The structure of the RBD-E77 Fab complex reveals neutralization and immune escape of SARS-CoV-2.

The spike protein (S) of SARS-CoV-2 is the major target of neutralizing antibodies and vaccines. Antibodies that target the receptor-binding domain (RBD) of S have high potency in preventing viral infection. The ongoing evolution of SARS-CoV-2, especially mutations occurring in the RBD of new variants, has severely challenged the development of neutralizing antibodies and vaccines. Here, a murine monoclonal antibody (mAb) designated E77 is reported which engages the prototype RBD with high affinity and potently neutralizes SARS-CoV-2 pseudoviruses. However, the capability of E77 to bind RBDs vanishes upon encountering variants of concern (VOCs) which carry the N501Y mutation, such as Alpha, Beta, Gamma and Omicron, in contrast to its performance with the Delta variant. To explain the discrepancy, cryo-electron microscopy was used to analyze the structure of an RBD-E77 Fab complex, which reveals that the binding site of E77 on RBD belongs to the RBD-1 epitope, which largely overlaps with the binding site of human angiotensin-converting enzyme 2 (hACE2). Both the heavy chain and the light chain of E77 interact extensively with RBD and contribute to the strong binding of RBD. E77 employs CDRL1 to engage Asn501 of RBD and the Asn-to-Tyr mutation could generate steric hindrance, abolishing the binding. In sum, the data provide the landscape for an in-depth understanding of immune escape of VOCs and rational antibody engineering against emerging variants of SARS-CoV-2.

Structural basis for receptor binding and broader interspecies receptor recognition of currently circulating Omicron sub-variants.

Multiple SARS-CoV-2 Omicron sub-variants, such as BA.2, BA.2.12.1, BA.4, and BA.5, emerge one after another. BA.5 has become the dominant strain worldwide. Additionally, BA.2.75 is significantly increasing in some countries. Exploring their receptor binding and interspecies transmission risk is urgently needed. Herein, we examine the binding capacities of human and other 28 animal ACE2 orthologs covering nine orders towards S proteins of these sub-variants. The binding affinities between hACE2 and these sub-variants remain in the range as that of previous variants of concerns (VOCs) or interests (VOIs). Notably, R493Q reverse mutation enhances the bindings towards ACE2s from humans and many animals closely related to human life, suggesting an increased risk of cross-species transmission. Structures of S/hACE2 or RBD/hACE2 complexes for these sub-variants and BA.2 S binding to ACE2 of mouse, rat or golden hamster are determined to reveal the molecular basis for receptor binding and broader interspecies recognition.

The mechanism of STING autoinhibition and activation.

2',3'-cGAMP, produced by the DNA sensor cGAS, activates stimulator of interferon genes (STING) and triggers immune response during infection. Tremendous effort has been placed on unraveling the mechanism of STING activation. However, little is known about STING inhibition. Here, we found that apo-STING exhibits a bilayer with head-to-head as well as side-by-side packing, mediated by its ligand-binding domain (LBD). This type of assembly holds two endoplasmic reticulum (ER) membranes together not only to prevent STING ER exit but also to eliminate the recruitment of TBK1, representing the autoinhibited state of STING. Additionally, we obtained the filament structure of the STING/2',3'-cGAMP complex, which adopts a bent monolayer assembly mediated by LBD and transmembrane domain (TMD). The active, curved STING polymer could deform ER membrane to support its ER exit and anterograde transportation. Our data together provide a panoramic vision regarding STING autoinhibition and activation, which adds substantially to current understanding of the cGAS-STING pathway.

Engineered Binding Microenvironments in Halogen Bonding Polymers for Enhanced Anion Sensing.

Mimicking Nature's polymeric protein architectures by designing hosts with binding cavities screened from bulk solvent is a promising approach to achieving anion recognition in competitive media. Accomplishing this, however, can be synthetically demanding. Herein we present a synthetically tractable approach, by directly incorporating potent supramolecular anion-receptive motifs into a polymeric scaffold, tuneable through a judicious selection of the co-monomer. A comprehensive analysis of anion recognition and sensing is demonstrated with redox-active, halogen bonding polymeric hosts. Notably, the polymeric hosts consistently outperform their monomeric analogues, with especially large halide binding enhancements of ca. 50-fold observed in aqueous-organic solvent mixtures. These binding enhancements are rationalised by the generation and presentation of low dielectric constant binding microenvironments from which there is appreciable solvent exclusion.

Based on molecular structures: Amyloid-ß generation, clearance, toxicity and therapeutic strategies.

