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According to previous research, turmeric seeds exhibit anti-inflammatory, anti-malignancy, and anti-aging properties due to an abundance of terpinen-4-ol (T4O). Although it is still unclear how T4O works on glioma cells, limited data exist regarding its specific effects. In order to determine whether or not glioma cell lines U251, U87, and LN229 are viable, CCK8 was used as an assay and a colony formation assay was performed using different concentrations of T4O (0, 1, 2, and 4 μM). The effect of T4O on the proliferation of glioma cell line U251 was detected through the subcutaneous implantation of the tumor model. Through high-throughput sequencing, a bioinformatic analysis, and real-time quantitative polymerase chain reactions, we identified the key signaling pathways and targets of T4O. Finally, for the measurement of the cellular ferroptosis levels, we examined the relationship between T4O, ferroptosis, and JUN and the malignant biological properties of glioma cells. T4O significantly inhibited glioma cell growth and colony formation and induced ferroptosis in the glioma cells. T4O inhibited the subcutaneous tumor proliferation of the glioma cells in vivo. T4O suppressed JUN transcription and significantly reduced its expression in the glioma cells. The T4O treatment inhibited GPX4 transcription through JUN. The overexpression of JUN suppressed ferroptosis in the cells rescued through T4O treatment. Taken together, our data suggest that the natural product T4O exerts its anti-cancer effects by inducing JUN/GPX4-dependent ferroptosis and inhibiting cell proliferation, and T4O will hope-fully serve as a prospective compound for glioma treatment.

Background The binding of the A1 domain of von Willebrand factor (VWF) to platelet receptor glycoprotein (GP)Ibα defines the VWF activity in hemostasis. Recent studies suggest that sequences flanking A1 form cooperatively an autoinhibitory module (AIM) that reduces the accessibility of the GPIbα binding site on A1. Application of a tensile force induces unfolding of the AIM. Desialylation induces spontaneous binding of plasma VWF to platelets. Most O-glycans in VWF are located around the A1 domain. Removing certain O-glycans in the flanking sequences by site-directed mutagenesis enhances A1 binding to GPIbα and produces an effect similar to type 2B von Willebrand disease in animals. Objectives To understand if and how desialylation of O-glycans in the flanking sequences increases A1 activity. Methods A recombinant AIM-A1 fragment encompassing VWF residues 1238-1493 and only O-glycans was treated with neuraminidase to produce desialylated protein. The glycan structure, dynamics, stability, and function of the desialylated protein was characterized by biochemical and biophysical methods and compared to the sialylated fragment. Results Asialo-AIM-A1 exhibited increased binding activity and induced more apparent platelet aggregation than its sialylated counterpart. It exhibited a lower melting temperature, and increased hydrogen-deuterium exchange rates at residues near the secondary GPIbα binding site and the N-terminal flanking sequence. Asialo-AIM-A1 is less mechanically stable than sialo-AIM-A1, with its unstressed unfolding rate approximately 3-fold greater than the latter. Conclusions Desialylation of O-glycans around A1 increases its activity by destabilizing the AIM.

Nicotinic acetylcholine receptors (nAChRs) are broadly expressed in the central and peripheral nervous systems, playing essential roles in cholinergic neurotransmission. The lynx family proteins, a subset of the Ly6/uPAR superfamily expressed in multiple brain regions, have been shown to bind to nAChRs and modulate their function via allosteric regulation. The binding interactions between lynx and nAChRs, however, have not been systematically quantified and compared. In this work, we characterized the interactions between lynx1 or lynx2 and α3β4‐ or α7‐nAChRs using single‐molecule atomic force microscopy (AFM). The AFM technique allows the quantification of the off‐rate of lynx‐nAChR binding and of the energetic barrier width between the bound state and transition state, providing a biophysical means to compare the selectivity of lynx proteins for nAChR subtypes. Results indicate that lynx1 has a marginal preference for α7‐ over α3β4‐nAChRs. Strikingly, lynx2 exhibits a two order of magnitude stronger affinity for α3β4‐ compared to α7‐nAChRs. Together, the AFM assay serves as a valuable tool for the biophysical characterization of lynx‐nAChR binding affinities. Revealing the differential affinities of lynx proteins for nAChR subtypes will help elucidate how lynx regulates nAChR‐dependent functions in the brain, including nicotine addiction and other critical pathways.

