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Proteins and polysaccharides have been frequently used in recent years to prepare environment-friendly packaging materials. However, films based on proteins or polysaccharides alone often have poor performance as packaging, so they need to be combined to improve properties. In this work, we applied enzyme technology to prepare sodium caseinate (SC)-carboxymethyl chitosan (CMC) films, incorporating epigallocatechin gallate (EGCG) as bridging molecules and antibacterial agents. SC-EGCG-CMC ternary conjugate was firstly synthesized by tyrosinase (Tyr), and the composite films were then prepared with the aid of glycerol. Under tyrosinase catalytic conditions, EGCG could cross-link with SC and CMC covalently. The effects of different concentrations of EGCG and tyrosinase on mechanical properties, water vapor permeability, antibacterial properties and free radical scavenging ability were studied. The crosslinking degree and mechanical properties were improved with the increase of EGCG and tyrosinase content. The film showed good antibacterial activity against Gram-positive bacteria. In addition, the antibacterial activity and free radical scavenging ability increased with the increase of EGCG concentration. This work provides an efficient enzymatic method to prepare films with good strength and antibacterial properties, which can be used to improve the storage quality of foods.

Research on pillar[n]arenes has witnessed a very quick expansion. This emerging class of functionalized macrocyclic oligoarenes not only offers host–guest properties due to the presence of the central cavity, but also presents a wide variety of covalent functionalization possibilities. This short review focuses on copillararenes, a subfamily of pillar[n]arenes. In copillararenes, at least one of the hydroquinone units bears different functional groups compared to the others. After having defined the particular features of copillararenes, this short review compares the different synthetic strategies allowing their construction. Some key applications and future perspectives are also described. 1 Introduction2 General Features of Pillar[5]arenes3 Synthesis of Functionalized Copillar[4+1]arenes4 Concluding Remarks

The original version of this Article contained a factual error within the discussion section, which incorrectly stated that SARS-CoV-2 expresses a hemagglutinin esterase. This has been corrected in the PDF and HTML version of the Article by the removal of the corresponding text. The deleted text is reproduced below: Besides the spike glycoprotein, coronaviruses, including SARS-CoV-2, express at their surface the hemagglutinin esterase (HE) that often contains an active lectin domain that mediates glycan binding in several other viruses. However, the HE lectin domains of HCoV-OC43 and HCoV-HKU1 lost their glycan binding capacity due to mutations and deletion during adaptation to human host62. Our result also suggests that HE might not be directly involved in 9-AcSA binding, as binding kinetics to AcSA is similar on purified S1 and at the virion level. However, as their esterase activity is maintained, HE could be involved in the deacetylation during release of viral progeny from the host cell surface and possibly also for breaking decoy interactions of the S protein with O-AcSA carrying mucins63.

An epidemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide. SARS-CoV-2 relies on its spike protein to invade host cells by interacting with the human receptor protein Angiotensin-Converting Enzymes 2 (ACE2). Therefore, designing an antibody or small-molecular entry blockers is of great significance for virus prevention and treatment. This study identified five potential small molecular anti-virus blockers via targeting SARS-CoV-2 spike protein by combining in silico technologies with in vitro experimental methods. The five molecules were natural products that binding to the RBD domain of SARS-CoV-2 was qualitatively and quantitively validated by both native Mass Spectrometry (MS) and Surface Plasmon Resonance (SPR). Anti-viral activity assays showed that the optimal molecule, H69C2, had a strong binding affinity (dissociation constant KD) of 0.0947 µM and anti-virus IC50 of 85.75 µM.

Cross-linking mass spectrometry methods have not been successfully applied to protein-protein interaction discovery at a proteome-wide level mainly due to the computation complexity (O (n2)) issue. In a previous report, we proposed a decision tree searching strategy (DTSS), which can reduce complexity by orders of magnitude. In this study, we further found that the monolinked peptides carry out the information on the retention time of the corresponding cross-linked pairs; therefore, the retention time of cross-linked peptide pairs can be predicted accurately. By utilizing the retention time as an extra filter, the false positive rate can be reduced by around 86% with a sensitivity loss of 10%. The method combined with DTSS (T-DTSS) not only benefits improving identification confidence but also leads to lower cutoff scores and facilitates substantially increasing inter-cross-link identification. T-DTSS was successfully applied to the identification of inter-cross-links obtained from Escherichia coli cell lysate cross-linked by a newly synthesized enrichable cross-linker, pDSBE. The approach can be applicable to both cleavable and noncleavable methods.