Amyloid-ß (Aß) has long been considered as one of the most important pathogenic factors in Alzheimer's disease (AD), but the specific pathogenic mechanism of Aß is still not completely understood. In recent years, the development of structural biology technology has led to new understandings about Aß molecular structures, Aß generation and clearance from the brain and peripheral tissues, and its pathological toxicity. The purpose of the review is to discuss Aß metabolism and toxicity, and the therapeutic strategy of AD based on the latest progress in molecular structures of Aß. The Aß structure at the atomic level has been analyzed, which provides a new and refined perspective to comprehend the role of Aß in AD and to formulate therapeutic strategies of AD.

SNX27 suppresses SARS-CoV-2 infection by inhibiting viral lysosome/late endosome entry.

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27-retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.

Real-time Voltammetric Anion Sensing Under Flow.

The development of real-life applicable ion sensors, in particular those capable of repeat use and long-term monitoring, remains a formidable challenge. Herein, we demonstrate, in a proof-of-concept, the real-time voltammetric sensing of anions under continuous flow in a 3D-printed microfluidic system. Electro-active anion receptive halogen bonding (XB) and hydrogen bonding (HB) ferrocene-isophthalamide-(iodo)triazole films were employed as exemplary sensory interfaces. Upon exposure to anions, the cathodic perturbations of the ferrocene redox-transducer are monitored by repeat square-wave voltammetry (SWV) cycling and peak fitting of the voltammograms by a custom-written MATLAB script. This enables the facile and automated data processing of thousands of SW scans and is associated with an over one order-of-magnitude improvement in limits of detection. In addition, this improved analysis enables tuning of the measurement parameters such that high temporal resolution can be achieved. More generally, this new flow methodology is extendable to a variety of other analytes, including cations, and presents an important step towards translation of voltammetric ion sensors from laboratory to real-world applications.

3D­printed Ti6Al4V scaffolds combined with pulse electromagnetic fields enhance osseointegration in osteoporosis.

The loosening and displacement of prostheses after dental implantation and arthroplasty is a substantial medical burden due to the complex correction surgery. Three­dimensional (3D)­printed porous titanium (pTi) alloy scaffolds are characterized by low stiffness, are beneficial to bone ingrowth, and may be used in orthopedic applications. However, for the bio­inert nature between host bone and implants, titanium alloy remains poorly compatible with osseointegration, especially in disease conditions, such as osteoporosis. In the present study, 3D­printed pTi scaffolds with ideal pore size and porosity matching the bone tissue, were combined with pulse electromagnetic fields (PEMF), an exogenous osteogenic induction stimulation, to evaluate osseointegration in osteoporosis. In vitro, external PEMF significantly improved osteoporosis­derived bone marrow mesenchymal stem cell proliferation and osteogenic differentiation on the surface of pTi scaffolds by enhancing the expression of alkaline phosphatase, runt­related transcription factor­2, osteocalcin, and bone morphogenetic protein­2. In vivo, Microcomputed tomography analysis and histological evaluation indicated the external PEMF markedly enhanced bone regeneration and osseointegration. This novel therapeutic strategy has potential to promote osseointegration of dental implants or artificial prostheses for patients with osteoporosis.

Reagentless Redox Capacitive Assaying of C-Reactive Protein at a Polyaniline Interface.

Methods that enable the sensitive and label-free detection of protein biomarkers are well-positioned to make potentially significant contributions to diagnostics and derived personalized healthcare. In support of this goal, a myriad of (electrochemical) methodologies have been developed; recently, electrochemical capacitance spectroscopy emerged as an impedance-derived approach which, in employing surface-confined redox-transducers, circumvents problems associated with the use of solution-phase redox-probes. Herein, we expand this scope by utilizing phytic acid-doped polyaniline as a novel redox-charging polymer support enabling the reagentless assaying of C-reactive protein in serum with good sensitivity. The construction of the sensory interface via electropolymerization allows facile tuning of the surface coverage and redox (capacitive) properties of the polymers, which, in turn, modulate both assay selectivity, fouling, and sensitivity. Significantly, this methodology is readily extendable to a wide range of electrode materials and analytes.

Enhanced voltammetric anion sensing at halogen and hydrogen bonding ferrocenyl SAMs.

Halogen bonding mediated electrochemical anion sensing has very recently been established as a potent platform for the selective and sensitive detection of anions, although the principles that govern binding and subsequent signal transduction remain poorly understood. Herein we address this challenge by providing a comprehensive study of novel redox-active halogen bonding (XB) and hydrogen bonding (HB) ferrocene-isophthalamide-(iodo)triazole receptors in solution and at self-assembled monolayers (SAMs). Under diffusive conditions the sensory performance of the XB sensor was significantly superior. In molecular films the XB and HB binding motifs both display a notably enhanced, but similar, response to specific anions. Importantly, the enhanced response of these films is rationalised by a consideration of the (interfacial) dielectric microenvironment. These effects, and the resolved relationship between anion binding and signal transduction, underpin an improved fundamental understanding of anion sensing at redox-active interfaces which will benefit not just the development of more potent, real-life relevant, sensors but also new tools to study host-guest interactions at interfaces.