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., Von Willebrand factor (VWF) is a large glycoprotein in the blood secreted from endothelial cells lining the blood vessel and activation of VWF leads to formation of VWF-platelet complexes or thrombi. Here authors use single-molecule force measurement, X-ray crystallography and functional measurements to monitor the activation of VWF via mechanical unfolding of the autoinhibitory module (AIM).

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.

It is challenging to deal with the Internet congestion problem because of several factors such as ever-growing traffic and distributed network architecture. The congestion problem can be solved or alleviated by various methods, including rate control, bandwidth-guarantee routing and bandwidth reservation. We use the term broad-sensed Internet congestion control and avoidance (BICC&A) to generally denote all of the above methods. Most BICC&A solutions depend on or benefit from the knowledge of network conditions, including traffic status (type and volume), available bandwidth and topology. In this paper, we present a comprehensive survey of the applications of machine learning to network condition acquirement methods for BICC&A and specific BICC&A methods. First, we provide an overview of the background knowledge of BICC&A and machine learning. Then, we provide detailed reviews on the applications of machine learning techniques to network condition acquirement methods for BICC&A and to specific BICC&A methods. Finally, we outline important research opportunities.

BACKGROUND. Proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 is crucial for normal hemostasis. Our previous studies demonstrate that binding of coagulation factor VIII (or FVIII) to VWF enhances the proteolytic cleavage of VWF by ADAMTS13 under shear. OBJECTIVES. Present study aims to determine the mechanism underlying FVIII-mediated enhancing effect on VWF proteolysis by ADAMTS13 under force. METHODS. Single molecular force spectroscopy, atomic force microscopy, and surface plasmon resonance are all employed. RESULTS. Using the single molecule force spectroscopy, we show that an addition of FVIII (~5 nM) to D’D3 or D’D3A1 does not significantly alter force-induced unfolding of these fragments; however, an addition of FVIII at the same concentration to D’D3A1A2 eliminates its long unfolding event at ~40 nm, suggesting that binding of FVIII to D’D3 and/or A2 may result in force-induced conformational changes in A2 domain. Atomic force spectroscopy further demonstrates the direct binding between FVIII and D’D3 (or A2) with an intrinsic 2D off-rate (k(0)) of 0.02 ± 0.01 s(−1) (or 0.3 ± 0.1 s(−1)). The direct binding interaction between FVIII and A2 is further confirmed with the surface plasmon resonance assay, with a dissociation constant (K(D)) of ~0.2 μM; no binding is detected between FVIII and A1 under the same conditions. CONCLUSIONS. Our results suggest that binding of FVIII to D’D3 and/or A2 may alter the mechanical property in the central A2 domain. The findings provide novel insight into the molecular mechanism underlying FVIII-dependent regulation of VWF proteolysis by ADAMTS13 under mechanical force.

Immunotherapy emerges as an effective avenue for tumor elimination and has many advantages compared with traditional surgery, radiotherapy, and chemotherapy. Tumor vaccines play an important role in cancer immunotherapy, while the application of tumor vaccines in clinical usage is limited because only limited response can be induced by primary tumor antigens. Accordingly, it is a key point to activate T-cell response with some novel tumor vaccines. Here, we applied phage display biopanning and screened a peptide (TY) that could combine with bone-marrow-derived-dendritic-cells (BMDCs) specifically and spleenic DCs. Then we developed mesoporous silica nanoparticles (MSN-TY/OVA/CpG), with peptide TY and OVA/CpG to target and activate DCs, respectively. Our results showed that the nanoparticles (NPs) could be specifically absorbed by DC

Intratumoral hypoxia is a common feature of pancreatic cancer (PC) and also plays a role in its progression. However, hypoxia-regulated signatures in PC are still not completely understood. This study aimed to identify core hypoxia-associated genes and determine their underlying molecular mechanisms in PC cells. Transformer 2 alpha homolog (TRA2A) was found to be an important hypoxia-associated gene, which was upregulated in PC tissues and in PC cells cultured under hypoxia. High TRA2A expression was associated with advanced stage, poor differentiation, and lymph node metastasis. Under normoxic and hypoxic conditions, knockdown of TRA2A both markedly suppressed PC cell proliferation and motility in vitro and in vivo, as well as activation of the AKT pathway. Hypoxia-inducible factor 1 subunit alpha (HIF1α) upregulated the transcription of TRA2A by directly binding to its promoter. TRA2A showed a co-expression relationship with HIF1α in PC tissues. Overexpression of TRA2A alleviated the pro-inhibitive functions of HIF1α-inhibition on PC cell proliferation and motility under hypoxia. In conclusion, TRA2A is a crucial downstream gene of HIF1α that accelerates the proliferation and motility of PC cells. TRA2A may be a novel and practical molecular target for investigating the hypoxic response of PC cells. Abbreviations: TRA2A, transformer 2A protein; PC, pancreatic cancer; HIF1α, hypoxia-inducible factor 1-alpha; GEO, Gene Expression Omnibus; IHC, immunohistochemical staining.

