Intrinsically disordered proteins and intrinsically disordered protein regions.

Intrinsically disordered proteins (IDPs) and IDP regions fail to form a stable structure, yet they exhibit biological activities. Their mobile flexibility and structural instability are encoded by their amino acid sequences. They recognize proteins, nucleic acids, and other types of partners; they accelerate interactions and chemical reactions between bound partners; and they help accommodate posttranslational modifications, alternative splicing, protein fusions, and insertions or deletions. Overall, IDP-associated biological activities complement those of structured proteins. Recently, there has been an explosion of studies on IDP regions and their functions, yet the discovery and investigation of these proteins have a long, mostly ignored history. Along with recent discoveries, we present several early examples and the mechanisms by which IDPs contribute to function, which we hope will encourage comprehensive discussion of IDPs and IDP regions in biochemistry textbooks. Finally, we propose future directions for IDP research.

Identification of trypsin-degrading commensals in the large intestine.

Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions1-3. However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells4,5. Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.

Monitoring Both Extended and Tryptic Forms of Stable Isotope-Labeled Standard Peptides Provides an Internal Quality Control of Proteolytic Digestion in Targeted Mass Spectrometry-Based Assays.

Targeted mass spectrometry (MS)-based proteomic assays, such as multiplexed multiple reaction monitoring (MRM)-MS assays, enable sensitive and specific quantification of proteotypic peptides as stoichiometric surrogates for proteins. Efforts are underway to expand the use of MRM-MS assays in clinical environments, which requires a reliable strategy to monitor proteolytic digestion efficiency within individual samples. Towards this goal, extended stable isotope-labeled standard (SIS) peptides (hE), which incorporate native proteolytic cleavage sites, can be spiked into protein lysates prior to proteolytic (trypsin) digestion, and release of the tryptic SIS peptide (hT) can be monitored. However, hT measurements alone cannot monitor the extent of digestion and may be confounded by matrix effects specific to individual patient samples; therefore, they are not sufficient to monitor sample-to-sample digestion variability. We hypothesized that measuring undigested hE, along with its paired hT, would improve detection of digestion issues compared to only measuring hT. We tested the ratio of the SIS pair measurements, or hE/hT, as a quality control (QC) metric of trypsin digestion for two MRM assays: a direct-MRM (398 targets) and an immuno-MRM (126 targets requiring immunoaffinity peptide enrichment) assay, with extended SIS peptides observable for 54% (216) and 62% (78) of the targets, respectively. We evaluated the quantitative bias for each target in a series of experiments that adversely affected proteolytic digestion (e.g., variable digestion times, pH, and temperature). We identified a subset of SIS pairs (36 for the direct-MRM, 7 for the immuno-MRM assay) for which the hE/hT ratio reliably detected inefficient digestion that resulted in decreased assay sensitivity and unreliable endogenous quantification. The hE/hT ratio was more responsive to a decrease in digestion efficiency than a metric based on hT measurements alone. For clinical-grade MRM-MS assays, this study describes a ready-to-use QC panel and also provides a road map for designing custom QC panels.

Site-Specific Activity-Based Protein Profiling Using Phosphonate Handles.

Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses immobilized metal-affinity chromatography-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe, enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID-ABPP (activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated afatinib derivative (PF-06672131). As PhosID-ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor, which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID-ABPP is highly complementary to biotin-based enrichment strategies in ABPP studies, with PhosID-ABPP providing the advantage of direct activity-based probe interaction site identification.

The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites.

Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.

Differentiation of Uc-MSCs into insulin secreting islet-like clusters by trypsin through TGF-beta signaling pathway.

Differentiation of human umbilical cord mesenchymal stem cells (Uc-MSCs) into islet-like clusters which are capable of synthesizing and secreting insulin can potentially serve as donors for islet transplantation in the patient deficiency in islet ß cell function both in type 1 or type 2 diabetic patients. Therefore, we developed an easy and higher efficacy approach by trypsinazing the Uc-MSCs and followed culture in differentiation medium to induce of Uc-MSCs differentiation into islet-like clusters, and the potential mechanism that in the early stage of differentiation was also investigated by using RNA-sequencing and bioinformatics. Results show that induction efficacy was reached to 98% and TGF-ß signaling pathway may play critical role in the early stage differentiation, it was further confirmed that the retardant effect of differentiation progress either in cell morphology or in islet specific genes expression can be observed upon blocking the activation of TGF-ß signaling pathway using specific inhibitor of LY2109761 (TßRI/II kinase inhibitor). Our current study, for the first time, development a protocol for differentiation of Uc-MSCs into islet-like clusters, and revealed the importance of TGF-ß signaling pathway in the early stage of differentiation of Uc-MSCs into islet-like clusters. Our study will provide alternative approach for clinical treatment of either type I or type II diabtes mellitus with dysfunctional pancreatic islets.