Cyclin Cyc2p is required for micronuclear bouquet formation in Tetrahymena thermophila.

Meiotic bouquet formation (known as crescent formation in Tetrahymena thermophila) is indispensable for homologous pairing and recombination, but the regulatory mechanism of bouquet formation remains largely unknown. As a conjugation specific cyclin gene, CYC2 knockout mutants failed to form an elongated crescent structure and aborted meiosis progress in T. thermophila. γ-H2A.X staining revealed fewer micronuclear DNA double-strand breaks (DSBs) in cyc2Δ cells than in wild-type cells. Furthermore, cyc2Δ cells still failed to form a crescent structure even though DSBs were induced by exogenous agents, indicating that a lack of DSBs was not completely responsible for failure to enter the crescent stage. Tubulin staining showed that impaired perinuclear microtubule structure may contribute to the blockage in micronuclear elongation. At the same time, expression of microtubule-associated kinesin genes, KIN11 and KIN141, was significantly downregulated in cyc2Δ cells. Moreover, micronuclear specific accumulation of heterochromatin marker trimethylated H3K23 abnormally increased in the cyc2Δ mutants. Together, these results show that cyclin Cyc2p is required for micronuclear bouquet formation via controlling microtubule-directed nuclear elongation in Tetrahymena.

Acid-Regulated Switching of Metal Cation and Anion Guest Binding in Halogen-Bonding Rotaxanes.

The ability of natural enzymes to regulate their guest binding affinities and preferences through the use of co-ligands which alter the features of the binding site is fundamental to biological homeostatic control. Herein, the rarely exploited orthosteric control of guest binding is demonstrated using neutral halogen bonding [2]rotaxanes, in which a chemical stimulus (acid) interacting with the interlocked host binding site switches the host's native guest preference from metal cations to anions. When neutral, the rotaxanes exhibit pronounced transition metal cation affinity and comparatively weak anion binding properties. However, the addition of acid attenuates the rotaxanes' ability to coordinate cations, while concurrently enabling strong binding of halides through charge assisted halogen bonding and hydrogen bonding interactions in competitive aqueous solvent media. The appendage of a fluorescent anthracene reporter group to the rotaxane framework also enables diagnostic sensory responses to cation/anion binding.

Unexpected solvent effects on the UV/Vis absorption spectra of o-cresol in toluene and benzene: in contrast with non-aromatic solvents.

Cresol is a prototype molecule in understanding intermolecular interactions in material and biological systems, because it offers different binding sites with various solvents and protonation states under different pH values. It is found that the UV/Vis absorption spectra of o-cresol in aromatic solvents (benzene, toluene) are characterized by a sharp peak, unlike the broad double-peaks in 11 non-aromatic solvents. Both molecular dynamics simulations and electronic structure calculations revealed the formation of intermolecular π-complexation between o-cresol and aromatic solvents. The thermal movements of solvent and solute molecules render the conformations of o-cresol changing between trans and cis isomers. The π-interaction makes the cis configuration a dominant isomer, hence leading to the single keen-edged UV/Vis absorption peak at approximately 283 nm. The free conformation changes between trans and cis in aqueous solution rationalize the broader absorption peaks in the range of 260-280 nm. The pH dependence of the UV/Vis absorption spectra in aqueous solutions is also rationalized by different protonation states of o-cresol. The explicit solvent model with long-ranged interactions is vital to describe the effects of π-complexation and electrostatic interaction on the UV/Vis absorption spectra of o-cresol in toluene and alkaline aqueous (pH > 10.3) solutions, respectively.

Cationic all-halogen bonding rotaxanes for halide anion recognition.

A family of cationic halogen bonding [2]rotaxanes have been synthesised via an active-metal template synthetic strategy. 1H NMR spectroscopic anion titration investigations reveal these interlocked host systems recognize halides selectively over oxoanions in aqueous-organic solvent media. Furthermore, systematically modulating the rigidity and size of the rotaxanes' anion binding cavities via metal complexation, as well as by varying the number of halogen bond-donor groups in the axle component, was found to dramatically influence halide anion selectivity.

Effects of AQP5 gene silencing on proliferation, migration and apoptosis of human glioma cells through regulating EGFR/ERK/ p38 MAPK signaling pathway.