Terpinen-4-ol (T4O), a compound isolated from the seeds of turmeric, has exhibited anti-malignancy, anti-aging, and anti-inflammatory properties in previous studies. However, the specific effects and molecular mechanisms of T4O on pancreatic cancer (PC) cells remain largely unknown. In this study, we demonstrated that T4O markedly suppressed PC cell proliferation and colony formation in vitro and induced apoptosis. Similarly, T4O significantly inhibited the migration and invasion of PC cells in vitro. Through RNA sequencing, 858 differentially expressed genes (DEGs) were identified, which were enriched in the Rhodopsin (RHO)/ Ras homolog family member A (RHOA) signaling pathway. Rho-associated coiled-coil containing protein kinase 2 (ROCK2), a DEG enriched in the RHO/RHOA signaling pathway, was considered as a key target of T4O in PC cells; it was significantly reduced after T4O treatment, highly expressed in PC tissues, and negatively associated with patient outcome. Overexpression of ROCK2 significantly reduced the inhibitory effects of T4O on PC cell proliferation and mobility. Moreover, T4O inhibited cell proliferation in vivo and decreased the Ki-67, cell nuclear antigen, EMT markers, and ROCK2 expression. In conclusion, we consider that T4O can suppress the malignant biological behavior of PC by reducing the expression of ROCK2, thus contributing to PC therapy.

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.

Various ligand-functionalized liposomes have been developed for targeted therapies. Typically, the binding properties of the ligands and targeted proteins are measured with surface plasmon resonance (SPR), where the proteins are immobilized on a rigid surface. However, the difference of protein-ligand binding kinetics between liposome-conjugated protein and rigid surface-conjugated protein is not fully understood. In this work, the binding kinetics of P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin conjugated on liposome and on rigid surfaces are investigated with Atomic Force Microscopy (AFM). The results suggest that protein orientation and diffusion on liposomal membrane can alter the binding kinetics of the protein-ligand interaction. Specifically, the association and dissociation rate constant of AFM probe-conjugated E-selectin and glass-conjugated PSGL-1 are measured as 9.32×10(4) M(−1)s(−1) and 1.54 s(−1), respectively. While for the liposome-conjugated E-selectin and glass-conjugated PSGL-1, the kinetic constants are measured as 5.00×10(7) M(−1)s(−1) and 2.76 s(−1), respectively. Thus, there is an order’s magnitude increase of binding affinity (from k(d) = 16.51 µM to k(d) = 0.06 µM) when protein is attached to liposome compared to attached to a rigid surface. The results might provide better understanding and pave the way for the future design of the ligand-targeted liposomes.

Pancreatic cancer (PC) is a lethal malignancy of the gastrointestinal tract. Previous studies have reported that microRNAs (miRNAs/miRs) are involved in the tumorigenesis of PC. Therefore, the present study aimed to determine the effects of miR‑7515 on PC cell proliferation, invasion and migration in vitro and in vivo, and investigate its underlying molecular mechanism using bioinformatics, double luciferase assay and western blotting. The results revealed that the expression levels of miR‑7515 were downregulated in PC, which predicted a poor clinical outcome. The overexpression of miR‑7515 significantly decreased the proliferation, invasive and migratory abilities of PC cells in vitro and in vivo, while the knockdown of miR‑7515 exerted the opposite effects. miR‑7515 was identified to directly bind to insulin‑like growth factor 1 (IGF‑1) and downregulate its expression, which subsequently downregulated the Ras/Raf/MEK/ERK signalling pathway. The overexpression of IGF‑1 reversed the inhibitory effects of miR‑7515 overexpression on PC cells. In conclusion, the findings of the present study indicated that miR‑7515 may act as a tumor suppressor in PC, as it repressed PC cell proliferation invasion and migration via downregulating the expression of IGF‑1 and the activity of the Ras/Raf/MEK/ERK signalling pathways.