Biodegradable films constructed from food ingredients are being developed for food coating and packaging applications to create more sustainable and environmentally friendly alternatives to plastics and other synthetic film-forming materials. In particular, there is a focus on the creation of active packaging materials from natural ingredients, especially plant-based ones. The film matrix is typically constructed from film-forming food components, such as proteins, polysaccharides and lipids. These matrices can be fortified with active ingredients, such as antioxidants and antimicrobials, so as to enhance their functional properties. Edible active films must be carefully designed to have the required optical, mechanical, barrier, and preservative properties needed for commercial applications. This review focuses on the fabrication, properties, and functional performance of edible films constructed from natural active ingredients. It provides an overview of the type of active ingredients that can be used, how they interact with the film matrix, how they migrate through the films, and how they are released. It also discusses the potential application of these active films for food preservation. Finally, future trends are highlighted and areas where further research are required are discussed.

Active biodegradable packaging are being developed from biodegradable biopolymers which may solve the environmental problems caused by petroleum-based materials (plastics), as well as improving the shelf life, quality, nutritional profile, and safety of packaged food. The functional performance of active ingredients in biodegradable packaging can be extended by controlling their release profiles. This can be achieved by incorporating active ingredients in sandwich-structured packaging including multilayer and composite packaging. In multilayer materials, the release profile can be controlled by altering the type, structure, and thickness of the different layers. In composite materials, the release profile can be manipulated by altering the interactions of active ingredients with the surrounding biopolymer matrix. This article reviews the preparation, properties, and applications of multilayer and composite packaging for controlling the release of active ingredients. Besides, the basic theory of controlled release is also elaborated, including diffusion, swelling, and biodegradation. Mathematical models are presented to describe and predict the controlled release of active ingredients from thin films, which may help researchers design packaging materials with improved functional performance.

Using T‐cell chimeric antigen receptors (CAR‐T) to activate and redirect T cells to tumors expressing the cognate antigen represents a powerful approach in cancer therapy. However, normal tissues with low expression of tumor‐associated antigens (TAAs) can be mistargeted, resulting in severe side effects. An approach using a collection of T cells expressing a diverse, 106‐member combinatorial cellular library of CARs, in which members can be specifically enriched based on avidity for cell membrane antigens, is reported. Using CD38 as the target antigen, an efficient and effective selection of CARs specifically recognizing CD38+ tumor cells is demonstrated. These selected CAR‐T's produce cytokines known to be associated with T cell activation in a CD38 expression‐dependent manner. This avidity‐based selection endows the engineered T cells with minimal off‐tumor effects, while retaining robust antitumor efficacy both in vitro and in vivo. The described method may facilitate the application of CAR‐T therapy to TAAs previously considered undruggable., A novel polyclonal combinatorial cellular library of chimeric antigen receptors provides a comprehensive coverage of immune responses to specific antigens in vitro. An efficient and effective avidity‐based selection strategy takes advantage of paracrine signaling systems in cell–cell interactions, which endow the engineered T cells with robust antitumor efficacy both in vitro and in vivo with minimal on‐target off‐tumor effects.

In this study, novel copillar[4+1]arenes were used as central heteromultivalent scaffolds via orthogonal couplings with a series of biologically relevant molecules such as carbohydrates, α-amino acids, biotin and phenylboronic acid. Further modifications by introducing maleimides or cyclooctyne groups provided molecular probes adapted to copper-free click chemistry. An octa-azidated fluorescent rotaxane bearing two distinct ligands was also generated in a fully controlled manner.

With the gradual maturity of the domestic financial quantitative trading market, more and more investors have recognized the quantitative investment because of its relatively stable income. Investment trading is not interfered by human beings. Analyzing the historical data of market trading has the characteristics of high probability of winning. This paper will take s300etf(159912) as an example, combine the double moving average and Counter-market operating indicator(CDP), explain the application law of the indicator, and discuss how to better grasp the market and achieve higher return on investment through the two indicators. Stock data is a classical financial time series, so Long Short-Term Memory(LSTM), as a classical model in the cyclic neural network model, can predict the trading index through LSTM. In this paper, the backtrader platform is used to simulate the transaction of historical data, and the benefits of the simulated transaction are discussed.

