The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that ...SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
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•SARS-CoV-2 uses ACE2 to enter target cells•SARS-CoV-2 and SARS-CoV bind with similar affinities to ACE2•Structures of SARS-CoV-2 spike glycoprotein in two conformations•SARS-CoV polyclonal antibodies inhibit SARS-CoV-2 spike-mediated entry into cells
SARS-CoV-2, a newly emerged pathogen spreading worldwide, binds with high affinity to human ACE2 and uses it as an entry receptor to invade target cells. Cryo-EM structures of the SARS-CoV-2 spike glycoprotein in two distinct conformations, along with inhibition of spike-mediated entry by SARS-CoV polyclonal antibodies, provide a blueprint for the design of vaccines and therapeutics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We developed a stand-alone, real-time optical detection device capable of reading fluorescence intensities from cell samples with high sensitivity and precision, for use as a portable fluorescent ...sensor for sensing fluorescently labeled enterohemorrhagic Escherichia coli (EHEC) Shiga toxins (Stxs). In general, the signal intensity from the fluorescently labeled Stxs was weak due to the small number of molecules bound to each cell. To address this technical challenge, we used a highly sensitive light detector (photomultiplier tube: PMT) to measure fluorescence, and designed a portable optical housing to align optical parts precisely; the housing itself was fabricated on a 3D printer. In addition, an electric circuit that amplified PMT output was designed and integrated into the system. The system shows the toxin concentration in the sample on a liquid crystal display (LCD), and a microcontroller circuit is used to read PMT output, process data, and display results. In contrast to other portable fluorescent detectors, the system works alone, without any peripheral computer or additional apparatus; its total size is about 17 x 13 x 9 cm.sup.3, and it weighs about 770 g. The detection limit was 0.01 ppm of Alexa Fluor 488 in PBS, which is ten thousand times lower than those of other smartphone-based systems and sufficiently sensitive for use with a portable optical detector. We used the portable real-time optical sensing system to detect Alexa Fluor 488-tagged Stx2B-subunits bound to monocytic THP-1 cells expressing the toxin receptor globotriaosylceramide (Gb3). The device did not detect a signal from Gb3-negative PD36 cells, indicating that it was capable of specifically detecting Stxs bound to cells expressing the toxin receptor. Following the development of a rapid and autonomous method for fluorescently tagging cells in food samples, the optical detection system described here could be used for direct detection of Shiga toxins in food in the field.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
BACKGROUND: The failure modes, time to development, and clinical relevance are known to differ between proximal junctional kyphosis (PJK) and proximal junctional failure (PJF). However, ...there are no reports that study the risk factors of PJK and PJF separately.
OBJECTIVE: The aim of this study was to investigate the risk factors for PJK and PJF separately.
METHODS: A retrospective study of 160 consecutive patients who underwent a long instrumented fusion to the sacrum for adult spinal deformity with a minimum follow-up of 2 years was conducted. A separate survivorship analysis of PJK and PJF was performed using the Cox proportional hazards model for the 3 categorical parameters of surgical, radiographic, and patient factors.
RESULTS: PJK developed in 27 patients (16.9%) and PJF in 29 patients (18.1%). The median survival time was 17.0 months for PJK and 3.0 months for PJF. Multivariate analyses revealed that a high body mass index was an independent risk factor for PJK (hazard ratio HR = 1.179), whereas the significant risk factors for PJF were older age, the presence of osteoporosis, the uppermost instrumented vertebra level at T11-L1, and a greater preoperative sagittal vertical axis (HR = 1.082, 6.465, 5.236, and 1.017, respectively). A large correction of sagittal deformity was shown to be a risk factor for PJF on univariate analyses, but not on multivariate analyses.
CONCLUSION: PJK developed at a median of 17 months and PJF at a median of 3 months. A high body mass index was an independent risk factor for PJK, whereas older age, osteoporosis, uppermost instrumented vertebra level at the thoracolumbar junction, and greater preoperative sagittal vertical axis were risk factors for PJF.