BackgroundHypoxia is associated with the development of pancreatic cancer (PC). However, genes associated with hypoxia response and their regulatory mechanism in PC cells were unclear. The current study aims to investigate the role of the hypoxia associated gene fucosyltransferase 11 (FUT11) in the progression of PC.MethodsIn the preliminary study, bioinformatics analysis predicted FUT11 as a key hypoxia associated gene in PC. The expression of FUT11 in PC was evaluated using quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. The effects of FUT11 on PC cells proliferation and migration under normoxia and hypoxia were evaluated using Cell Counting Kit 8, 5-ethynyl-2’-deoxyuridine (EDU) assay, colony formation assay and transwell assay. The effects of FUT11 in vivo was examined in mouse tumor models of liver metastasis and subcutaneous xenograft. Furthermore, Western blot, luciferase assay and immunoprecipitation were performed to explore the regulatory relationship among FUT11, hypoxia-inducible factor 1α (HIF1α) and pyruvate dehydrogenase kinase 1 (PDK1) in PC.ResultsFUT11 was markedly increased of PC cells with hypoxia, upregulated in the PC clinical tissues, and predicted a poor outcome of PC patients. Inhibition of FUT11 reduced PC cell growth and migratory ability of PC cells under normoxia and hypoxia conditions in vitro, and growth and tumor cell metastasis in vivo. FUT11 bound to PDK1 and regulated the expression PDK1 under normoxia and hypoxia. FUT11 interacted with PDK1 and decreased the ubiquitination of PDK1, lead to the activation of AKT/mTOR signaling pathway. FUT11 knockdown significantly increased the degradation of PDK1 under hypoxia, while treatment with MG132 can relieve the degradation of PDK1 induced by FUT11 knockdown. Overexpression of PDK1 in PC cells under hypoxia conditions reversed the suppressive impacts of FUT11 knockdown on PC cell growth and migration. In addition, HIF1α bound to the promoter of FUT11 and increased its expression, as well as co-expressed with FUT11 in PC tissues. Furthermore, overexpression of FUT11 partially rescued the suppressive effects of HIF1α knockdown on PC cell growth and migration in hypoxia condition.ConclusionOur data implicate that hypoxia-induced FUT11 contributes to proliferation and metastasis of PC by maintaining the stability of PDK1, thus mediating activation of AKT/mTOR signaling pathway, and suggest that FUT11 could be a novel and effective target for the treatment of pancreatic cancer.

Background: Hypoxia participated in the occurrence and development of pancreatic cancer (PC). However, genes associated with hypoxia respond and their regulated mechanism in PC cells were unclear. The current research was aimed to illuminate the role and hypoxia regulated mechanism of fucosyltransferase 11 (FUT11) in the progression of PC.Methods: After predicting FUT11 as a key hypoxia associated gene in PC using bioinformatics analysis. The expression of FUT11 in PC using quantitative real-time fluorescent PCR, western blot and immunohistochemistry. The effects of FUT11 on PC cells proliferation, migration and invasion under normoxia and hypoxia were detected using Cell Counting Kit 8, 5-ethynyl-2’-deoxyuridine assay, colony formation assay and transwell assay. Spleen capsule injected liver metastasis and subcutaneously injected model were performed to confirm the effects of FUT11 in vivo. Furthermore, western blot, luciferase assay and immunoprecipitation were performed to explore the regulated relationship among FUT11, hypoxia-inducible factor 1α (HIF1α) and pyruvate dehydrogenase kinase 1 (PDK1) in PC.Results: FUT11 was markedly increased of PC cells in hypoxia, up-regulated in the PC clinical tissues, and predicted a poor outcome. Inhibition of FUT11 reduced PC cell growth and mobility of PC cells under normoxia and hypoxia conditions in vitro, and growth and mobility in vivo. FUT11 bind with PDK1 and regulated the expression PDK1 under normoxia and hypoxia. FUT11 knockdown significantly increased the degradation rate of PDK1 under hypoxia, while treatment with MG132 can relieve the degradation of PDK1 induced by FUT11 knockdown. Overexpression of PDK1 in PC cells under hypoxia conditions reversed the suppressiv impacts of FUT11 knockdown on PC cell growth and mobility. In addition, HIF1α bound to the enhancer of FUT11 and increased its expression, as well as co-expressing with FUT11 in PC tissues. Furthermore, overexpress of FUT11 partially rescued the suppressiv effects of HIF1α knockdown on PC cell growth and mobility in hypoxia conditions.Conclusion: Our data further implicate that hypoxia-induced FUT11 in PC contributes to proliferation and metastasis by maintaining the stability of PDK1, and suggest FUT11 maybe a novel and effective target for treatment of pancreatic cancer.

