Molecular Cross-Talk between Integrins and Cadherins Leads to a Loss of Vascular Barrier Integrity during SARS-CoV-2 Infection

The vascular barrier is heavily injured following SARS-CoV-2 infection and contributes enormously to life-threatening complications in COVID-19. This endothelial dysfunction is associated with the phlogistic phenomenon of cytokine storms, thrombotic complications, abnormal coagulation, hypoxemia, and multiple organ failure. The mechanisms surrounding COVID-19 associated endotheliitis have been widely attributed to ACE2-mediated pathways. However, integrins are emerging as possible receptor candidates for SARS-CoV-2, and their complex intracellular signaling events are essential for maintaining endothelial homeostasis. Here, we showed that the spike protein of SARS-CoV-2 depends on its RGD motif to drive barrier dysregulation by hijacking integrin αVβ3, expressed on human endothelial cells. This triggers the redistribution and internalization of major junction protein VE-Cadherin which leads to the barrier disruption phenotype. Both extracellular and intracellular inhibitors of integrin αVβ3 prevented these effects, similarly to the RGD-cyclic peptide compound Cilengitide, which suggests that the spike protein—through its RGD motif—binds to αVβ3 and elicits vascular leakage events. These findings support integrins as an additional receptor for SARS-CoV-2, particularly as integrin engagement can elucidate many of the adverse endothelial dysfunction events that stem from COVID-19.     

Comparative Evaluation of the Foot-and-Mouth Disease Virus Permissive LF-BK αVβ6 Cell Line for Senecavirus A Research

Senecavirus A (SVA) is a member of the family Picornaviridae and enzootic in domestic swine. SVA can induce vesicular lesions that are clinically indistinguishable from Foot-and-mouth disease, a major cause of global trade barriers and agricultural productivity losses worldwide. The LF-BK α_Vβ₆ cell line is a porcine-derived cell line transformed to stably express an α_Vβ₆ bovine integrin and primarily used for enhanced propagation of Foot-and-mouth disease virus (FMDV). Due to the high biosecurity requirements for working with FMDV, SVA has been considered as a surrogate virus to test and evaluate new technologies and countermeasures. Herein we conducted a series of comparative evaluation in vitro studies between SVA and FMDV using the LF-BK α_Vβ₆ cell line. These include utilization of LF-BK α_Vβ₆ cells for field virus isolation, production of high virus titers, and evaluating serological reactivity and virus susceptibility to porcine type I interferons. These four methodologies utilizing LF-BK α_Vβ₆ cells were applicable to research with SVA and results support the current use of SVA as a surrogate for FMDV.     

The Spike Glycoprotein of SARS-CoV-2 Binds to β1 Integrins Expressed on the Surface of Lung Epithelial Cells

The spike glycoprotein attached to the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to and exploits angiotensin-converting enzyme 2 (ACE2) as an entry receptor to infect pulmonary epithelial cells. A subset of integrins that recognize the arginyl–glycyl–aspartic acid (RGD) sequence in the cognate ligands has been predicted in silico to bind the spike glycoprotein and, thereby, to be exploited for viral infection. Here, we show experimental evidence that the β1 integrins predominantly expressed on human pulmonary epithelial cell lines and primary mouse alveolar epithelial cells bind to this spike protein. The cellular β1 integrins support adhesive interactions with the spike protein independently of ACE2, suggesting the possibility that the β1 integrins may function as an alternative receptor for SARS-CoV-2, which could be targeted for the prevention of viral infections.     

A natural variant of the cysteine protease virulence factor of group A Streptococcus with an arginine-glycine-aspartic acid (RGD) motif preferentially binds human integrins αvβ3 and αIIbβ3

The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin α_vβ₃ (also known as the vitronectin receptor) or α_IIbβ₃ (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin α_IIbβ₃. Defined β₃ mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing α_vβ₃ and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of host-pathogen interactions.     