Targeting the interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor is a promising ...therapeutic strategy. We designed inhibitors using two de novo design approaches. Computer-generated scaffolds were either built around an ACE2 helix that interacts with the spike receptor binding domain (RBD) or docked against the RBD to identify new binding modes, and their amino acid sequences were designed to optimize target binding, folding, and stability. Ten designs bound the RBD, with affinities ranging from 100 picomolar to 10 nanomolar, and blocked SARS-CoV-2 infection of Vero E6 cells with median inhibitory concentration (IC
) values between 24 picomolar and 35 nanomolar. The most potent, with new binding modes, are 56- and 64-residue proteins (IC
~ 0.16 nanograms per milliliter). Cryo-electron microscopy structures of these minibinders in complex with the SARS-CoV-2 spike ectodomain trimer with all three RBDs bound are nearly identical to the computational models. These hyperstable minibinders provide starting points for SARS-CoV-2 therapeutics.
Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. ...In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.
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•SARS-CoV-2 RBD is immunodominant and accounts for 90% of serum neutralizing activity•RBD antibodies decline with a half-life of ∼50 days, but their avidity increases•Structural definition of a SARS-CoV-2 RBD antigenic map using monoclonal antibodies•ACE2-binding site dominates SARS-CoV-2 polyclonal neutralizing antibody responses
Serological analyses of ∼650 SARS-CoV-2-exposed individuals show that 90% of the serum or plasma neutralizing activity targets the virus receptor-binding domain, with structural insights revealing how distinct types of neutralizing antibodies targeting the ACE2-binding site dominate the immune response against SARS-CoV-2 spike.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Recent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways ...to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism.
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•MERS-CoV/SARS-CoV S composite glycan shields analyzed by cryo-EM and mass spectrometry•Structures of MERS-CoV/SARS-CoV S with neutralizing antibodies from survivors•LCA60 inhibits receptor binding by interacting with MERS-CoV S protein/glycans•S230 blocks receptor binding and triggers fusogenic rearrangements via functional mimicry
Structural analysis of the SARS-CoV S and MERS-CoV S glycoproteins in complex with neutralizing antibodies from human survivors sheds light into the mechanisms of membrane fusion and neutralization
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Current clinical therapeutic efficacy for the treatment of osteo‐ and rheumatoid‐arthritis is obviously limited. Although mesenchymal stem cells (MSCs) are considered as a source of promising ...regenerative therapy, un‐modified or genetically engineered MSCs injected in vivo restrict their clinical utility because of the low drug efficacy and unpredicted side effect, respectively. Herein, a strategy to enhance the migration efficacy of MSCs to inflamed joints via an inflammation‐mediated education process is demonstrated. To reinforce the limited anti‐inflammatory activity of MSCs, gold nanostar loaded with triamcinolone is conjugated to MSC. Furthermore, near‐infrared laser‐assisted photothermal therapy (PTT) induced by gold nanostar significantly elevates the anti‐inflammatory efficacy of the developed drugs, even in advanced stage arthritis model. An immunological regulation mechanism study of PTT is first suggested in this study; the expression of the interleukin 22 receptor, implicated in the pathogenesis of arthritis, is downregulated in T lymphocytes by PTT, and Th17 differentiation from naïve CD4 T cell is inhibited. Collectively, inflammation‐targeting MSCs conjugated with triamcinolone‐loaded gold nanostar (Edu‐MSCs‐AuS‐TA) promote the repolarization of macrophages and decrease neutrophil recruitment in joints. In addition, Edu‐MSCs‐AuS‐TA significantly alleviate arthritis‐associated pain, improve general locomotor activity, and more importantly, induce cartilage regeneration even for severe stages of arthritis model.