Boosted by the visions of 5G technology, along with the proliferation of IoT devices, we are lucky to bear witness of a tremendous shift within the Internet, mobile computing, and ubiquitous applications pervading people’s daily life. To catch up with the speed of this evolution, new architectures, which enable us to decentralize and focus more on the edge of the network, have to show up. To effectively address the record-breaking growth of data traffic, we also have to come up with new caching mechanisms in coping with users’ demanding QoE along with other aspects, such as data privacy and energy efficiency. In this paper, we intend to begin with reviews on edge caching. First, we make a detailed overview of mobile edge caching. Then, we move on to the QoS and QoE part, discussing the related literature concerning these topics. We then start to address factors concerning edge caching and QoE. Four cutting-edge applications utilizing these technologies with challenges posed by current network architectures are made clear. Our paper closes with some future research directions.

Muscle fatigue detection based on surface Electromyography (sEMG) is one of the essential goals of human–computer interaction. The main challenge is that the sEMG signal is unstable and complex. Meanwhile, the individual’s difference in fatigue tolerance will increase the detection difficulty. In order to reduce the impact of the above challenges, in this article, we use the sEMG signal to detect muscle fatigue based on the Multi-dimensional Feature Fusion Network (MFFNet), which is composed of Attention Frequency domain Network (AFNet) and Attention Time domain Network (ATNet). Precisely, AFNet consists of the convolutional neural network, channel attention network and spatial attention network. ATNet is composed of a two-way long and short-term memory network and time attention network. Furthermore, through the filter and Gaussian short-time Fourier transform, we can analyze the feature of the time domain and frequency domain of sEMG. Subsequently, fuse features of different dimensions are used to predict fatigue detection in many muscle fatigue detection experiments based on sEMG. The proposed method has better performance and interpretability. Experimental results prove that the proposed method can promote the development of sEMG in the field of muscle fatigue detection.

The optical responses of molecules and materials provide a basis for chemical measurement and imaging. The optical diffraction limit in conventional light microscopy is exceeded by mechanically probing optical absorption through the photothermal effect with atomic force microscopy (AFM). However, the spatial resolution of AFM-based photothermal optical microscopy is still limited, and the sample surface is prone to damage from scratching due to tip contact, particularly for measurements on soft matter. In this article, we develop peak force visible (PF-vis) microscopy for the measurement of visible optical absorption of soft matter. The spatial resolution of PF-vis microscopy is demonstrated to be 3 nm on green fluorescent protein-labeled virus-like particles, and the imaging sensitivity may approach a single protein molecule. On organic photovoltaic polymers, the spatial distribution of the optical absorption probed by PF-vis microscopy is found to be dependent on the diffusion ranges of excitons in the donor domain. Through finite element modeling and data analysis, the exciton diffusion range of organic photovoltaics can be directly extracted from PF-vis images, saving the need for complex and delicate sample preparations. PF-vis microscopy will enable high-resolution nano-imaging based on light absorption of fluorophores and chromophores, as well as deciphering the correlation between the spatial distribution of photothermal signals and underlying photophysical parameters at the tens of nanometer scale.

The current COVID-19 pandemic has led to a devastating impact across the world. Severe acute respiratory syndrome coronavirus 2 (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 SARS-CoV-2 or SARS-CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between SARS-CoV-2 RBD and ACE2 range from 70 to 105 pN and are 30-40% higher than those of SARS-CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that SARS-CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the SARS-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 SARS-CoV-2 RBD decreases to a similar level of the SARS-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.

CCL25, CXCL10, and Mn2+ induce three distinct active conformations of integrin α4β7, which have selective high affinity for either MAdCAM-1, VCAM-1, or nonselective high affinity for both ligands. Via this mechanism, integrin α4β7 adopts different active conformations to switch its ligand-binding specificity., Chemokine (C-C motif) ligand 25 (CCL25) and C-X-C motif chemokine 10 (CXCL10) induce the ligand-specific activation of integrin α4β7 to mediate the selective adhesion of lymphocytes to mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) or vascular cell adhesion molecule-1 (VCAM-1). However, the mechanism underlying the selective binding of different ligands by α4β7 remains obscure. In this study, we demonstrate that CCL25 and CXCL10 induce distinct active conformers of α4β7 with a high affinity for either MAdCAM-1 or VCAM-1. Single-cell force measurements show that CCL25 increases the affinity of α4β7 for MAdCAM-1 but decreases its affinity for VCAM-1, whereas CXCL10 has the opposite effect. Structurally, CCL25 induces a more extended active conformation of α4β7 compared with CXCL10-activated integrin. These two distinct intermediate open α4β7 conformers selectively bind to MAdCAM-1 or VCAM-1 by distinguishing their immunoglobulin domain 2. Notably, Mn2+ fully opens α4β7 with a high affinity for both ligands. Thus, integrin α4β7 adopts different active conformations to switch its ligand-binding specificity.

