mice.To summarize,we have obtained a SARS-CoV-2 vaccine based on the RBD of the S glycoprotein expressed in glycoengineered Pichia pastoris,which stimulates neutralizing and protective antibody responses.A technical route for fucose-free complex-type N-glycosylation modified recombinant subunit vaccine preparation has been established.

Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E.coli . Signal sequence of the outer membrane protein A (OmpA) of E.coli was attached to the 5′ end of the gene encoding protective antigen receptor-binding domain (the 4 th domain of PA, PAD4). The plasmid carrying the fusion gene was then transformed into E.coli and induced to express recombinant PAD4 by IPTG. The recombinant protein was purified by chromatography and then identified by N-terminal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E.coli . The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.

Characterization of the receptor-binding domain(RBD)of 2019 novel coronavirus:implication for development of RBD protein as a viral attachment inhibitor and vaccine

The outbreak of Coronavirus Disease 2019(COVID-19)has posed a serious threat to global public health,calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),also known as 2019 novel coronavirus(2019-nCoV).The CoV spike(S)protein plays the most important roles in viral attachment,fusion and entry,and serves as a target for development of antibodies,entry inhibitors and vaccines.Here,we identified the receptor-binding domain(RBD)in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2(ACE2)receptors.SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and,hence,attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells,thus inhibiting their infection to host cells.SARS-CoV RBD-specific antibodies could crossreact with SARS-CoV-2 RBD protein,and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2,suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

Identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against Middle East respiratory syndrome coronavirus

Middle East respiratory syndrome （MERS）, an emerging infectious disease caused by MERS coronavirus （MERS-CoV）, has garnered worldwide attention as a consequence of its continuous spread and pandemic potential, making the development of effective vaccines a high priority. We previously demonstrated that residues 377-588 of MERS-CoV spike （S） protein receptor-binding domain （RBD） is a very promising MERS subunit vaccine candidate, capable of inducing potent neutralization antibody responses. In this study, we sought to identify an adjuvant that optimally enhanced the immunogenicity of S377-588 protein fused with Fc of human IgG （S377-588-Fc）. Specifically, we compared several commercially available adjuvants, including Freund＇s adjuvant, aluminum, Monophosphoryl lipid A, Montanide ISA51 and MF59 with regard to their capacity to enhance the immunogenicity of this subunit vaccine. In the absence of adjuvant, S377-588-Fc alone induced readily detectable neutralizing antibody and T-cell responses in immunized mice. However, incorporating an adjuvant improved its immunogenicity. Particularly, among the aforementioned adjuvants evaluated, MF59 is the most potent as judged by its superior ability to induce the highest titers of IgG, IgG1 and IgG2a subtypes, and neutralizing antibodies. The addition of MF59 significantly augmented the immunogenicity of S377-588-Fcto induce strong IgG and neutralizing antibody responses as well as protection against MERS-CoV infection in mice, suggesting that MF59 is an optimal adjuvant for MERS-CoV RBD-based subunit vaccines.

An ultrapotent pan-β-coronavirus lineage B(β-CoV-B)neutralizing antibody locks the receptor-binding domain in closed conformation by targeting its conserved epitope

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design.Here,we identified a receptorbinding domain(RBD)-binding antibody,XG014,which potently neutralizesβ-coronavirus lineage B(β-CoV-B),including SARS-CoV-2,its circulating variants,SARSCoV and bat SARSr-CoV WIV1.Interestingly,antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibodydependent SARS-CoV-2 spike(S)protein-mediated cellcell fusion,suggesting a unique mode of recognition by XG014.Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional“down”conformation,while its family member XG005 directly competes with ACE2 binding and position the RBD“up”.Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo.Our findings suggest the potential to develop XG014 as pan-β-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines againstβ-CoV-B and newly emerging SARS-CoV-2 variants of concern.