Integrin αvβ1 promotes infection by human metapneumovirus

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes lower respiratory infections in children and adults worldwide. The hMPV fusion (F) protein is a membrane-anchored glycoprotein and major protective antigen. All hMPV F protein sequences determined to date contain an Arg-Gly-Asp (RGD) sequence, suggesting that F engages RGD-binding integrins to mediate cell entry. The divalent cation chelator EDTA, which disrupts heterodimeric integrin interactions, inhibits infectivity of hMPV but not the closely related respiratory syncytial virus (RSV), which lacks an RGD motif. Function-blocking antibodies specific for αvβ1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with αv or β1 cDNAs confers hMPV infectivity, whereas reduction of αv and β1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas Arg-Gly-Glu (RGE)-mutant F protein does not. These data suggest that αvβ1 integrin is a functional receptor for hMPV.     

β3 integrins mediate the cellular entry of hantaviruses that cause respiratory failure

Newly emerged hantaviruses replicate primarily in the pulmonary endothelium, cause acute platelet loss, and result in hantavirus pulmonary syndrome (HPS). We now report that specific integrins expressed on platelets and endothelial cells permit the cellular entry of HPS-associated hantaviruses. Infection with HPS-associated hantaviruses, NY-1 and Sin Nombre virus (SNV), is inhibited by antibodies to β₃ integrins and by the β₃-integrin ligand, vitronectin. In contrast, infection with the nonpathogenic (no associated human disease) Prospect Hill virus was inhibited by fibronectin and β₁-specific antibodies but not by β₃-specific antibodies or vitronectin. Transfection with recombinant α_IIbβ₃ or α_vβ₃ integrins rendered cells permissive to NY-1 and SNV but not Prospect Hill virus infection, indicating that α_IIbβ₃ and α_vβ₃ integrins mediate the entry of NY-1 and SNV hantaviruses. Furthermore, entry is divalent cation independent, not blocked by arginine-glycine-aspartic acid peptides and still mediated by, ligand-binding defective, α_IIbβ₃-integrin mutants. Hence, NY-1 and SNV entry is independent of β₃ integrin binding to physiologic ligands. These findings implicate integrins as cellular receptors for hantaviruses and indicate that hantavirus pathogenicity correlates with integrin usage.     

Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

G protein–coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β₂-adrenergic receptors (β₂AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling—a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β₂AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S. These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S. C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β₂AR actions favorable for treating obstructive lung disease.

