An efficient strategy with a synergistic effect of hydrophilic and electrostatic interactions for simultaneous enrichment of N- and O-glycopeptides.

N- and O-glycosylation modifications of proteins are closely linked to the onset and development of many diseases and have gained widespread attention as potential targets for therapy and diagnosis. However, the low abundance and low ionization efficiency of glycopeptides as well as the high heterogeneity make glycosylation analysis challenging. Here, an enrichment strategy, using Knoevenagel copolymers modified with polydopamine-adenosine (denoted as PDA-ADE@KCP), was firstly proposed for simultaneous enrichment of N- and O-glycopeptides through the synergistic effects of hydrophilic and electrostatic interactions. The adjustable charged surface and hydrophilic properties endow the material with the capability to achieve effective enrichment of intact N- and O-glycopeptides. The experimental results exhibited excellent selectivity (1 : 5000) and sensitivity (0.1 fmol µL-1) of the prepared material for N-glycopeptides from standard protein digest samples. Moreover, it was further applied to simultaneous capturing of N- and O-glycopeptides from mouse liver protein digests. Compared to the commercially available zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) material, the number of glycoproteins corresponding to all N- and O-glycopeptides enriched with PDA-ADE@KCP was much more than that with ZIC-HILIC. Furthermore, PDA-ADE@KCP captured more O-glycopeptides than ZIC-HILIC, revealing its superior performance in O-glycopeptide enrichment. All these results indicated that the strategy holds immense potential in characterizing N- and O-intact glycopeptides in the field of proteomics.

Dual-Valence Characteristics of Be11: Tin/Lead-like Superatom.

To enhance the superatom family, the new superatom analogue Be11 of group IVA elements has been developed. Be11 can exhibit multiple valence states (+2 and +4), similar to carbon-group elements, and is capable of forming stable ionic compounds with other atoms such as carbon, chalcogen, (super)halogen, and hydroxyl. This resembles how tin and lead atoms combine with these elements to form stable molecules. Their special stability can be rationalized from the perspective of a cluster shell model. Sn or Pb could be the nearest atomic analogue to Be11 in group IVA, as the +2 oxidation state is more stable than the +4 oxidation state. This comparative investigation highlights the resemblance between Be11 and carbon-group elements, which encourages additional exploration within the superatom family.

Antimicrobial Peptides Targeting Streptococcus mutans: Current Research on Design, Screening and Efficacy.

Antimicrobial peptides (AMPs) are small-molecule peptides that play a vital role in the nonspecific immune defense system of organisms. They mainly kill microorganisms by physically destroying the cell membrane and causing the leakage of contents. AMPs have attracted much attention as potential alternatives to antibiotics due to their low susceptibility to resistance. Streptococcus mutans (S. mutans) is one of the main causative agents of human dental caries. The design, screening, and efficacy evaluation of AMPs targeting S. mutans offer new possibilities for the prevention and treatment of oral diseases, especially dental caries, in the future. This article reviews AMPs from different sources that have inhibitory effects on S. mutans, discusses the mechanism of action of AMPs against S. mutans biofilms, and focuses on the research progress of screening methods, design modification, and biological activity evaluation of AMPs. We hope to provide insights and reference value for the development of new biologics.

A novel hydrophilic hydrogel with a 3D network structure for the highly efficient enrichment of N-glycopeptides.

Protein glycosylation is of great significance in various physiological processes. Nevertheless, it remains a huge challenge to identify glycopeptides in complex biosamples by the direct mass spectrometry analysis due to the low ion efficiency and low abundance of glycopeptides. In this study, a novel hydrogel (denoted as ZIF-8/SAP) with a stable three-dimensional (3D) network structure and excellent hydrophilicity was successfully fabricated to capture glycopeptides with high efficiency. Owing to the unique characteristics, ZIF-8/SAP exhibited great selectivity (1 : 1000), great sensitivity (1 fmol µL-1), large binding capacity (300 mg g-1) and satisfactory reusability (seven cycles). Inspired by the great enrichment performance of the as-prepared material toward glycopeptides, ZIF-8/SAP was further applied to capture glycopeptides from a real human serum sample. The experimental results demonstrated that 217 N-glycosylation sites were identified in 283 N-glycopeptides, corresponding to 95 glycoproteins identified from 10 µL human serum by the nano-LC-MS/MS analysis, revealing the great potential of the novel ZIF-8/SAP hydrogel for glycopeptide enrichment and glycoproteomic research.

A novel graphene oxide/chitosan foam incorporated with metal-organic framework stationary phase for simultaneous enrichment of glycopeptide and phosphopeptide with high efficiency.