Exploring Options for Proximity-Dependent Biotinylation Experiments: Comparative Analysis of Labeling Enzymes and Affinity Purification Resins.

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.

Accelerating Proteomics Using Broad Specificity Proteases.

Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000× less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow "on-the-fly" digestion of samples in the future.

Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences.

Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.

Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands.

Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1 × 1010 clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a Kd of <80 nM, a Tm of >70 °C, and a t1/2 in 0.1 mg/ml trypsin of >20 h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10 min of cleaning-in-place with 0.1 M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets.

Metformin inhibits digestive proteases and impairs protein digestion in mice.

Metformin is among the most prescribed medications worldwide and the first-line therapy for type 2 diabetes. However, gastrointestinal side effects are common and can be dose limiting. The total daily metformin dose frequently reaches several grams, and poor absorption results in high intestinal drug concentrations. Here, we report that metformin inhibits the activity of enteropeptidase and other digestive enzymes at drug concentrations predicted to occur in the human duodenum. Treatment of mouse gastrointestinal tissue with metformin reduces enteropeptidase activity; further, metformin-treated mice exhibit reduced enteropeptidase activity, reduced trypsin activity, and impaired protein digestion within the intestinal lumen. These results indicate that metformin-induced protein maldigestion could contribute to the gastrointestinal side effects and other impacts of this widely used drug.

The anti-inflammatory mechanism of SAHA in acute pancreatitis through HDAC5/SLIT2/Akt/ß-catenin axis.

Acute pancreatitis (AP) is widely recognized to be an inflammation-related disease, in which HDAC was upregulated. The anti-inflammatory role of suberoylanilide hydroxamic acid (SAHA), a HDAC inhibitor, has been documented. In this context, this research was implemented to figure out whether SAHA manipulated inflammation in AP. Subsequent to induction of AP mouse model, HDAC5 expression was detected. The binding of HDAC5 and SLIT2 was detected by Co-Immunoprecipitation and Chromatin immunoprecipitation assays. SAHA treatment and gain- and loss-of-function approaches were used in AP mice and lipopolysaccharide (LPS)-induced pancreatic acinar cells. In mice, biochemical methods were implemented to measure activities of pancreatic lipase, trypsin, myeloperoxidase (MPO) and pancreatic edema, TUNEL staining to determine pancreatic cell apoptosis, and flow cytometry to assess the total number of leukocytes and neutrophils in pancreas. In pancreatic acinar cells, CCK-8 was performed to evaluate cell viability. HDAC5 exhibited overexpression in AP mice. Mechanical analysis showed that HDAC5 facilitated SLIT2 deacetylation to downregulate SLIT2, thus activating Akt/ß-catenin pathway in pancreatic acinar cells. SAHA treatment, HDAC5 silencing or SLIT2 overexpression diminished inflammation in AP in vivo and in vitro. SAHA treatment, HDAC5 silencing or SLIT2 overexpression reduced activities of pancreatic lipase, trypsin, MPO, pancreatic edema and cell apoptosis in AP mice as well as elevated viability of LPS-induced pancreatic acinar cells. SAHA might exert anti-inflammatory effects in AP mice via HDAC5/SLIT2/Akt/ß-catenin axis.

Proteome-Wide Mendelian Randomization Identifies Causal Links Between Blood Proteins and Acute Pancreatitis.

BACKGROUND & AIMS: Acute pancreatitis (AP) is a complex disease and the leading cause of gastrointestinal disease-related hospital admissions. Few therapeutic options exist for AP prevention. Blood proteins with causal evidence may represent promising drug targets, but few have been causally linked with AP. Our objective was to identify blood proteins linked with AP by combining genome-wide association meta-analysis and proteome-wide Mendelian randomization (MR) studies. METHODS: We performed a genome-wide association meta-analysis totalling 10,630 patients with AP and 844,679 controls and a series of inverse-variance weighted MR analyses using cis-acting variants on 4719 blood proteins from the deCODE study (N = 35,559) and 4979 blood proteins from the Fenland study (N = 10,708). RESULTS: The meta-analysis identified genome-wide significant variants (P <5 × 10-8) at 5 loci (ABCG5/8, TWIST2, SPINK1, PRSS2 and MORC4). The proteome-wide MR analyses identified 68 unique blood proteins that may causally be associated with AP, including 29 proteins validated in both data sets. Functional annotation of these proteins confirmed expression of many proteins in metabolic tissues responsible for digestion and energy metabolism, such as the esophagus, adipose tissue, and liver as well as acinar cells of the pancreas. Genetic colocalization and investigations into the druggable genome also identified potential drug targets for AP. CONCLUSIONS: This large genome-wide association study meta-analysis for AP identified new variants linked with AP as well as several blood proteins that may be causally associated with AP. This study provides new information on the genetic architecture of this disease and identified pathways related to AP, which may be further explored as possible therapeutic targets for AP.