Most G protein–coupled receptors (GPCRs) are now recognized as multisignal transducers (1, 2). Early concepts of agonist–receptor interactions were based on the idea that there was a single “active” receptor conformation induced by the binding of any agonist, resulting in an interaction with the heterotrimeric G protein and a universal, singular signal. Generally, the α-subunit of the G protein, upon its dissociation, was considered the primary activator (or inhibitor) of the effector, resulting in the intracellular signal. Subsequently, it became clear that multiple signaling outcomes from activation of a given GPCR can occur from a single agonist due to specific molecular determinants of the receptor triggering independent mechanisms (3–5). As these multiple functions were being identified, it was apparent that agonists with different structures could act at a given receptor to preferentially activate one signal with minimal engagement of others, a property later termed signal biasing (6–8). Biased agonists, then, could represent important advantages over nonbiased agonists due to this signal selectivity, activating a specified therapeutic pathway while minimally evoking unnecessary or deleterious signaling. The pathway selectivity of biased agonists is thought to be established by the stabilization of specific conformation(s) of the agonist–receptor complex via a set of interactions that differ from those of unbiased (also called balanced) agonists (9–12). While it is conceivable that small modifications of established cognate agonists might yield such specialized signaling, significant deviation from common agonist structures may be necessary to meet this goal (13).The signals/functions of a given GPCR that might be sought for selective activation are defined by the cell type, disease, and desired final physiologic function. In asthma and chronic obstructive pulmonary disease (COPD), active human airway smooth muscle (HASM) cellular contraction limits airflow, representing a major cause of morbidity and mortality. β₂-adrenergic receptors (β₂ARs) expressed on HASM cells are the targets for binding of therapeutically administered β-agonists, which relax the cells via a cyclic adenosine monophosphate–mediated mechanism (14). β-agonists are used for treating acute bronchospasm as well as for long-term prevention. However, the HASM bronchodilator response to acute β-agonist is attenuated by receptor desensitization (15), with typical treatments of humans, or isolated HASM cells, leading to a loss of receptor function over time (16–18), clinically termed tachyphylaxis.Agonist-promoted desensitization of β₂AR (and other GPCRs) is due to partial uncoupling of the receptor to the G protein, which is initiated by phosphorylation of intracellular Ser/Thr residues of the receptor by G protein–coupled receptor kinases (GRKs) (19, 20). The GRK-phosphorylated β₂AR recruits β-arrestin1 or β-arrestin2 to these receptors, with subsequent interactions that appear to compete with the receptor for its binding to the Gα subunit, thus attenuating the intracellular response (11, 21). Such competition has been strongly inferred for the β₂AR (22, 23) and is compelling for rhodopsin–arrestin interactions (24). In addition, β-arrestin binding to GPCRs can initiate receptor internalization and other events such as receptor activation of ERK1/2 (25) through its multiprotein adapter functions. Thus β-arrestin engagement can be considered an early “second signal” of the β₂AR as well as a desensitization initiator for attenuating the Gs signal. An agonist that is biased toward Gαs coupling (cAMP production and airway smooth muscle [ASM] relaxation) and away from β-arrestin binding (desensitization) would be desirable in treating obstructive lung diseases, since efficacy would not be attenuated acutely, nor would tachyphylaxis be experienced from extended treatment. While biased agonists favoring either G protein or β-arrestin (6) signaling have been described for some GPCRs (such as μ-opioid and type 1 angiotensin II receptors), Gαs biasing has not been apparent from most studies with catecholamine-like compounds for the β₂AR. Thus, we have little information as to whether the two β₂AR pathways can be differentially activated in a selective manner by an efficacious agonist, nor is it apparent from a structural standpoint what strategy might be employed to design agonists biased in this manner for this receptor.In order to find this type of biasing for the β₂AR, we screened a 40-million-compound scaffold ranking (SR) library that was agnostic to known β₂AR agonist structures. We found a scaffold in which substitutions of certain R groups led to individual compounds that are apparently Gαs-biased agonists for β₂AR with no apparent engagement of β-arrestin in model systems. Additional studies in HASM cells revealed a lack of tachyphylaxis of the relaxation effect by the lead compound compared with the most widely utilized β₂AR agonist, albuterol. The structure of this biased agonist is very different from that of catecholamine-like agonists. To ascertain the mechanism that may underlie this biased activity, we used structural modeling and molecular simulations and studied homologous compounds with different R groups and receptor mutagenesis to predict the interaction sites with the activated β₂AR. Such studies uncovered distinct structural characteristics that may be responsible for the biasing effect.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Cross-α/β polymorphism of PSMα3 fibrils