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β3integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β5/β3integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbβ3integrins and (RGD)P2Y2R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbβ3integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα13protein from binding to the cytoplasmic domain of the β3integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation.

Diamond-like carbon (DLC) thin films were prepared by hydrothermal electrochemical method in one-step process. The structural characterization of these films was carried out by scanning electron microscopy (SEM), Raman spectroscopy, and infrared reflectance spectroscopy (IR). It was found that there was an increased sp2 carbon content but decreased sp3 carbon and hydrogen contents with an increase in current density. The flexibility and internal stresses of the DLC films were affected by hydrogen, sp3 amorphous carbon and ordered crystalline sp2 carbon contents. The highly flexible DLC films with high sp3 carbon and hydrogen contents were prepared at a current density of 0.001 mA/cm2.

Background: Von Willebrand factor (VWF), is a large, multimeric plasma glycoprotein that plays a critical role in hemostasis. VWF is synthesized and secreted as ultra large (UL) multimers that contain up to 100 protomers. If not processed by ADAMTS13, a plasma metalloprotease, ULVWF may initiate the spontaneous formation of life-threatening thrombi, as seen in thrombotic thrombocytopenic purpura (TTP). The cleavage site is buried under the central β-sheet within the VWF-A2 domain and tensile force is required to expose the cleavage site for ADAMTS13. Our prior studies demonstrate that several VWF-binding proteins, including coagulation factor VIII, apoB100/LDL, as well as the ectodomains of platelet glycoprotein Iba (GPIba), appear to function as cofactors that facilitate the proteolytic cleavage of VWF by ADAMTS13 under shear. However, the mechanism underlying GPIba enhancing effect on ADAMTS13-mediated VWF proteolysis is yet to be determined. Methods: Recombinant human GPIbα was purchased from Sino Biological. The recombinant VWF-A2 domain fragment with a SpyTag on the N-terminus and an AviTag-HisTag on the C-terminus was expressed in the HEK 293T cells and affinity-purified by the Ni-NTA affinity chromatography. Biotinylation was performed in vitro using a biotin-labeling kit. The binding between VWF-A2 and GPIbα was studied by a custom-built atomic force microscope (AFM) using our established single-molecule binding study protocol. MicroScale Thermophoresis was conducted by a Monolith device to detect the binding affinity between the VWF-A2 and GPIbα. A dual-beam mini-tweezers instrument was utilized to characterize the force-induced conformational changes of the A2 domain in the absence and presence of GPIbα with a pulling speed of 200 nm/s. Results: AFM results indicated that specific binding interactions occurred between GPIbα and VWF-A2. Monolith MST assay revealed a strong binding affinity (Kd of ~20 nM) between GPIbα and the VWF-A2 fragment. In the optical tweezer study, pulling on a single VWF-A2 resulted in an unfolding event at 10-30 pN with an extension ranging from 30 to 40 nm (Fig. 1A). Gaussian fits of the unfolding extension distributions revealed a most probable force-induced extension of 34.87 ± 2.2 nm (mean ± SEM) (Fig. 1B). Addition of 100 nM of GPIbα led to a noticeable decrease in both unfolding force and extension of VWF-A2 (Fig. 1A). The most probable unfolding extension reduced to 16.05 ± 0.2 nm in the presence of 100 nM of GPIbα (Fig. 1B), indicating the binding of GPIbα may influence mechanical unfolding of VWF-A2. Further, the unfolding results were analyzed by a worm-like chain model fit, which yielded a contour length for the initially folded structure of VWF-A2 at 58.83 ± 2.0 nm (mean ± SEM) and 24.53 ± 0.2 nm in the absence and presence of GPIbα, respectively (Fig. 1C), indicating that the specific interactions between GPIbα and A2 domain may partially unfold the A2 domain. Conclusions: These results demonstrate for the first time that binding of GPIbα to VWF-A2 may alter the force-induced conformational changes in the A2 domain. Under physiological conditions, the glycocalicin (or soluble GP1bα) may bind the VWF-A2 and cause A2 partial unfolding, which may result in excessive cleavage of VWF by ADAMTS13, thus regulating hemostasis. Figure legend: Fig.1. GPIbα influences the mechanical unfolding of the A2 domain of VWF. (A) Typical optical tweezer pulling traces of A2 domain in the absence (red) and presence (blue) of GPIbα (100 nM). The arrows point to the unfolding events. The pulling speed is 200 nm/s. (B) The histograms of the unfolding extension of pulling VWF-A2 in the absence (red) or presence (blue) of 100 nM of GPIbα at 200 nm/s. Solid lines are Gaussian fits to the distributions. (C) The relationship between unfolding force (pN) and unfolding extension (nm) of pulling the VWF-A2 in the absence (red) or presence (blue) of 100 nM of GPIbα. The data are fitted to the worm-like chain model (solid lines). Horizontal and vertical error bars are one standard deviation for force and half width of the half bin width for extension, respectively. Figure Disclosures Cao: Ivygen: Consultancy; Bayer: Research Funding. Zheng:Sanofi: Consultancy, Speakers Bureau; Clotsolution: Other: Co-Founder; Alexion: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.

