The current COVID-19 pandemic has led to a devastating impact across the world. SARS-CoV-2 (the virus causing COVID-19) is known to use the receptor-binding domain (RBD) at viral surface spike (S) protein to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD−ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has a higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS outbreak. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approaches to quantify the specific interactions between CoV-2 or CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between CoV-2 RBD and ACE2 range from 70 to 105 pN and are 30-40% higher than those of CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the CoV-1 RBD−ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After removing N-linked glycans on ACE2, its mechanical binding strength with CoV-2 RBD decreases to a similar level of the CoV-1 RBD−ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1 and could help develop new strategies to block SARS-CoV-2 entry.

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease characterized by autoantibody-induced platelet clearance. Some ITP patients are refractory to standard immunosuppressive treatments such as intravenous immunoglobulin (IVIg). These patients often have autoantibodies that target the ligand-binding domain (LBD) of glycoprotein Ibα (GPIbα), a major subunit of the platelet mechanoreceptor complex GPIb-IX. However, the molecular mechanism of this Fc-independent platelet clearance is not clear. Here, we report that many anti-LBD monoclonal antibodies such as 6B4, but not AK2, activated GPIb-IX in a shear-dependent manner and induced IVIg-resistant platelet clearance in mice. Single-molecule optical tweezer measurements of antibodies pulling on full-length GPIb-IX demonstrated that the unbinding force needed to dissociate 6B4 from the LBD far exceeds the force required to unfold the juxtamembrane mechanosensory domain (MSD) in GPIbα, unlike the AK2-LBD unbinding force. Binding of 6B4, not AK2, induced shear-dependent unfolding of the MSD on the platelet, as evidenced by increased exposure of a linear sequence therein. Imaging flow cytometry and aggregometry measurements of platelets and LBD-coated platelet-mimetic beads revealed that 6B4 can sustain crosslinking of platelets under shear, whereas 6B4 Fab and AK2 cannot. These results suggest a novel mechanism by which anti-LBD antibodies can exert a pulling force on GPIb-IX via platelet crosslinking, activating GPIb-IX by unfolding its MSD and inducing Fc-independent platelet clearance.

Von Willebrand factor (VWF) activates in response to shear flow to initiate hemostasis, while aberrant activation could lead to thrombosis. Above a critical shear force, the A1 domain of VWF becomes activated and captures platelets via the GPIb-IX complex. Here we show that the shear-responsive element controlling VWF activation resides in the discontinuous autoinhibitory module (AIM) flanking A1. Application of tensile force in a single-molecule setting induces cooperative unfolding of the AIM to expose A1. The AIM-unfolding force is lowered by truncating either N- or C-terminal AIM region, type 2B VWD mutations, or binding of a ristocetin-mimicking monoclonal antibody, all of which could activate A1. Furthermore, the AIM is mechanically stabilized by the nanobody that comprises caplacizumab, the only FDA-approved anti-thrombotic drug to-date that targets VWF. Thus, the AIM is a mechano-regulator of VWF activity. Its conformational dynamics may define the extent of VWF autoinhibition and subsequent activation under force.

Dendritic cells (DCs) are professional antigen-presenting cells with antigen recognition molecules on the surface. Clec9a is selectively expressed on mouse CD8a+ DCs and CD103+ DCs subsets, which are functionally similar to human BDCA3+ DCs. It is reported that Clec9a is responsible for the antigen cross-presentation of these DC subsets. In the present study, by using phage display technique, we discovered a novel peptide WH, which can selectively bind to mouse Flt3L induced Clec9a+ DCs or Clec9a over-expressed HEK-293T cells. Furthermore, by using computer-aided docking model and mutation assay, we observed that Asp248 and Trp250 are two key residues for Clec9a to bind with peptide WH. When coupled with OVA257-264 epitope, peptide WH can significantly enhance the ability of Clec9a+ DCs to activate OVA-specific CD8+ T cells, which elicit strong ability to secret IFN-γ, express perforin and granzyme B mRNA. In B16-OVA lung metastasis mouse model, WH-OVA257-264 fusion peptide can also enhance the activation of CD8+ T cells and decrease the lung metastasis loci. All these results suggested that peptide WH could be considered as an antigen delivery carrier targeting Clec9a+ DCs for cancer immunotherapy.

Atomic force microscopy is used to establish that low-affinity integrins bind in cis to P2Y2R. Integrin activation is initiated by a membrane-normal switchblade motion triggered by integrin priming after the virus binds to the integrin PSI domain. Tensile force between the P2Y2R and unbending integrin stimulates outside-in signaling.

The current COVID-19 pandemic has already had a devastating impact across the world. SARS-CoV-2 (the virus causing COVID-19) is known to use its surface spike (S) protein's receptor binding domain (RBD) to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD–ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS epidemic. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes a combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approach to quantify the specific interactions between CoV-2 or CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between CoV-2 RBD and ACE2 range from 70 to 110 pN, and are 30-50% higher than those of CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the CoV-1 RBD–ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After the removal of N-linked glycans on ACE2, its mechanical binding strength with CoV-2 RBD decreases to a similar level of the CoV-1 RBD–ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1, and could aid in the development of new strategies to block SARS-CoV-2 entry.

The wavelength-tunable interlayer exciton (IE) from layered semiconductor materials has not been achieved. van der Waals heterobilayers constructed using single-layer transition metal dichalcogenides can produce continuously changed interlayer band gaps, which is a feasible approach to achieve tunable IEs. In this work, we design a series of van der Waals heterostructures composed of a WSe2 layer with a fixed band gap and another WS2(1–x)Se2x alloy layer with continuously changed band gaps. The existence of IEs and tunable interlayer band gaps in these heterobilayers is verified by steady-state photoluminescence experiments. By tuning the composition of the WS2(1–x)Se2x alloy layers, we realized a very wide tunable band gap range of 1.97–1.40 eV with a wavelength-tunable IE emission range of 1.52–1.40 eV from the heterobilayers. The time-resolved photoluminescence experiments show the IE emission lifetimes over nanoseconds.

Pancreatic stellate cells (PSCs) are important components of the tumor microenvironment in pancreatic cancer (PC) and contribute to its development and metastasis through mechanisms that remain incompletely characterized. Tumor hypoxia affects the function and behavior of PC and stromal cells, and can alter exosomal content to modify cell-cell communication. The present study explored the effects of exosomal miRNAs produced by hypoxia-preconditioned PSCs on the growth and metastatic potential of PC cells. Subcutaneous xenografts and liver metastasis mouse models revealed increased tumorigenic potential upon co-implantation of PC cells and PSCs as compared to PC cells alone. Screening miRNA profiles of mouse plasma exosomes and cultured PSCs, followed by miRNA overexpression and inhibition assays, enabled us to identify miR-4465 and miR-616-3p as prominent hypoxia-induced, PSC-derived, exosomal miRNAs promoting PC cell proliferation, migration, and invasion. Proteomics analysis of PC cells incubated with exosomes derived from hypoxic PSCs showed significant downregulation of PTEN. Dual-luciferase reporter assays and western blotting showed that both miR-4465 and miR-616-3p target PTEN and activate AKT signaling in PC cells. We conclude that hypoxia upregulates miR-4465 and miR-616-3p expression in PSC-derived exosomes. Following exosome uptake, these miRNAs promote PC progression and metastasis by suppressing the PTEN/AKT pathway.