Table S1. sgRNA-targeted genomic sites in this study. Table S2. Primer list.

Figure S1. Comparison of the in vitro activity of M13 and f1 phage G8PPD. Figure S2. MS analyses of the interface between SpCas9 and M13 G8PPD. Figure S3. Construction and purification of SpCas9 K1158 and K1176 mutants. Figure S4. Profile of SpCas9-induced mutations in the absence and presence of G8PPD. Figure S5. G8PPD peptides derived from inoviridae bacteriophages. Figure S6. NGS analyses of the effects of G8P pre-incubation on NmCas9 and SaCas9 in HEK293 cells. Figure S7. NGS analyses of the effects of G8Ps on the specificity of SpCas9 at HBB site in HEK293 cells. Figure S8. Cytotoxicity of CRISPR inhibitors. Figure S9. ChIP-qPCR analyses of the effects of M13 G8Ps on Cas9 binding at AAVS1 on target and pre-determined off-target sites in Hela cells.

Review history.

A number of vinylsulfonamides were synthesized and screened to identify reagents that can be used to modify octreotide under biological pH and room temperature with improved efficiency. N-Phenyl-N-aceto-vinylsulfonamide exhibits higher reactivity and has emerged as an efficient reagent that has the ability to realize the selective modification of peptides and proteins at the N-terminus via aza-Michael addition. We showed that, after conjugation of peptides and proteins with the reagent containing a bioorthogonal functional group, the derivatives could be further labelled by functionalities, including fluorescent tags, modified drugs and polyethylene glycol (PEG) polymers without the need for prior treatment. Somatostatin, lysozyme, and RNaseA were selectively modified at the N-terminus, which illustrated the application of the method.

Inducing macromolecular interactions with small molecules to activate cellular signaling is a challenging goal. PROTACs (proteolysis-targeting chimeras) are bifunctional molecules that recruit a target protein in proximity to an E3 ubiquitin ligase to trigger protein degradation. Structural elucidation of the key ternary ligase-PROTAC-target species and its impact on target degradation selectivity remain elusive. We solved the crystal structure of Brd4 degrader MZ1 in complex with human VHL and the Brd4 bromodomain (Brd4BD2). The ligand folds into itself to allow formation of specific intermolecular interactions in the ternary complex. Isothermal titration calorimetry studies, supported by surface mutagenesis and proximity assays, are consistent with pronounced cooperative formation of ternary complexes with Brd4BD2. Structure-based-designed compound AT1 exhibits highly selective depletion of Brd4 in cells. Our results elucidate how PROTAC-induced de novo contacts dictate preferential recruitment of a target protein into a stable and cooperative complex with an E3 ligase for selective degradation.

Background CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies. Results We report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity. Conclusion G8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

A novel water soluble, phosphate-based enrichable, retro-Michael addition-driven MS-cleavable and multi-targeting cross-linker was developed., Chemical cross-linking mass spectrometry (XL-MS) is a powerful technology for obtaining protein structural information and studying protein–protein interactions. We report phospho-bisvinylsulfone (pBVS) as a novel water-soluble, MS-cleavable, phosphate-based enrichable and multi-targeting cross-linker. In this approach, the fragmentation of pBVS cross-linked peptides occurs in situ through retro-Michael addition. The phosphate group is successfully used as a small affinity tag to isolate cross-linked peptides from the highly abundant non-cross-linked peptides. In addition, the linker targets multiple types of amino acid residues, including cysteine, lysine and histidine. This method was applied to cross-link bovine serum albumin (BSA), myoglobin and Lbcpf1 demonstrating the ability to yield accurate and abundant information to facilitate protein structure elucidation.

Protein S-glutathionylation is a reversible post-translational modification regulating sulfhydryl homeostasis. However, little is known about the proteins and pathways regulated by S-glutathionylation in whole organisms and current approaches lack the sensitivity to examine this modification under basal conditions. We now report the quantification and identification of S-glutathionylated proteins from animal tissue, using a highly sensitive methodology combining high-accuracy proteomics with tandem mass tagging to provide precise, extensive coverage of S-glutathionylated targets in mouse liver. Critically, we show significant enrichment of S-glutathionylated mitochondrial and Krebs cycle proteins, identifying that S-glutathionylation is heavily involved in energy metabolism processes in vivo. Furthermore, using mice nulled for GST Pi (GSTP) we address the potential for S-glutathionylation to be mediated enzymatically. The data demonstrate the impact of S-glutathionylation in cellular homeostasis, particularly in relation to energy regulation and is of significant interest for those wishing to examine S-glutathionylation in an animal model.