Accelerated Screening of Protein-Ligand Interactions via Parallel T2-Weighted 19F-MRI.

In drug discovery, ligands are sought that modulate the (mal-)function of medicinally relevant target proteins. In order to develop new drugs, typically a multitude of potential ligands are initially screened for binding and subsequently characterized for their affinity. Nuclear magnetic resonance (NMR) is a well-established and highly sensitive technology for characterizing such interactions. However, it has limited throughput, because only one sample can be measured at a time. In contrast, magnetic resonance imaging (MRI) is inherently parallel and MR parameters can conveniently be encoded in its images, potentially offering increased sample throughput. We explore this application using a custom-built 9-fold sample holder and a 19F-MRI coil. With this setup, we show that ligand binding can be detected by T2-weighted 19F-MRI using 4-(trifluoromethyl)benzamidine (TFBA) and trypsin as the reporter ligand and target protein, respectively. Furthermore, we demonstrate that the affinity of nonfluorinated ligands can be determined in a competition format by monitoring the dose-dependent displacement of TFBA. By comparing 19F-T2-weighted MR images of TFBA in the presence of different benzamidine (BA) concentrations-all recorded in parallel-the affinity of BA could be derived. Therefore, this approach promises parallel characterization of protein-ligand interactions and increased throughput of biochemical assays, with potential for increased sensitivity when combined with hyperpolarization techniques.

Modulation of Capsid Dynamics in Bromoviruses by the Host and Heterologous Viral Replicase.

The three genomic and a single subgenomic RNA of Cowpea chlorotic mottle virus (CCMV), which is pathogenic to plants, is packaged into three morphologically indistinguishable icosahedral virions with T=3 symmetry. The two virion types, C1V and C2V, package genomic RNAs 1 (C1) and 2 (C2), respectively. The third virion type, C3+4V, copackages genomic RNA3 and its subgenomic RNA (RNA4). In this study, we sought to evaluate how the alteration of native capsid dynamics by the host and viral replicase modulate the general biology of the virus. The application of a series of biochemical, molecular, and biological assays revealed the following. (i) Proteolytic analysis of the three virion types of CCMV assembled individually in planta revealed that, while retaining the structural integrity, C1V and C2V virions released peptide regions encompassing the N-terminal arginine-rich RNA binding motif. In contrast, a minor population of the C3+4V virion type was sensitive to trypsin-releasing peptides encompassing the entire capsid protein region. (ii) The wild-type CCMV virions purified from cowpea are highly susceptible to trypsin digestion, while those from Nicotiana benthamiana remained resistant, and (iii) finally, the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis evaluated the relative dynamics of C3+4V and B3+4V virions assembled under the control of the homologous versus heterologous replicase. The role of viral replicase in modulating the capsid dynamics was evident by the differential sensitivity to protease exhibited by B3+4V and C3+4V virions assembled under the homologous versus heterologous replicase. Our results collectively conclude that constant modulation of capsid dynamics by the host and viral replicase is obligatory for successful infection. IMPORTANCE Infectious virus particles or virions are considered static structures and undergo various conformational transitions to replicate and infect many eukaryotic cells. In viruses, conformational changes are essential for establishing infection and evolution. Although viral capsid fluctuations, referred to as dynamics or breathing, have been well studied in RNA viruses pathogenic to animals, such information is limited among plant viruses. The primary focus of this study is to address how capsid dynamics of plant-pathogenic RNA viruses, namely, Cowpea chlorotic mottle (CCMV) and Brome mosaic virus (BMV), are modulated by the host and viral replicase. The results presented have improved and transformed our understanding of the functional relationship between capsid dynamics and the general biology of the virus. They are likely to provide stimulus to extend similar studies to viruses pathogenic to eukaryotic organisms.

Human astrovirus capsid protein releases a membrane lytic peptide upon trypsin maturation.