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Amyloids are elongated fibers of proteins or peptides typically composed of stacked cross β-sheets (1, 2). Self-assembling amyloids are notorious for their involvement in human neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases (1, 2). Phenol soluble modulins (PSMs) are amyloid peptides secreted by the bacteria Staphylococcus aureus (S. aureus) (3–5). Of the PSM family, PSMα3 is of recent interest due to its unique secondary structure upon fibrillation. Whereas other PSM variants undergo conformational changes with aggregation, the α-helical PSMα3 peptide retains its secondary structure while stacking in a manner reminiscent of β-sheets, forming what has been termed cross-α fibrils (3, 4, 6). Although “α-sheet” amyloid fibrils have been previously observed in two-dimensional infrared (2DIR) (7) and associated with PSMs (8), the novel cross-α fibril is distinct from that class of structures. To avoid confusion between these two similarly named but distinct secondary structures, a comparison between the α-sheet domain in cytosolic phosphatase A2 (9) (Protein Data Bank [PDB] identification:1rlw) (10) and cross-α fibrils adopted by PSMα3 (PDB ID:5i55) (3) has been highlighted in SI Appendix, Fig. S1. Interestingly, shorter terminations of PSMα3 have been shown to exhibit β-sheet polymorphs (11). The proposed cross-α fibril structure of the full-length PSMα3 peptide has been confirmed with X-ray diffraction and circular dichroism (4). The present study aims to further characterize these fibrils with linear and nonlinear infrared spectroscopies.S. aureus is an infectious human pathogen with the ability to form communities of microorganisms called biofilms that hinder traditional treatment methods (12–14). PSMs contribute to inflammatory response and play a crucial role in structuring and detaching biofilms (11, 12, 14). While biofilm growth requires the presence of multiple PSMs (14, 15), Andreasen and Zaman have demonstrated that PSMα3 acts as a scaffold, seeding the amyloid formation of other PSMs (5). To effectively inhibit S. aureus biofilm growth, a better understanding of PSMα3 aggregation is needed.The α-helical structure of PSMα3 (12) presents a challenge for probing the vibrational modes and secondary structure of both the monomer and the fibrils. While IR spectroscopy has been used extensively to characterize β-sheets (16–19), the spectral features associated with α-helices are difficult to distinguish from those of the random coil secondary structure (20, 21). This limitation has left researchers to date with an incomplete picture of the spectroscopic features unique to cross-α fibers. The present work combines a variety of 2DIR methods to remove these barriers and probe the active infrared vibrational modes of cross-α fibers.The full-length, 22-residue PSMα3 peptide was synthesized and prepared for aggregation studies following reported methods (3, 4, 11). A total of 10 mM PSMα3 was incubated in D₂O at room temperature over 7 d. These data were compared to the monomer treated under similar conditions. Monomeric samples were prepared at a significantly lower concentration of 0.5 mM to prevent aggregation. Fiber formation was confirmed by transmission electron microscopy (see SI Appendix, Fig. S2 for details). Fourier transform infrared (FTIR) spectra were taken for both the fibrils in solution as well as the low concentration monomers. Spectroscopic simulations of the PSMα3 monomer and fibers were performed on previously reported PDB structures (PDB identification: 5i55) (3) (Fig. 1).Open in a separate windowFig. 1.PDB structures of PSMα3 (A) monomers and (B) cross-α fibers extended along the screw axis. (C) FTIR spectra of 0.5 mM monomeric PSMα3 (blue) compared to the 10 mM PSMα3 fibril (red) in D₂O upon aggregation.     

Reinforced 3D Composite Structures of γ-, α-Al2O3 with Carbon Nanotubes and Reduced GO Ribbons Printed from Boehmite Gels

The ability of boehmite to form printable inks has sparked interest in the manufacturing of 3D alumina (Al₂O₃) and composite structures by enabling direct ink writing methods while avoiding the use of printing additives. These materials may exhibit high porosity due to the printing and sintering procedures, depending on the intended application. The 3D-printed porous composite structures of γ-Al₂O₃ and α-Al₂O₃ containing 2 wt.% of carbon nanotubes or reduced graphene oxide ribbons were fabricated from boehmite gels, followed by different heat treatments. The reinforcing effect of these carbon nanostructures was evidenced by compression tests carried out on the different alumina structures. A maximum relative increase of 50% in compressive strength was achieved for the γ-Al₂O₃ composite structure reinforced with reduced graphene oxide ribbons, which was also accompanied by an increase in the specific surface area.     

β2 Adrenergic receptors mediate important electrophysiological effects in human ventricular myocardium

OBJECTIVE—To define the effects of β₂ adrenergic receptor stimulation on ventricular repolarisation in vivo.
DESIGN—Prospective study.
SETTING—Tertiary referral centre.
PATIENTS—85 patients with coronary artery disease and 22 normal controls.
INTERVENTIONS—Intravenous and intracoronary salbutamol (a β₂ adrenergic receptor selective agonist; 10-30 µg/min and 1-10 µg/min), and intravenous isoprenaline (a mixed β₁/β₂ adrenergic receptor agonist; 1-5 µg/min), infused during fixed atrial pacing.
MAIN OUTCOME MEASURES—QT intervals, QT dispersion, monophasic action potential duration.
RESULTS—In patients with coronary artery disease, salbutamol decreased QT_onset and QT_peak but increased QT_end duration; QT_onset-QT_peak and QT_peak-QT_end intervals increased, resulting in T wave prolongation (mean (SEM): 201 (2) ms to 233 (2) ms; p < 0.01). There was a large increase in dispersion of QT_onset, QT_peak, and QT_end which was more pronounced in patients with coronary artery disease—for example, QT_end dispersion: 50 (2) ms baseline v 98 (4) ms salbutamol (controls), and 70 (1) ms baseline v 108 (3) ms salbutamol (coronary artery disease); p < 0.001. Similar responses were obtained with isoprenaline. Monophasic action potential duration at 90% repolarisation shortened during intracoronary infusion of salbutamol, from 278 (4.1) ms to 257 (3.8) ms (p < 0.05).
CONCLUSIONS—β₂ adrenergic receptors mediate important electrophysiological effects in human ventricular myocardium. The increase in dispersion of repolarisation provides a mechanism whereby catecholamines acting through this receptor subtype may trigger ventricular arrhythmias.