Gold nanostars‐conjugated mesenchymal stem cells, functionalized with triamcinolone, serve as a potent corticosteroids. Their therapeutic effectiveness is enhanced by near‐infrared laser irradiation, generating localized heat. Thermo‐responsive T lymphocytes exhibit diminished activity, attribute to interleukin 22 receptor inactivation under NIR laser exposure. This multifaceted approach adds therapeutic modulation in the treatment of severe arthritis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The development of an efficient catalytic activation (cleavage) system for C−H and C−C bonds is an important challenge in organic synthesis, because these bonds comprise a variety of organic ...molecules such as natural products, petroleum oils, and polymers on the earth. Among many elegant approaches utilizing transition metals to activate C−H and C−C bonds facilely, chelation-assisted protocols based on the coordinating ability of an organic moiety have attracted great attention, though they have often suffered from the need for an intact coordinating group in a substrate. In this Account, we describe our entire efforts to activate C−H or C−C bonds adjacent to carbonyl groups by employing a new concept of metal−organic cooperative catalysis (MOCC), which enables the temporal installation of a 2-aminopyridyl group into common aldehydes or ketones in a catalytic way. Consequently, a series of new catalytic reactions such as alcohol hydroacylation, oxo-ester synthesis, C−C triple bond cleavage, hydrative dimerization of alkynes, and skeletal rearrangements of cyclic ketones was realized through MOCC. In particular, in the quest for an optimized MOCC system composed of a Wilkinson’s catalyst (Ph3P)3RhCl and an organic catalyst (2-amino-3-picoline), surprising efficiency enhancements could be achieved when benzoic acid and aniline were introduced as promoters for the aldimine formation process. Furthermore, a notable accomplishment of C–C bond activation has been made using 2-amino-3-picoline as a temporary chelating auxiliary in the reactions of unstrained ketones with various terminal olefins and Wilkinson’s catalyst. In the case of seven-membered cyclic ketones, an interesting ring contraction to five- or six-membered ones takes place through skeletal rearrangements initiated by the C−C bond activation of MOCC. On the other hand, the fundamental advances of these catalytic systems into recyclable processes could be achieved by immobilizing both metal and organic components using a hydrogen-bonded self-assembled system as a catalyst support. This catalyst-recovery system provides a homogeneous phase at high temperature during the reaction and a heterogeneous phase at room temperature after the reaction. The product could be separated conveniently from the self-assembly support system by decanting the upper layer. The immobilized catalysts of both 2-aminopyridine and rhodium metal species sustained high catalytic activity for up to the eight catalytic reactions. In conclusion, the successful incorporation of an organocatalytic cycle into a transition metal catalyzed reaction led us to find MOCC for C−H and C−C bond activation. In addition, the hydrogen-bonded self-assembled support has been developed for an efficient and effective recovery system of homogeneous catalysts and could be successful in immobilizing both metal and organic catalysts.
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IJS, KILJ, NUK, PNG, UL, UM
Despite the lysosomal "proton sponge hypothesis" being considered to be an additional factor for stimulating the cellular toxicity of nanoparticle-based drug delivery systems, a clear relationship ...between the massive influx of calcium ions and the proton sponge effect, both of which are associated with cancer cell apoptosis, has still not been elucidated. Cetrimonium bromide (CTAB: cationic quaternary amino group based) gold nanorods possessed a more effective electric surface charge for inducing the lysosomal proton sponge effect than anionic gold nanoparticles. In this aspect, identifying released cytoplasmic Cl
−
, arising from the ruptured lysosomal compartment, in the cytoplasm is critical for supporting the "proton sponge hypothesis". This study clarified that the burst release of Cl
−
, as a result of lysosomal swelling by CTAB gold nanorods, stimulates the transient receptor potential channels melastatin 2 (TRPM2) channels, and subsequently induces a massive Ca
2+
influx, which independently increases apoptosis of cancer cells. Although the previous concept of elevated cancer apoptosis acting through the proton sponge effect is unclear, this study supports the evidence that a massive Ca
2+
influx mediated in response to a burst release of Cl
−
significantly influenced cytotoxicity of cancer cells in tumor tissues.
Schematic image illustrates the mechanism of Cl
−
release through the proton sponge by the cationic AuNR induced by TRPM2 activation.
The rapid and sensitive classification of bacteria is the first step of bacterial community research and the treatment of infection. Herein, a fluorescent probe BacGO is presented, which shows the ...best universal selectivity for Gram‐positive bacteria among known probes with a minimum staining procedure for sample detection and enrichment of the live bacteria. BacGO could also be used to assess of the Gram status in the bacterial community from wastewater sludge. Furthermore, BacGO could sensitively and selectively detect a Gram‐positive bacterial infection, not only in vitro but also using an in vivo keratitis mouse model. BacGO provides an unprecedented research tool for the study of dynamic bacterial communities and for clinical application.
BacGO, a novel Gram‐positive bacterial probe, was developed from a library of fluorescent molecules with a boronic‐acid motif that binds to peptidoglycan on the Gram‐positive bacterial cell wall. BacGO can be used to identify Gram‐positive bacteria in diverse, highly complex samples, and is an attractive alternative to Gram staining.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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