We have previously shown that N-methyl- N'-nitro- N-nitrosoguanidine (MNNG), a well-known DNA alkylating agent and carcinogen, can induce multiple cellular responses with dynamic characteristics, including such responses as nontargeted mutations (NTM) at undamaged bases in DNA, up-regulation of low fidelity DNA polymerases, clustering of epidermal growth factor receptor (EGFR) and interference with its downstream signaling pathway. A dose-related analysis also revealed that different concentrations of MNNG can trigger diverse proteome changes associated with different cytotoxic effects. To further understand the dynamic cellular responses and hazardous effects caused by environmental carcinogen, a proteomic time-course study of whole cellular proteins from human amniotic epithelial cells after MNNG treatment was performed. Analysis at three different time points (3, 12 and 24 h after exposure) revealed that the major changes were taking place around 3 and 12 h after exposure. Using MALDI-TOF MS coupled with a micro solid-phase extraction (SPE) device, 90% ( n = 70) differentially expressed proteins were identified. Functional assignment revealed that many important pathways were affected, including the protein biosynthesis pathway and Ran GTPase system. We also carried out a network analysis of these proteins and the data suggest a central role for some key regulators in different pathways.

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of approximately 2%. An apparently identical disease occurs in multiple species including but not limited to cats, dogs, and rabbits. Although the precise aetiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here we use a bottom up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG – a consistently affected tissue) from EGS affected patients and appropriate control cases postmortem. In what appears to be the first proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease oflarge animals (not a model of human disease), we identified 2311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls.Further examination of selected proteomic candidates by quantitative fluorescent western blotting (QFWB) and sub-cellular expression profiling by immunohistochemistry, highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein(Tau) and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: 1. Diseases & disorders including; neurological disease, skeletal & muscular disorders; 2. Molecular and cellular functions: including cellular assembly & organisation, cell-to-cell signalling and interaction (including epinephrine, dopamine & adrenergic signalling and receptor function) and small molecule biochemistry. Interestingly,whilst the biofunctions identified in this study may represent pathways underpinning EGS induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to smallanimal models with altered neuronal vulnerability, and human neurological conditions.Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals.

The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signaling. In cardiac muscle, caveolae concentrate signaling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation–contraction coupling, and consequently cardiac contractility. Proteomic analysis of cardiac caveolae is hampered by the presence of contaminants that have sometimes, erroneously, been proposed to be resident in these domains. Here we present the first unbiased analysis of the proteome of cardiac caveolae, and investigate dynamic changes in their protein constituents following adrenoreceptor (AR) stimulation. Rat ventricular myocytes were treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae. Buoyant caveolin-enriched microdomains (BCEMs) were prepared from MβCD-treated and control cell lysates using a standard discontinuous sucrose gradient. BCEMs were harvested, pelleted, and resolubilized, then alkylated, digested, and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as BCEM resident if they were consistently depleted from the BCEM fraction following MβCD treatment. Selective activation of α-, β1-, and β2-AR prior to preparation of BCEMs was achieved by application of agonist/antagonist pairs for 10 min in populations of field-stimulated myocytes. We typically identified 600–850 proteins per experiment, of which, 249 were defined as high-confidence BCEM residents. Functional annotation clustering indicates cardiac BCEMs are enriched in integrin signaling, guanine nucleotide binding, ion transport, and insulin signaling clusters. Proteins possessing a caveolin binding motif were poorly enriched in BCEMs, suggesting this is not the only mechanism that targets proteins to caveolae. With the notable exception of the cavin family, very few proteins show altered abundance in BCEMs following AR activation, suggesting signaling complexes are preformed in BCEMs to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.