Keywords: β₂ adrenergic receptors; ventricular repolarisation; QT dispersion; salbutamol; isoprenaline     

A Simple and Efficient Method for Preparing High-Purity α-CaSO4·0.5H2O Whiskers with Phosphogypsum

A simple and efficient approach for the high-purity CaSO₄·2H₂O (DH) whiskers and α-CaSO₄·0.5H₂O (α-HH) whiskers derived from such phosphogypsum (PG) was proposed. The impact of different experimental parameters on supersaturated dissolution–recrystallization and preparation processes of α-CaSO₄·0.5H₂O was elaborated. At 3.5 mol/L HCl concentration, the dissolution temperature and time were 90 °C and 20 min, respectively. After eight cycles and 5–8 times cycles, total crystallization amount of CaSO₄·2H₂O was 21.75 and 9.97 g/100 mL, respectively, from supersaturated HCl solution. The number of cycles affected the shape and amount of the crystal. Higher HCl concentration facilitated CaSO₄·2H₂O dissolution and created a much higher supersaturation, which acted as a larger driving force for phase transformation of CaSO₄·2H₂O to α-CaSO₄·0.5H₂O. The HCl solution system’s optimum experimental conditions for HH whiskers preparation involved acid leaching of CaSO₄·2H₂O sample, with HCl concentration 6.0 mol/L, reaction temperature 80 °C, and reaction time 30 min–60 min. Under the third cycle conditions, α-CaSO₄·0.5H₂O whiskers were uniform in size, clear, and distinct in edges and angles. The length range of α-CaSO₄·0.5H₂O whiskers was from 106 μm to 231 μm and diameter range from 0.43 μm to 1.35 μm, while the longest diameter ratio was 231. Purity of α-CaSO₄·0.5H₂O whiskers was approximately 100%, where whiteness reached 98.6%. The reuse of the solution enables the process to discharge no waste liquid. It provides a new reference direction for green production technology of phosphogypsum.     

A First-Principles Study of the Cu-Containing β″ Precipitates in Al-Mg-Si-Cu Alloy

The nanostructured β″ precipitates are critical for the strength of Al-Mg-Si-(Cu) aluminum alloys. However, there are still controversial reports about the composition of Cu-containing β″ phases. In this work, first-principles calculations based on density functional theory were used to investigate the composition, mechanical properties, and electronic structure of Cu-containing β″ phases. The results predict that the Cu-containing β″ precipitates with a stoichiometry of Mg_4+xAl_2−xCuSi₄ (x = 0, 1) are energetically favorable. As the concentration of Cu atoms increases, Cu-containing β″ phases with different compositions will appear, such as Mg₄AlCu₂Si₄ and Mg₄Cu₃Si₄. The replacement order of Cu atoms in β″ phases can be summarized as one Si3/Al site → two Si3/Al sites → two Si3/Al sites and one Mg1 site. The calculated elastic constants of the considered β″ phases suggest that they are all mechanically stable, and all β″ phases are ductile. When Cu atoms replace Al atoms at Si3/Al sites in β″ phases, the values of bulk modulus (B), shear modulus (G), and Young’s modulus (E) all increase. The calculation of the phonon spectrum shows that Mg_4+xAl_2−xCuSi₄ (x = 0, 1) are also dynamically stable. The electronic structure analysis shows that the bond between the Si atom and the Cu atom has a covalent like property. The incorporation of the Cu atom enhances the electron interaction between the Mg2 and the Si3 atom so that the Mg2 atom also joins the Si network, which may be one of the reasons why Cu atoms increase the structure stability of the β″ phases.     