A novel hydrophilic porous biocomposite was fabricated by incorporating graphene oxide (GO) @chitosan (CS) foam substrate (GO@CS@ZIF-8 foam) with ZIF-8 crystals in situ via a facile stirring method for simultaneous enrichment of glycopeptides and phosphopeptides from complex biological samples. The experimental results demonstrated that GO@CS@ZIF-8 foam exhibited favorable specificity for simultaneous enrichment of N-glycopeptides and phosphopeptides under the same condition for HRP and ß-casein tryptic digest mixtures. The novel material was further applied to enriching both glycopeptides and phosphopeptides simultaneously from 4 µL complex human serum, and 423 N-glycopeptides and 40 phosphopeptides corresponding to 133 glycoproteins and 29 phosphoproteins were identified, respectively.

FGF-21 biomarker detection at the sub-nanogram per mL level in human serum using normal-flow liquid chromatography/tandem mass spectrometry.

RATIONALE: Quantitative detection of the FGF-21 biomarker at the sub-nanogram per mL level in human serum has generally been achieved using nanoflow liquid chromatography/tandem mass spectrometry (LC/MS/MS) due to its high sensitivity. However, a nano-LC/MS/MS-based assay can suffer from limited reproducibility and MS signal instability making it challenging to employ it as a robust analytical method for routine clinical applications. METHODS: To tackle these limitations, parallel reaction monitoring (PRM)-based targeted protein quantification using normal-flow liquid chromatography coupled with high-resolution, accurate mass instrumentation was evaluated as a possible alternative. Different from the conventional selected reaction monitoring (SRM) using triple quadrupole MS, the proposed strategy used high-resolution orbitrap MS coupled with conventional normal-flow liquid chromatography. The primary goal of this assay development effort is to significantly improve the robustness of the LC/MS/MS-based assay while maintaining high sensitivity by the use of high-resolution MS and a large sample loading volume. RESULTS: The performance of the normal-flow LC/MS/MS assay was evaluated by using it to quantify the FGF-21 protein, a potential biomarker for non-alcoholic fatty liver disease, in serum samples. Multiple replicated PRM sample quantification results demonstrated the excellent reproducibility and operational robustness of the assay. A limit of quantification of less than 0.4 ng/mL for FGF-21 in a complex serum matrix could be achieved by using the heavy-isotope-labeled peptide technique, a result which is comparable with the sensitivity obtained using the nano-LC/SRM MS-based assay. CONCLUSIONS: The strategy offered an effective alternative to nano-LC/SRM MS for the quantification of protein biomarkers in a complex biomatrix with much improved reproducibility and operational robustness.

Preclinical development of GR1501, a human monoclonal antibody that neutralizes interleukin-17A.

Interleukin-17A (IL-17A) is a soluble pro-inflammatory cytokine, which is mainly secreted by Th17 cells. In humans, IL-17A mRNA and protein levels are increased in several autoimmune diseases, including psoriasis and rheumatoid arthritis. This study describes the preclinical in vitro and in vivo characterization of GR1501, a human IL-17A-neutralizing IgG4 monoclonal antibody. GR1501 binds human, rhesus and cynomolgus IL-17A with high affinity but does not bind to mouse IL-17A or other IL-17 family members. GR1501 effectively blocks the interaction between IL-17A and its receptor IL-17RA, thereby inhibiting IL-17A-induced CXCL1 and IL-6 release in cells and mice. In mouse air pouch model, GR1501 effectively inhibits IL-17A induced leukocytes infiltration into the air pouch. GR1501 also reduces joint swelling and inflammation in mouse arthritis model. These data suggest that GR1501 is a potent and selective IL-17A-neutralizing monoclonal antibody and will support the clinical investigation of this monoclonal antibody in psoriasis and rheumatoid arthritis.

Nonpeptide-Based Small-Molecule Probe for Fluorogenic and Chromogenic Detection of Chymotrypsin.

We report herein a nonpeptide-based small-molecule probe for fluorogenic and chromogenic detection of chymotrypsin, as well as the primary application for this probe. This probe was rationally designed by mimicking the peptide substrate and optimized by adjusting the recognition group. The refined probe 2 exhibits good specificity toward chymotrypsin, producing about 25-fold higher enhancement in both the fluorescence intensity and absorbance upon the catalysis by chymotrypsin. Compared with the most widely used peptide substrate (AMC-FPAA-Suc) of chymotrypsin, probe 2 shows about 5-fold higher binding affinity and comparable catalytical efficiency against chymotrypsin. Furthermore, it was successfully applied for the inhibitor characterization. To the best of our knowledge, probe 2 is the first nonpeptide-based small-molecule probe for chymotrypsin, with the advantages of simple structure and high sensitivity compared to the widely used peptide-based substrates. This small-molecule probe is expected to be a useful molecular tool for drug discovery and chymotrypsin-related disease diagnosis.