High detection sensitivity and resolution are two critical parameters for recording good peptide mass fingerprints (PMF) of low abundance proteins. This paper reports a mass spectrometry (MS) sample preparation technique that could improve sensitivity and resolution. By coating the MS steel target with a thin layer of pentadecafluorooctamido propyltrimethoxysilane, which was both polar and nonpolar solvent repellent, the transferred sample droplets on its surface were significantly smaller. As a result, the analyte of the peptide mixture became more concentrated and homogeneous, which helped to improve the sensitivity. The advantages of a modified MS target were documented by mass spectra improvement of attomole level standard peptides and silver-stained proteins from polyacrylamide gels. The mass signal of angiotensin II at 100 attomole was difficult to record on the conventional support, whereas it was easily detected on the modified one. The PMF of cytochrome C was also better recorded on the modified support, in terms of both signal-to-noise ratio and the number of detected peptides. When silver-stained proteins from two-dimensional electrophoresis gels were analyzed, in most cases more satisfactory peptide mass spectra were obtained from the modified support. Searching protein databases with more mass data from the improved PMFs, several unknown proteins were successfully identified.

The tetranuclear molybdenum cluster {[Mo 4 S 4 (μ-OAc) 2 (dtp) 4 ·H](CH 3 ) 2 CO} ( 4 ) (dtp=S 2 P(OEt) 2 ) was obtained by the reaction of [Mo 3 S 4 (dtp) 4 (H 2 O)] with Mo(CO) 6 in HOAc/Ac 2 O medium and then by recrystallization from acetone. The crystal belongs to the space group C 4 2n - P 2/ n with two molecules in a unit cell whose dimensions are a =17.271(3), b =11.985(3), c =13.061(2) A and β=105.94(1)°, V =2599(2) A 3 , D c =1.826 g/cm 3 . On the basis of 3613 independent reflections with I ⩾3σ( I ), the structure was refined to R =0.040, R w =0.055. The cluster anion [Mo 4 S 4 (μ-OAc) 2 (dtp) 4 ] − contains a [Mo 4 S 4 ] 5+ cubane type core. This formulation is supported by XPS, ESR and 1 H NMR measurements of 4 . There are 11 electrons which make up six MoMo bonds.

This paper expands the application of the newly developed highly sensitive microfluidic chip-based Edman degradation system. Comparison between the MS/MS spectra of a native peptide and its N-terminus truncated counterpart after carrying out one cycle of Edman degradation in a microfluidic chip can not only provide N-terminal residue information, but also facilitate the identification of different series of fragment ions. Manual peptide sequencing is more feasible and rapid using this method as demonstrated with three peptide examples including one neuropeptide. Furthermore, two cycles of Edman degradation allow the determination of the exact value of b 2 ion of the intact peptide, which can serve as an internal calibrant to increase the mass accuracy of the MS/MS spectrum.

A nano-scale solid-phase extraction (SPE) device was developed for the detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with a simplified microfabrication technology. By using SU-8 photoresist instead of epoxy glue to connect the microchannel and transfer capillary, polymeric contaminant signals in MS analysis were significantly reduced. Micro SPE columns with different capacities and geometric characteristics were investigated in order to increase the detection sensitivity and decrease spot size for MALDI-TOF-MS analysis. It is shown that enhancements in sensitivities for the detection of proteins in low abundance were correlated with the reduction in column capacity and increase in column aspect ratio. Fifty nanoliters of matrix solution were sufficient to elute the sample completely from the optimized micro SPE column with 3.5 nL capacity. The mass spectrum of a 5 fmol in-gel tryptic digest of bovine serum albumin (BSA), processed by the micro SPE column, demonstrated that 29 peptides matched the protein giving a sequence coverage of 51%, which was better than that obtained from analysis of 25 fmol of the same sample prepared by the dried-droplet method. With the micro SPE column treatment of 2 µL of digestion supernatant of a gel spot of the IQGAP1 protein, 15 peptides were detected from the mass spectrum with the highest individual score of 111, while, with a ZipTip procedure, only nine peaks were detected with the highest individual score of 71. Analytical results demonstrated that this approach greatly improved the sequence coverage and identification specificity for the tested protein. It can serve as a very useful tool in proteomics studies, especially for low abundance proteins. Copyright © 2006 John Wiley & Sons, Ltd.

A novel microfluidic chip based Edman degradation system is developed, in which Edman degradation can be carried out with peptide at a subfemtomole level; combined with MALDI-TOF-MS detection, the identification specificity of gel-separated protein in low abundance has been drastically improved.