Viewing rare conformations of the β2 adrenergic receptor with pressure-resolved DEER spectroscopy

The β₂ adrenergic receptor (β₂AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The β₂AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free β₂AR. Here we employ spin-labeling and pressure-resolved double electron–electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in β₂AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

Many aspects of physiology in health and disease are regulated by signal transduction through G protein coupled receptors (GPCRs). Among these, the β₂ adrenergic receptor (β₂AR) is an archetype for the subset of family A GPCRs activated by hormones and neurotransmitters, as well as a pharmaceutical target for asthma and chronic obstructive pulmonary disease. The activity of β₂AR and other ligand-binding GPCRs can be finely tuned by ligands of varying efficacy, with agonists stimulating an increase in activation of cognate G proteins and inverse agonists decreasing G protein activation below a basal level.β₂AR crystal structures (1) have defined the outward movement of transmembrane helix 6 (TM6) as the largest structural rearrangement associated with activation of the receptor, as was originally found for rhodopsin (2) and subsequently for other receptors. This movement of TM6 is required for β₂AR to productively couple to its signaling partners (3).Ligand-independent or basal activity has been observed in many GPCRs. The level of basal receptor activity is highly receptor-specific and is important in maintaining homeostasis in physiologic systems independent of agonist stimulation (4). Basal activity in the β₂AR (5–7) and other receptors (4, 8) could arise from distinct receptor conformations that promote weak activation of the G protein, possibly involving an induced fit mechanism for receptor–G protein interaction. Alternatively, the basal activity could arise from a preexisting equilibrium (9, 10) between inactive and active conformations, where a small fraction of active receptors could account for baseline levels of signaling in physiologic systems. In support of this mechanism, sparse NMR data on the unliganded adenosine receptor provides evidence for the existence of an equilibrium between the inactive and active conformations (11).Although data from single-molecule fluorescence on β₂AR (12) have been interpreted in terms of the presence of the active form of the unliganded receptor, direct structural evidence for the existence of an active-like conformation in equilibrium with the unliganded, inactive conformation is lacking for this pharmacologically important GPCR. Indeed, structural studies of equilibrium populations by single-molecule fluorescence resonance energy transfer (FRET) (13) and double electron–electron resonance (DEER) spectroscopy (5) did not detect the presence of the active conformation in the ensemble of the unliganded β₂AR. This leaves the mechanism of basal activity for β₂AR unresolved. If true conformational selection plays a role, then a population of active form must exist in the equilibrium manifold of the unliganded receptor. The goal of the present work is to investigate the possibility that an active conformation in fact exists but is too sparsely populated to detect with the methods employed.Detection and characterization of sparsely populated (rare or excited) conformations is a general problem in elucidating the molecular mechanisms of protein function. Such low-lying excited states may play important functional roles despite being sparsely populated (14). Extensive empirical evidence indicates that the application of pressure may provide a solution to this problem by reversibly increasing the population of excited states (15, 16), allowing the use of standard experimental techniques to characterize these states and elucidate their functional roles. The mechanisms underlying the pressure-dependent conformational shifts are discussed in recent literature (17–20). It is important to note that as long as the pressure-induced shift in population is reversible, the rare state must also exist at atmospheric pressure and is not an artifact of pressure itself.Recently, site-directed spin labeling (SDSL) together with variable pressure electron paramagnetic resonance (EPR) was developed to monitor protein conformational shifts due to applied hydrostatic pressure (21). Of particular interest for the present study is SDSL with DEER (22). DEER provides a probability distribution of the distances between site-specifically introduced spin labels. Thus, each conformation in an ensemble generates a different distance in the distribution with a probability proportional to the population of the associated conformation, provided that the spin labels are properly placed to monitor the conformational equilibrium. In this respect, DEER is well suited to provide a structural definition to excited states revealed by pressure because it does not require a single conformation to be fully populated but instead reveals each conformation in the ensemble that is sufficiently populated (above ∼5 to 10%). Here we employ pressure-resolved DEER (23) to identify and characterize sparsely populated states (below 1%) in the conformational ensemble of unliganded β₂AR. The data provide direct structural evidence for the presence of an active-like conformation in the equilibrium ensemble of the unliganded receptor. The addition of agonists and inverse agonists increase and decrease the equilibrium population of the active form, respectively. The results indicate a mechanism for basal activity as well as that for inverse agonists in the β₂AR. Equally important, the variable pressure data provide a thermodynamic characterization of the active state including the partial molar volume, free energy, and compressibility relative to the ground (inactive) state.     