Estimation-free spatial-domain image reconstruction of structured illumination microscopy

Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain,it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary,besides,the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts.Here,we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets,and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets(the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function(OTF)).Experiments on reconstructing raw datasets including nonbiological,biological,and simulated samples demonstrate that our method has SR capability,high reconstruction speed,and high robustness to aberration and noise.

Delineating homogeneous domains of fractured rocks using topological manifolds and deep learning

Determining homogeneous domains statistically is helpful for engineering geological modeling and rock mass stability evaluation.In this text,a technique that can integrate lithology,geotechnical and structural information is proposed to delineate homogeneous domains.This technique is then applied to a high and steep slope along a road.First,geological and geotechnical domains were described based on lithology,faults,and shear zones.Next,topological manifolds were used to eliminate the incompatibility between orientations and other parameters(i.e.trace length and roughness)so that the data concerning various properties of each discontinuity can be matched and characterized in the same Euclidean space.Thus,the influence of implicit combined effect in between parameter sequences on the homogeneous domains could be considered.Deep learning technique was employed to quantify abstract features of the characterization images of discontinuity properties,and to assess the similarity of rock mass structures.The results show that the technique can effectively distinguish structural variations and outperform conventional methods.It can handle multisource engineering geological information and multiple discontinuity parameters.This technique can also minimize the interference of human factors and delineate homogeneous domains based on orientations or multi-parameter with arbitrary distributions to satisfy different engineering requirements.

Consistency between domain wall oscillation modes and spin wave modes in nanostrips

Investigations on domain wall(DW) and spin wave(SW) modes in a series of nanostrips with different widths and thicknesses have been carried out using micromagnetic simulation. The simulation results show that the frequencies of SW modes and the corresponding DW modes are consistent with each other if they have the same node number along the width direction. This consistency is more pronounced in wide and thin nanostrips, favoring the DW motion driven by SWs.Further analysis of the moving behavior of a DW driven by SWs is also carried out. The average DW speed can reach a larger value of ~ 140 m/s under two different SW sources. We argue that this study is very meaningful for the potential application of DW motion driven by SWs.

Anomalous photovoltaic effect in Na_(0.5)Bi_(0.5)TiO_(3)-based ferroelectric ceramics based on domain engineering

The anomalous photovoltaic(APV)effect is promising for high-performance ferroelectric materials and devices in photoelectric applications.However,it is a challenge how to tune the APV effect by utilizing the characteristic structure of ferroelectrics.Here,a domain engineering strategy is proposed to enhance the APV effect in lead-free 0.88(Na_(0.5)Bi_(0.5)TiO_(3))-0.12(Ba_(1–1.5x)S_(mx)TiO_(3))(NBT-BST)ferroelectric ceramics.By tuning the domain size based on Sm^(3+)doping,a maximum open-circuit voltage(VOC)of 18.1 V is obtained when Sm^(3+)content is 0.75%,which is much larger than its bandgap(Eg).The mechanism of this large VOC originates from the multiple positive effects induced by the small-size domain,where decreasing domain size enhances ferroelectric polarization and net interface barrier potential,leading to a large driving electric field.Moreover,the APV effect exhibits a giant temperature sensitivity due to the dramatic evolution of small-size domain in the temperature field.This work sheds light on the exploration of ferroelectrics with APV effect and inspires their future high-performance optoelectronic device applications.

Mapping the antiparallel aligned domain rotation by microwave excitation

The evolution process of magnetic domains in response to external fields is crucial for the modern understanding and application of spintronics.In this study,we investigated the domain rotation in stripe domain films of varying thicknesses by examining their response to microwave excitation in four different orientations.The resonance spectra indicate that the rotation field of stripe domain film under an applied magnetic field approaches the field where the resonance mode of sample changes.The saturation field of the stripe domain film corresponds to the field where the resonance mode disappears when measured in the stripe direction parallel to the microwave magnetic field.The results are reproducible and consistent with micromagnetic simulations,providing additional approaches and techniques for comprehending the microscopic mechanisms of magnetic domains and characterizing their rotation.