The human astrovirus (HAstV) is a non-enveloped, single-stranded RNA virus that is a common cause of gastroenteritis. Most non-enveloped viruses use membrane disruption to deliver the viral genome into a host cell after virus uptake. The virus-host factors that allow for HAstV cell entry are currently unknown but thought to be associated with the host-protease-mediated viral maturation. Using in vitro liposome disruption analysis, we identified a trypsin-dependent lipid disruption activity in the capsid protein of HAstV serotype 8. This function was further localized to the P1 domain of the viral capsid core, which was both necessary and sufficient for membrane disruption. Site-directed mutagenesis identified a cluster of four trypsin cleavage sites necessary to retain the lipid disruption activity, which is likely attributed to a short stretch of sequence ending at arginine 313 based on mass spectrometry of liposome-associated peptides. The membrane disruption activity was conserved across several other HAstVs, including the emerging VA2 strain, and effective against a wide range of lipid identities. This work provides key functional insight into the protease maturation process essential to HAstV infectivity and presents a method to investigate membrane penetration by non-enveloped viruses in vitro. IMPORTANCE Human astroviruses (HAstVs) are an understudied family of viruses that cause mild gastroenteritis but have recent cases associated with a more severe neural pathogenesis. Many important elements of the HAstV life cycle are not well understood, and further elucidating them can help understand the various forms of HAstV pathogenesis. In this study, we utilized an in vitro liposome-based assay to describe and characterize a previously unreported lipid disruption activity. This activity is dependent on the protease cleavage of key sites in HAstV capsid core and can be controlled by site-directed mutagenesis. Our group observed this activity in multiple strains of HAstV and in multiple lipid conditions, indicating this may be a conserved activity across the AstV family. The discovery of this function provides insight into HAstV cellular entry, pathogenesis, and a possible target for future therapeutics.

Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats.

While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation in vitro. IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.

Comparative evaluation of trypsin and elastase digestion techniques for isolation of murine retinal vasculature.

Dysfunctional pericytes and disruption of adherens or tight junctions are related to many microvascular diseases, including diabetic retinopathy. In this context, visualizing retinal vascular architecture becomes essential for understanding retinal vascular disease pathophysiology. Although flat mounts provide a demonstration of the retinal blood vasculature, they often lack a clear view of microaneurysms and capillary architecture. Trypsin and elastase digestion are the two techniques for isolating retinal vasculatures in rats, mice, and other animal models. Our observations in the present study reveal that trypsin digestion impacts the association between pericytes and endothelial cells. In contrast, elastase digestion effectively preserves these features in the blood vessels. Furthermore, trypsin digestion disrupts endothelial adherens and tight junctions that elastase digestion does not. Therefore, elastase digestion emerges as a superior technique for isolating retinal vessels, which can be utilized to collect reliable and consistent data to comprehend the pathophysiology of disorders involving microvascular structures.

Enzymatic Dissociation of DNA-Histone Condensates in an Electrophoretic Setting: Modulating DNA Patterning and Hydrogel Viscoelasticity.

Development of an energy-driven self-assembly process is a matter of interest for understanding and mimicking diverse ranges of biological and environmental patterns in a synthetic system. In this article, first we demonstrate transient and temporally controlled self-assembly of a DNA-histone condensate where trypsin (already present in the system) hydrolyzes histone, resulting in disassembly. Upon performing this dynamic self-assembly process in a gel matrix under an electric field, we observe diverse kinds of DNA patterning across the gel matrix depending on the amount of trypsin, incubation time of the reaction mixture, and gel porosity. Notably, here, the micrometer-sized DNA-histone condensate does not move through the gel and only free DNA can pass; therefore, transport and accumulation of DNA at different zones depend on the release rate of DNA by trypsin. Furthermore, we show that the viscoelasticity of the native gel increases in the presence of DNA and a pattern over gel viscoelasticity at different zones can be achieved by tuning the amount of enzyme, i.e., the dissociation rate of the DNA-histone condensate. We believe enabling spatiotemporally controlled DNA patterning by applying an electric field will be potentially important in designing different kinds of spatiotemporally distinct dynamic materials.

Ultrafast protein digestion using an immobilized enzyme reactor following high-resolution mass spectrometry analysis for rapid identification of abrin toxin.

Abrin toxin, highly dangerous with an estimated human lethal dose of 0.1-1 µg per kg body weight, has attracted much attention regarding criminal and terroristic misuse over the past decade. Therefore, developing a rapid detection method for abrin toxin is of great significance in the field of biosecurity. In this study, based on the specific dissociation method of an immobilized enzyme reactor, the trypsin immobilized reactor Fe3O4@CTS-GA-Try was prepared to replace free trypsin, and the immobilized enzyme digestion process was systematically investigated and optimized by using bovine serum albumin as the simulant of abrin. After 5 min one-step denaturation and reduction, a satisfactory peptide number and coverage were yielded with only 15 s assisted by an ultrasound probe to identify model proteins. Subsequently, abrin was rapidly digested using the established method, resulting in a stable and highly reproducible characteristic peptide number of 39, which can be analyzed by nanoelectrospray ionization coupled with high-resolution mass spectrometry. With the acquisition mode of full MS scan coupled with PRM, not only MS spectroscopy of total abrin peptides but also the corresponding MS/MS spectroscopy of specific abrin peptides can achieve the characteristic detection of abrin toxin and its different isoforms in less than 10 minutes, with high repeatability. This assay provides a universal platform and has great potential for the development of on-site detection and rapid mass spectrometric analysis techniques for macromolecular protein toxins and can further be applied to the integrated detection of chemical and biological agents.