β-Glucans from Trametes versicolor (L.) Lloyd Is Effective for Prevention of Influenza Virus Infection

Coriolus versicolor (C. versicolor) is a higher fungi or mushroom which is now known by its accepted scientific names as Trametes versicolor (L.) Lloyd. Many studies have shown that β-glucans from C. versicolor have various physiological activities, including activating macrophages to protect against Salmonella infection. However, whether β-glucans have antiviral effects has not been reported. Hence, the objective of this study was to confirm whether β-glucans could boost the immune response to combat influenza virus in mouse and chick models. The results show that β-glucans induced the expression of Dectin-1, costimulatory molecules (CD80/86) and cytokines IL-6, IL-1β, IFN-β and IL-10 in murine bone marrow dendritic cells (BMDCs). In addition, orally administered β-glucans reduced weight loss, mortality and viral titers in the lungs of mice infected with influenza virus and attenuated pathological lung damage caused by the virus in the mice. Orally administered β-glucans improved survival and reduced lung viral titers in chickens infected with H9N2 avian influenza virus. These results suggest that β-glucans have a significant antiviral effect. Therefore, β-glucans could become a potential immunomodulator against influenza virus.     

Pin-Pointing the Key Hubs in the IFN-γ Pathway Responding to SARS-CoV-2 Infection

Interferon gamma (IFN-γ) may be potential adjuvant immunotherapy for COVID-19 patients. In this work, we assessed gene expression profiles associated with the IFN-γ pathway in response to SARS-CoV-2 infection. Employing a case-control study from SARS-CoV-2-positive and -negative patients, we identified IFN-γ-associated pathways to be enriched in positive patients. Bioinformatics analyses showed upregulation of MAP2K6, CBL, RUNX3, STAT1, and JAK2 in COVID-19-positive vs. -negative patients. A positive correlation was observed between STAT1/JAK2, which varied alongside the patient’s viral load. Expression of MX1, MX2, ISG15, and OAS1 (four well-known IFN-stimulated genes (ISGs)) displayed upregulation in COVID-19-positive vs. -negative patients. Integrative analyses showcased higher levels of ISGs, which were associated with increased viral load and STAT1/JAK2 expression. Confirmation of ISGs up-regulation was performed in vitro using the A549 lung cell line treated with Poly (I:C), a synthetic analog of viral double-stranded RNA; and in different pulmonary human cell lines and ferret tracheal biopsies infected with SARS-CoV-2. A pre-clinical murine model of Coronavirus infection confirmed findings displaying increased ISGs in the liver and lungs from infected mice. Altogether, these results demonstrate the role of IFN-γ and ISGs in response to SARS-CoV-2 infection, highlighting alternative druggable targets that can boost the host response.     

The Characteristic of Fe as a β-Ti Stabilizer in Ti Alloys

It is well known that adding elements, especially β-Ti stabilizers, are holding a significant effect on titanium alloy strength due to the solution and precipitate strengthening mechanisms. In order to reveal the Fe strengthening mechanism in titanium, this study investigate the effect of Fe on the stability of β-Ti and the phase transition between α, β and ω phase with first-principle calculations. According to our study, Fe is a strong β-Ti phase stabilizer could owe to the 3d orbital into e_g and t_2g states which results in strong hybridization between Fe-d orbital and Ti-d orbital. The phase transition from ω to β or from α to β becomes easier for Fe-doped Ti compared to pure titanium. Based on our results, it is found that one added Fe atom can lead the phase transition (ω → β) of at least nine titanium atoms, which further proves that Fe has a strong stabilizing effect on β-Ti phase. This result provides a solid guide for the future design of high-strength titanium with the addition of Fe.     