We investigated the effects of aquaporin 5 (AQP5) gene silencing on the proliferation, migration and apoptosis of human glioma cells through regulating the EGFR/ERK/p38MAPK signaling pathway. qRT-PCR was applied to examine the mRNA expressions of AQP5 in five human glioma cell lines. U87-MG, U251 and LN229 cells were selected and assigned into blank, vector, AQP5 siRNA and FlagAQP5 groups. MTT assay was used to measure cell proliferation. Flow cytometry (FCM) with AnnexinV-FITC/PI double staining and PI staining were employed to analyze cell apoptosis and cell cycle respectively. Scratch test was used to detect cell migration. Western blotting was performed to determine the EGFR/ERK/p38 MAPK signaling pathway-related proteins. Results showed that the positive expression of AQP5 in primary glioblastoma was associated with the tumor size and whether complete excision was performed. The mRNA expressions of AQP5 in cell lines of U87-MG, U251 and LN229 were significantly higher than in U373 and T98G. The proliferation rates of U87-MG, U251 and LN229 cells in the AQP5 siRNA group were lower than in the vector and blank groups. The apoptosis rate increased in the AQP5 siRNA group compared with the vector group. Scratch test demonstrated that AQP5 gene silencing could suppress cell migration. Compared with the vector and blank groups, the AQP5 siRNA group showed decreased expressions of the ERK1/2, p38 MAPK, p-ERK1/2 and p-p38 MAPK proteins. AQP5 gene silencing could inhibit the cell proliferation, reduce cell migration and promote the cell apoptosis of U87-MG, U251 and LN229 by suppressing EGFR/ERK/p38 MAPK signaling pathway.

Two bisthienylethene-Ir(III) complexes showing acid/base-induced structural transformation and on-off luminescence switching in solution.

Complexes [Ir(dfppy)2(pbdtiH)](PF6)·2CHCl3 (1-H) and [Ir(dfppy)2(pbdti)] (1) were synthesized by the reaction of bisthienylethene pbdtiH and an [Ir(dfppy)2Cl]2 dimer under neutral and basic conditions, respectively. Thus, the {Ir(dfppy)2}(+) unit is coordinated by pbdtiH in 1-H, and by pbdti(-) in 1, which are confirmed by their crystal structures. The structures of 1-H and 1 could be interconverted in solution, upon alternately adding NEt3 and TFA, thus resulting in reversible luminescence switching between the on-state of 1-H and the off-state of 1 at room temperature. In addition, both 1-H and 1 show solid-state luminescence, with a broad emission at 534 nm and 525 nm, respectively. The free pbdtiH ligand shows photochromic behavior in CH2Cl2 solution. However, no photochromism has been observed in 1-H and 1, indicating that the coordination of the pbdtiH/pbdti(-) ligand to the {Ir(dfppy)2}(+) unit could suppress their photochromic behaviors.

IDH1R¹³²H decreases the proliferation of U87 glioma cells through upregulation of microRNA-128a.

Mutations in isocitrate dehydrogenase 1 (IDH1) are found in >70% of secondary glioblastomas and lower-grade gliomas (grades II-III). Among the numerous phenotypic differences between IDH1 mutant and wild-type glioma patients, the most salient is an improved survival rate for patients with a mutation. MicroRNAs (miRNAs) are a class of small, non­coding, single­stranded RNAs that can negatively regulate gene expression at the post­transcriptional level, predominantly by binding to the 3'­untranslated region of their target mRNAs. The dysregulated expression of several miRNAs has been reported to modulate glioma progression; however, it is unclear whether mutations in IDH1 regulate glioma cell proliferation through miRNA dysregulation. In the present study, stable overexpression of IDH1WT or IDH1R132H was established in the U87 glioma cell line. It was found that IDH1R132H decreased cell proliferation of U87 glioma cells by inducing the expression of the miRNA miR­128a. This process was dependent on the transcription factor hypoxia inducible factor­1α (HIF­1α), which binds to a hypoxia response element in the promoter of miR­128a. Furthermore, miR­128a negatively regulated the expression of B­cell­specific Moloney murine leukemia virus integration site 1 protein (Bmi­1), which is involved in suppressing cell proliferation. These findings suggest that the IDH1R132H­HIF­1α­miR­128a­Bmi­1 pathway is involved in glioma cell proliferation.

Early-stage hemangioblastoma presenting as a small lesion with significant edema in the cerebellum.

Hemangioblastomas are benign tumors that are frequently associated with peritumoral cysts; however, their early characteristics before cyst formation remain unclear. In this article, the authors present a novel case of a cerebellar hemangioblastoma presenting as a small solid lesion with significant edema. Surgery was performed to resect the tumor, and a follow-up magnetic resonance imaging scan revealed complete excision of the mass and resolution of the cerebellar edema. Histological examination confirmed that the lesion was a hemangioblastoma. This is the only report in the literature to describe the imaging and histopathologic characteristics of an initial hemangioblastoma in the cerebellum.