// Zhongyi Yan 1 , Yahong Wu 1 , Jiangfeng Du 1 , Guodong Li 1 , Shengdian Wang 2 , Wenpeng Cao 1 , Xiuman Zhou 1 , Chunjing Wu 1 , Dan Zhang 1 , Xueli Jing 1 , Yifan Li 1 , Hongfei Wang 1 , Yanfeng Gao 1, 3 , Yuanming Qi 1, 3 1 School of Life Sciences, Zhengzhou University, Zhengzhou, China 2 Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 3 Collaborative Innovation Center of New Drug Research and Safety Evaluation, Zhengzhou, China Correspondence to: Yanfeng Gao, email: gaoyf@zzu.edu.cn Yuanming Qi, email: qym@zzu.edu.cn Keywords: Clec9a, dendritic cells, cancer immunotherapy, peptide Received: February 11, 2016 Accepted: May 09, 2016 Published: May 26, 2016 ABSTRACT 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 Asp 248 and Trp 250 are two key residues for Clec9a to bind with peptide WH. When coupled with OVA 257-264 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-OVA 257-264 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.

Cycle-life tests of commercial 22650-type olivine-type lithium iron phosphate (LiFePO4)/graphite lithium-ion batteries were performed at room and elevated temperatures. A number of non-destructive electrochemical techniques, i.e., capacity recovery using a small current density, electrochemical impedance spectroscopy, and differential voltage and differential capacity analyses, were performed to deduce the degradation mechanism of these batteries. To further characterize their internal materials, we disassembled the batteries, and material analyses were performed. All results indicated that loss in active lithium was the main reason for battery aging, and the cells showed diverse recession of active materials at different temperatures. In addition, high discharge rate and growing impedance lead to a capacity fall down at 25 °C at approximately 300–500 cycles.

Enveloped viruses are viruses that have a lipid membrane. Many enveloped viruses, such as the emerging virus Ebola, are pathogenic and of great clinical importance. Recent studies have shown that the outer lipid leaflets of viral envelopes contain phosphatidylserine (PS), which serves as an important attachment factor for enveloped virus entry into host cells. The envelope PS interacts with cellular PS receptors in a manner that mimics apoptotic body-PS receptor interactions during the engulfment of dead cell debris by phagocytes. The PS receptors utilized by enveloped viruses for viral internalization are designated phosphatidylserine-mediated virus entry enhancing receptors (PVEERs). While the importance of PVEERs in mediating viral entry has been demonstrated, the biophysical properties of the PVEER-virus interactions, including the detailed kinetics of PVEER-virion-associated PS binding, have not been comprehensively evaluated and compared. Using Ebola virus (EBOV) as the model system, this study investigated and compared the interactions between model Ebola viral particles and PVEER family proteins that are known to mediate enveloped virus attachment and entry. We used single-molecule force spectroscopy to quantify the specific interaction forces between PS or model EBOV viral particles and PVEERs. Based on the dynamic force spectra of various PS-receptor interactions, our results suggest that PS-PVEER interactions are mechanically comparable to antibody-antigen interactions, and that Gas6 and MFG-E8 may generate more force-resistant intersections than TIM-1 and ProteinS. The findings can help explain the biophysical mechanisms underlying the PVEER-mediated entry of enveloped virus into cells.