Research on Three-Dimensional Porous Composite Nano-Assembled α-MnO2/Reduced Graphene Oxides and Their Super-Capacitive Performance

A series of three-dimensional porous composite α-MnO₂/reduced graphene oxides (α-MnO₂/RGO) were prepared by nano-assembly in a hydrothermal environment at pH 9.0–13.0 using graphene oxide as the precursor, KMnO₄ and MnCl₂ as the manganese sources and F⁻ as the control agent of the α-MnO₂ crystal form. The α-MnO₂/RGO composites prepared at different hydrothermal pH levels presented porous network structures but there were significant differences in these structures. The special pore structure promoted the migration of ions in the electrolyte in the electrode material, and the larger specific surface area promoted the contact between the electrode material and the electrolyte ions. The introduction of graphene solved the problem of poor conductivity of MnO₂, facilitated the rapid transfer of electrons, and significantly improved the electrochemical performance of materials. When the pH was 12.0, the specific surface area of the 3D porous composite material αMGs-12.0 was 264 m²·g⁻¹, and it displayed the best super-capacitive performance; in Na₂SO₄ solution with 1.0 mol·L⁻¹ electrolyte, the specific capacitance was 504 F·g⁻¹ when the current density was 0.5 A·g⁻¹ and the specific capacitance retention rate after 5000 cycles was 88.27%, showing that the composite had excellent electrochemical performance.     

Influence of TiO2 and ZnO Nanoparticles on α-Synuclein and β-Amyloid Aggregation and Formation of Protein Fibrils

The most common neurological disorders, i.e., Parkinson’s disease (PD) and Alzheimer’s disease (AD), are characterized by degeneration of cognitive functions due to the loss of neurons in the central nervous system. The aggregation of amyloid proteins is an important pathological feature of neurological disorders.The aggregation process involves a series of complex structural transitions from monomeric to the formation of fibrils. Despite its potential importance in understanding the pathobiology of PD and AD diseases, the details of the aggregation process are still unclear. Nanoparticles (NPs) absorbed by the human circulatory system can interact with amyloid proteins in the human brain and cause PD. In this work, we report the study of the interaction between TiO₂ nanoparticles (TiO₂-NPs) and ZnO nanoparticles (ZnO-NPs) on the aggregation kinetics of β-amyloid fragment 1-40 (βA) and α-synuclein protein using surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS). The characterizations of ZnO-NPs and TiO₂-NPs were evaluated by X-ray diffraction (XRD) spectrum, atomic force microscopy (AFM), and UV-Vis spectroscopy. The interaction of nanoparticles with amyloid proteins was investigated by SERS. Our study showed that exposure of amyloid protein molecules to TiO₂-NPs and ZnO-NPs after incubation at 37 °C caused morphological changes and stimulated aggregation and fibrillation. In addition, significant differences in the intensity and location of active Raman frequencies in the amide I domain were found. The principal component analysis (PCA) results show that the effect of NPs after incubation at 4 °C does not cause changes in βA structure.     

Liquid-Phase-Assisted Catalytic Nitridation of Silicon and In Situ Growth of α-Si3N4

Si₃N₄ powders were synthesized with Fe, Co, or Ni as catalysts using Si powder at 1250 °C in a nitrogen atmosphere by liquid-phase-assisted catalytic nitridation synthesis (LPA–CNS). The catalytic effects of the metals on the nitridation of silicon powder were investigated by mixing the powder with 2 wt% by mass of Fe, Co, or Ni in a high-temperature liquid phase in flowing nitrogen. The α-Si₃N₄ micro-morphology could be effectively changed by adjusting the type of catalyst in the initial reaction mixtures. Fe, Co, and Ni promoted the formation of α-Si₃N₄ at 1250 °C and controlled the morphology of the α-Si₃N₄ particles. The hexagonal flakes of α-Si₃N₄ with a better defined morphology were obtained using Ni as the catalyst, compared to that obtained from the other two catalysts.