Low-Rank Optimal Transport for Robust Domain Adaptation

When encountering the distribution shift between the source(training) and target(test) domains, domain adaptation attempts to adjust the classifiers to be capable of dealing with different domains. Previous domain adaptation research has achieved a lot of success both in theory and practice under the assumption that all the examples in the source domain are welllabeled and of high quality. However, the methods consistently lose robustness in noisy settings where data from the source domain have corrupted labels or features which is common in reality. Therefore, robust domain adaptation has been introduced to deal with such problems. In this paper, we attempt to solve two interrelated problems with robust domain adaptation:distribution shift across domains and sample noises of the source domain. To disentangle these challenges, an optimal transport approach with low-rank constraints is applied to guide the domain adaptation model training process to avoid noisy information influence. For the domain shift problem, the optimal transport mechanism can learn the joint data representations between the source and target domains using a measurement of discrepancy and preserve the discriminative information. The rank constraint on the transport matrix can help recover the corrupted subspace structures and eliminate the noise to some extent when dealing with corrupted source data. The solution to this relaxed and regularized optimal transport framework is a convex optimization problem that can be solved using the Augmented Lagrange Multiplier method, whose convergence can be mathematically proved. The effectiveness of the proposed method is evaluated through extensive experiments on both synthetic and real-world datasets.

Multiple Matching Attenuation Based on Curvelet Domain Extended Filtering

The paper develops a multiple matching attenuation method based on extended filtering in the curvelet domain,which combines the traditional Wiener filtering method with the matching attenuation method in curvelet domain.Firstly,the method uses the predicted multiple data to generate the Hilbert transform records,time derivative records and time derivative records of Hilbert transform.Then,the above records are transformed into the curvelet domain and multiple matching attenuation based on least squares extended filtering is performed.Finally,the attenuation results are transformed back into the time-space domain.Tests on the model data and field data show that the method proposed in the paper effectively suppress the multiples while preserving the primaries well.Furthermore,it has higher accuracy in eliminating multiple reflections,which is more suitable for the multiple attenuation tasks in the areas with complex structures compared to the time-space domain extended filtering method and the conventional curvelet transform method.

BIG HANKEL OPERATORS ON HARDY SPACES OF STRONGLY PSEUDOCONVEX DOMAINS

In this article,we investigate the(big) Hankel operator H_(f) on the Hardy spaces of bounded strongly pseudoconvex domains Ω in C^(n).We observe that H_(f ) is bounded on H~p(Ω)(1 <p <∞) if f belongs to BMO and we obtain some characterizations for Hf on H^(2)(Ω) of other pseudoconvex domains.In these arguments,Amar's L^(p)-estimations and Berndtsson's L^(2)-estimations for solutions of the ■_(b)-equation play a crucial role.In addition,we solve Gleason's problem for Hardy spaces H^(p)(Ω)(1 ≤p≤∞) of bounded strongly pseudoconvex domains.

Complementary-Label Adversarial Domain Adaptation Fault Diagnosis Network under Time-Varying Rotational Speed and Weakly-Supervised Conditions

Recent research in cross-domain intelligence fault diagnosis of machinery still has some problems,such as relatively ideal speed conditions and sample conditions.In engineering practice,the rotational speed of the machine is often transient and time-varying,which makes the sample annotation increasingly expensive.Meanwhile,the number of samples collected from different health states is often unbalanced.To deal with the above challenges,a complementary-label(CL)adversarial domain adaptation fault diagnosis network(CLADAN)is proposed under time-varying rotational speed and weakly-supervised conditions.In the weakly supervised learning condition,machine prior information is used for sample annotation via cost-friendly complementary label learning.A diagnosticmodel learning strategywith discretized category probabilities is designed to avoidmulti-peak distribution of prediction results.In adversarial training process,we developed virtual adversarial regularization(VAR)strategy,which further enhances the robustness of the model by adding adversarial perturbations in the target domain.Comparative experiments on two case studies validated the superior performance of the proposed method.

ON THE SOBOLEV DOLBEAULT COHOMOLOGY OF A DOMAIN WITH PSEUDOCONCAVE BOUNDARIES

In this note,we mainly make use of a method devised by Shaw[15]for studying Sobolev Dolbeault cohomologies of a pseudoconcave domain of the type Ω=Ω\∪_(j=1^(m))Ω_(j),where Ω and {Ω_(j)}_(j=1^(m)■Ω are bounded pseudoconvex domains in ℂ^(n) with smooth boundaries,and Ω_(1),…,Ω_(m) are mutually disjoint.The main results can also be quickly obtained by virtue of[5].

Diabetic retinopathy identification based on multi-sourcefree domain adaptation

AIM:To address the challenges of data labeling difficulties,data privacy,and necessary large amount of labeled data for deep learning methods in diabetic retinopathy(DR)identification,the aim of this study is to develop a source-free domain adaptation(SFDA)method for efficient and effective DR identification from unlabeled data.METHODS:A multi-SFDA method was proposed for DR identification.This method integrates multiple source models,which are trained from the same source domain,to generate synthetic pseudo labels for the unlabeled target domain.Besides,a softmax-consistence minimization term is utilized to minimize the intra-class distances between the source and target domains and maximize the inter-class distances.Validation is performed using three color fundus photograph datasets(APTOS2019,DDR,and EyePACS).RESULTS:The proposed model was evaluated and provided promising results with respectively 0.8917 and 0.9795 F1-scores on referable and normal/abnormal DR identification tasks.It demonstrated effective DR identification through minimizing intra-class distances and maximizing inter-class distances between source and target domains.CONCLUSION:The multi-SFDA method provides an effective approach to overcome the challenges in DR identification.The method not only addresses difficulties in data labeling and privacy issues,but also reduces the need for large amounts of labeled data required by deep learning methods,making it a practical tool for early detection and preservation of vision in diabetic patients.

Magnetic domain structures in ultrathin Bi_(2)Te_(3)/CrTe_(2) heterostructures

Chromium tellurium compounds are important two-dimensional van der Waals ferromagnetic materials with high Curie temperature and chemical stability in air,which is promising for applications in spintronic devices.Here,highquality spin-orbital-torque(SOT)device,Bi_(2)Te_(3)/CrTe_(2)heterostructure was epitaxially grown on Al_(2)O_(3)(0001)substrates.Anomalous Hall measurements indicate the existence of strong ferromagnetism in this device with the CrTe_(2)thickness down to 10 nm.In order to investigate its micromagnetic structure,cryogenic magnetic force microscope(MFM)was utilized to measure the magnetic domain evolutions at various temperatures and magnetic fields.The virgin domain state of the device shows a worm-like magnetic domain structure with the size around 0.6μm-0.8μm.Larger irregular-shape magnetic domains(>1μm)can be induced and pinned,after the field is increased to coercive field and ramped back to low fields.The temperature-dependent MFM signals exhibit a nice mean-field-like ferromagnetic transition with Curie temperature around 201.5 K,indicating a robust ferromagnetic ordering.Such a device can be potentially implemented in future magnetic memory technology.

MINIMIZERS OF L^(2)-SUBCRITICAL VARIATIONAL PROBLEMS WITH SPATIALLY DECAYING NONLINEARITIES IN BOUNDED DOMAINS

This paper is concerned with the minimizers of L^(2)-subcritical constraint variar tional problems with spatially decaying nonlinearities in a bounded domain Ω of R~N(N≥1).We prove that the problem admits minimizers for any M> 0.Moreover,the limiting behavior of minimizers as M→∞ is also analyzed rigorously.