Background: Proteolytic cleavage of von Willebrand factor (VWF) by a plasma metalloprotease ADAMTS13 is critical for normal hemostasis. Our previous studies demonstrate that binding of coagulation factor VIII (FVIII) through its light chain to VWF significantly accelerates the proteolytic cleavage of VWF by ADAMTS13 under fluidic shear. However, the mechanism underlying the FVIII-cofactor activity enhancing VWF proteolysis by ADAMTS13 under shear is not fully understood. Present study aims to test a hypothesis that FVIII binding to the D'D3 and/or other adjacent domains may facilitate a mechanic-induced conformational change in the central A2 domain of VWF, resulting in an increased accessibility and the cleavage of Tyr1605-Met1606 bond by ADAMTS13. Methods: Recombinant B-domainless FVIII and various VWF fragments (D'D3, D'D3A1, D'D3A1A2, and A2) with an Avi-His-tag on their N-termini and a Spy-tag on their C-termini were expressed and affinity-purified to homogeneity. Laser optical tweezers analysis determined the force-induced conformational changes of VWF fragments in the absence or presence of FVIII. Atomic force microscopy (AFM) and surface plasmon resonance (SPR) determined the binding events and kinetics between FVIII and various VWF domains. Results: Laser optical tweezers analysis demonstrated a Gaussian distribution of unfolding extension events upon pulling the single D'D3A1 fragment with the most probable extension distance of 22.8 ± 1.9 nm (mean ± SEM). Addition of 5 nM FVIII to the D'D3A1 or D'D3 fragment resulted in only a small decrease (~25%) or no change in the pulling extension profile. However, two peaks of Gaussian distribution were detected when pulling the D'D3A1A2 with one peak at ~18 nm and the other at ~40 nm. Addition of 5 nM FVIII to the D'D3A1A2 fragment, only one unfolding event was detected with the most probable unfolding extension distance of 24.5 ± 0.4 nm. Moreover, a single Gaussian distribution of unfolding extension profile was detected when pulling an isolated A2 fragment with the most probable unfolding extension of 31 ± 1 nm. Interestingly, addition of 5 nM FVIII resulted in a significant reduction of the most probable unfolding extension to 16.0 ± 0.4 nm, suggesting that FVIII may directly interact with A2 domain. AFM and SPR demonstrated the binding kinetics between FVIII and various VWF fragments. As shown, the intrinsic off-rate (k0) between FVIII and D'D3 was 0.19 ± 0.9 s-1 and the barrier width (Δx‡) between the naive state and the transition state was 1.5 ± 0.2 Å; the k0 between FVIII and D'D3A1A2 was 2.8 ± 1.0 s-1 and the barrier width (Δx‡) between the naive state and the transitional state was 1.7 ± 0.2 Å. Surprisingly, there was a detectable, but relatively weak binding between FVIII and A2 domain using AFM. SPR experiments confirmed the binding of FVIII to D'D3, D'D3A1A2, and A2 with the dissociation constant (KD) of ~2.9 nM, ~5.2 nM and ~1.6 nM, respectively. These results demonstrate that the specific interaction between FVIII and D'D3 or A2 domain. Conclusions: We have demonstrated for the first time using single-molecule force spectroscopy that binding of FVIII to the D'D3 and/or A2 domain of VWF may facilitate the force-induced conformational changes of the A2 domain, resulting in the enhanced proteolysis by ADAMTS13 under force. Our findings shed new light on how FVIII may regulate VWF proteolysis and function under (patho) physiological conditions. Disclosures Zheng: Shire/Takeda: Research Funding; Alexion: Speakers Bureau; Clotsolution: Other: Co-Founder; Ablynx/Sanofi: Consultancy, Speakers Bureau.

Black carbon-stabilized anatase particles were prepared by a simple one-step hydrothermal–electrochemical method using glucose and titanium citrate as the carbon and titanium source, respectively. Morphological, chemical, structural, and electrochemical characterizations of these powders were carried out by Raman spectroscopy, Fourier-transform infrared spectroscopy, x-ray diffraction, scanning electron microscopy, and cyclic voltammetry. It was revealed that 200-nm carbon/anatase TiO2 was homogeneously dispersed, and the powders exhibited excellent cyclic performance at high current rates of 0.05 V/s. The powders are interesting potential materials that could be used as anodes for lithium-ion batteries.

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.

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., Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β5/β3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbβ3 integrins and (RGD)P2Y2R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbβ3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα13 protein from binding to the cytoplasmic domain of the β3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation.