1The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated 2 efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health 3 emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion 4 protein that is metastable and difficult to produce recombinantly in large quantities. Here, we 5 designed and expressed over 100 structure-guided spike variants based upon a previously 6 determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical 7 and structural characterization of these variants identified numerous individual substitutions that 8 increased protein yields and stability. The best variant, HexaPro, has six beneficial proline 9 substitutions leading to ~10-fold higher expression than its parental construct and is able to 10 withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution 11 cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-12 yield production of a stabilized prefusion spike protein will accelerate the development of 13 vaccines and serological diagnostics for SARS-CoV-2. 14 3 INTRODUCTION 15 Coronaviruses are enveloped viruses containing positive-sense RNA genomes. Four human 16 coronaviruses generally cause mild respiratory illness and circulate annually. However, SARS-17 CoV and MERS-CoV were acquired by humans via zoonotic transmission and caused outbreaks 18 of severe respiratory infections with high case-fatality rates in 2002 and 2012, respectively 1,2 . 19 SARS-CoV-2 is a novel betacoronavirus that emerged in Wuhan, China in December 2019 and 20 is the causative agent of the ongoing COVID-19 pandemic 3,4 . As of May 26, 2020, the WHO has 21 reported over 5 million cases and 350,000 deaths worldwide. Effective vaccines, therapeutic 22 antibodies and small-molecule inhibitors are urgently needed, and the development of these 23 interventions is proceeding rapidly. 24 Coronavirus virions are decorated with a spike (S) glycoprotein that binds to host-cell 25 receptors and mediates cell entry via fusion of the host and viral membranes 5 . S proteins are 26 trimeric class I fusion proteins that are expressed as a single polypeptide that is subsequently 27cleaved into S1 and S2 subunits by cellular proteases 6,7 . The S1 subunit contains the receptor-28 binding domain (RBD), which, in the case of SARS-CoV-2, recognizes the angiotensin-29 converting enzyme 2 (ACE2) receptor on the host-cell surface [8][9][10] . The S2 subunit mediates 30 membrane fusion and contains an additional protease cleavage site, referred to as S2′, that is 31 adjacent to a hydrophobic fusion peptide. Binding of the RBD to ACE2 triggers S1 dissociation, 32 allowing for a large rearrangement of S2 as it transitions from a metastable prefusion 33 conformation to a highly stable postfusion conformation 6,11 . During this rearrangement, the 34 fusion peptide is inserted into the host-cell membrane after cleavage at S2′, and two h...

Background and Purpose-We measured the temporal evolution of the T2 and diffusion tensor imaging parameters after transient and permanent cerebral middle cerebral artery occlusion (MCAo) in macaques, and compared it to standard histological analysis at the study end point. Methods-Stroke was created in adult male macaques by occluding a middle cerebral artery branch for 3 hours (transient MCAo, nϭ4 or permanent occlusion, nϭ3). Conventional MRI and diffusion tensor imaging scans were performed 0 (acute day), 1, 3, 7, 10, 17, and 30 days after MCAo. Animals were euthanized after the final scan and the brains removed for histological analysis. Results-Apparent diffusion coefficient in the lesion was decreased acutely, fractional anisotropy was elevated, and T2 remained normal. Thereafter, apparent diffusion coefficient increased above normal, fractional anisotropy decreased to below normal, T2 increased to a maximum and then declined. Reperfusion at 3 hours accelerated these MRI changes.Only the fractional anisotropy value was significantly different between transient and permanent groups at 30 days. Final MRI-defined fractional lesion volumes were well correlated with corresponding histological lesion volumes. Permanent MCAO animals showed more severe histological damage than their transient MCAO counterparts, especially myelin damage and axonal swelling. Conclusions-Overall, the MRI evolution of stroke in macaques was closer to what has been observed in humans than in rodent models. This work supports the use of serial MRI in stroke studies in nonhuman primates.

The development of luminescent materials with concurrent multimodal emissions is a great challenge to improve security and data storage density. Lanthanide‐doped nanocrystals are particularly appropriate for such applications for their abundant intermediate energy states and distinguishable spectroscopic profiles. However, traditional lanthanide luminescent nanoparticles have a limited capacity for information storage or complexity to shield against counterfeiting. Herein, it is demonstrated that the combination of upconverting and downshifting emissions in a particulate designed lanthanide‐doped core@multishell nanoarchitecture allows the generation of multicolor dual‐modal luminescence over a wide spectral range for complex information storage. Precise control of lanthanide dopants distribution in the core and distinct shells enables simultaneous excitation of 980/808 nm focusing/defocusing laser and 254 nm light and produces complex upconverting emissions from Er, Tm, Eu, and Tb via multiphoton energy transfer processes and downshifting emissions from Eu and Tb via efficient energy transfer from Ce to Eu/Tb in Gd‐assisted lattices. It is experimentally proven that multiple visualized anti‐counterfeit and information encryption with facile decryption and authentication using screen‐printing inks containing the present core@multishell nanocrystals are practically applicable by selecting different excitation modes.

RATIONALE: Long-acting slow effective release antiretroviral therapy (LASER ART) was developed to improve patient regimen adherence, prevent new infections, and facilitate drug delivery to human immunodeficiency virus cell and tissue reservoirs. In an effort to facilitate LASER ART development, “multimodal imaging theranostic nanoprobes” were created. These allow combined bioimaging, drug pharmacokinetics and tissue biodistribution tests in animal models.METHODS: Europium (Eu3+)- doped cobalt ferrite (CF) dolutegravir (DTG)- loaded (EuCF-DTG) nanoparticles were synthesized then fully characterized based on their size, shape and stability. These were then used as platforms for nanoformulated drug biodistribution.RESULTS: Folic acid (FA) decoration of EuCF-DTG (FA-EuCF-DTG) nanoparticles facilitated macrophage targeting and sped drug entry across cell barriers. Macrophage uptake was higher for FA-EuCF-DTG than EuCF-DTG nanoparticles with relaxivities of r2 = 546 mM-1s-1 and r2 = 564 mM-1s-1 in saline, and r2 = 850 mM-1s-1 and r2 = 876 mM-1s-1 in cells, respectively. The values were ten or more times higher than what was observed for ultrasmall superparamagnetic iron oxide particles (r2 = 31.15 mM-1s-1 in saline) using identical iron concentrations. Drug particles were detected in macrophage Rab compartments by dual fluorescence labeling. Replicate particles elicited sustained antiretroviral responses. After parenteral injection of FA-EuCF-DTG and EuCF-DTG into rats and rhesus macaques, drug, iron and cobalt levels, measured by LC-MS/MS, magnetic resonance imaging, and ICP-MS were coordinate.CONCLUSION: We posit that these theranostic nanoprobes can assess LASER ART drug delivery and be used as part of a precision nanomedicine therapeutic strategy.

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Due to the moist environment and inevitable movement, efficient wound closure and healing of vulnerable joint skin remains a great challenge. Herein, a poly(γ‐glutamic acid)‐crosslinked amino‐functionalized PEGylated poly(glycerol sebacate) (γ‐PGA/PEGS‐NH2) adhesive hydrogel is reported. PEGS‐NH2 and γ‐PGA not only forms covalent amide bonds with biological tissue surfaces to achieve strong moist adhesion but also establishes a stable chemically crosslinked network in bulk hydrogels to resist deformation. Furthermore, bioinspired gallic acid‐modified chitosan (CS‐GA) is introduced to enhance moist adhesion via multiple hydrogen bonds and establish a dynamic physically crosslinked network to dissipate energy. Consequently, this adhesive hydrogel strongly adheres to moist biological tissue, showing an adhesion six times higher than that of fibrin glue and comparable to that of strong cyanoacrylate glue. Moreover, benefiting from high mechanical resilience and effective energy dissipation, 200 cycles of loading–unloading mechanical tests conducted under an adhesive state and a full‐thickness rat skin incision model applied on a dynamic nape further confirmed the desirable dynamic tissue adhesion and wound healing performance. Combining the above ideal features with their good injectability and shape‐adaptability to complex contours, such adhesive hydrogels are demonstrated to be promising candidates for joint wound closure and healing in moist and dynamic physiological environment.

Summary Under dehydration in plants, antagonistic activities of histone 3 lysine 4 (H3K4) methyltransferase and histone demethylase maintain a dynamic and homeostatic state of gene expression by orientating transcriptional reprogramming toward growth or stress tolerance. However, the histone demethylase that specifically controls histone methylation homeostasis under dehydration stress remains unknown. Here, we document that a histone demethylase, JMJ17, belonging to the KDM5/JARID1 family, plays crucial roles in response to dehydration stress and abscisic acid (ABA) in Arabidopsis thaliana. jmj17 loss‐of‐function mutants displayed dehydration stress tolerance and ABA hypersensitivity in terms of stomatal closure. JMJ17 specifically demethylated H3K4me1/2/3 via conserved iron‐binding amino acids in vitro and in vivo. Moreover, H3K4 demethylase activity of JMJ17 was required for dehydration stress response. Systematic combination of genome‐wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP‐seq) and RNA‐sequencing (RNA‐seq) analyses revealed that a loss‐of‐function mutation in JMJ17 caused an ectopic increase in genome‐wide H3K4me3 levels and activated a plethora of dehydration stress‐responsive genes. Importantly, JMJ17 bound directly to the chromatin of OPEN STOMATA 1 (OST1) and demethylated H3K4me3 for the regulation of OST1 mRNA abundance, thereby modulating the dehydration stress response. Our results demonstrate a new function of a histone demethylase under dehydration stress in plants.

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