N-linked glycoproteome analysis reveals central glycosylated proteins involved in response to wheat yellow mosaic virus in wheat.

Glycosylation is an important proteins post-translational modification and is involved in protein folding, stability and enzymatic activity, which plays a crucial role in regulating protein function in plants. Here, we report for the first time on the changes of N-glycoproteome in wheat response to wheat yellow mosaic virus (WYMV) infection. Quantitative analyses of N-linked glycoproteome were performed in wheat without and with WYMV infection by ZIC-HILIC enrichment method combined with LC-MS/MS. Altogether 1160 N-glycopeptides and 971 N-glycosylated sites corresponding to 734 N-glycoproteins were identified, of which 64 N-glycopeptides and 64 N-glycosylated sites in 60 N-glycoproteins were significantly differentially expressed. Two conserved typical N-glycosylation motifs N-X-T and N-X-S and a nontypical motifs N-X-C were enriched in wheat. Gene Ontology analysis showed that most differentially expressed proteins were mainly enriched in metabolic process, catalytic activity and response to stress. Kyoto Encyclopedia of Genes and Genomes analysis indicated that two significantly changed glycoproteins were specifically related to plant-pathogen interaction. Furthermore, we found that over-expression of TaCERK reduced WYMV accumulation. Glycosylation site mutation further suggested that N-glycosylation of TaCERK could regulate wheat resistance to WYMV. This study provides a new insight for the regulation of protein N-glycosylation in defense response of plant.

A Gas Flow Measurement System Based on Lead Zirconate Titanate Piezoelectric Micromachined Ultrasonic Transducer.

Ultrasonic flowmeter is one of the most widely used devices in flow measurement. Traditional bulk piezoelectric ceramic transducers restrict their application to small pipe diameters. In this paper, we propose an ultrasonic gas flowmeter based on a PZT piezoelectric micromachined ultrasonic transducer (PMUT) array. Two PMUT arrays with a resonant frequency of 125 kHz are used as the sensitive elements of the ultrasonic gas flowmeter to realize alternate transmission and reception of ultrasonic signals. The sensor contains 5 × 5 circular elements with a size of 3.7 × 3.7 mm2. An FPGA with a resolution of ns is used to process the received signal, and a flow system with overlapping acoustic paths and flow paths is designed. Compared with traditional measurement methods, the sensitivity is greatly improved. The flow system achieves high-precision measurement of gas flow in a 20 mm pipe diameter. The flow measurement range is 0.5-7 m/s and the relative error of correction is within 4%.

Structural characterization of a polysaccharide from Trametes sanguinea Lloyd with immune-enhancing activity via activation of TLR4.

A bioactive polysaccharide (TS2-2A) with a molecular weight of 15 kDa was isolated from Trametes sanguinea Lloyd, a medicinal food homologous fungus, by water extraction-alcohol precipitation and chromatographic separation. NMR analysis of polysaccharide and MS/MS analysis of its oligosaccharide indicated that TS2-2A featured a novel straight chain with a backbone of 1,3-α-d-glucopyranose and 1,4-ß-d-glucopyranose at a molar ratio of 1:4. Moreover, TS2-2A, recognized by Toll-like receptor 4 (TLR4), stimulated RAW 264.7 macrophages to release related cytokines and contributed to immune-enhancing effects. Briefly, with remarkable immune-enhancing activity and noncytotoxicity, TS2-2A was proposed to be a potential immune enhancer for supplementing drugs or functional foods.

Tackling environmental challenges in pollution controls using artificial intelligence: A review.

This review presents the developments in artificial intelligence technologies for environmental pollution controls. A number of AI approaches, which start with the reliable mapping of nonlinear behavior between inputs and outputs in chemical and biological processes in terms of prediction models to the emerging optimization and control algorithms that study the pollutants removal processes and intelligent control systems, have been developed for environmental clean-ups. The characteristics, advantages and limitations of AI methods, including single and hybrid AI methods, were overviewed. Hybrid AI methods exhibited synergistic effects, but with computational heaviness. The up-to-date review summarizes i) Various artificial neural networks employed in wastewater degradation process for the prediction of removal efficiency of pollutants and the search of optimizing experimental conditions; ii) Evaluation of fuzzy logic used for intelligent control of aerobic stage of wastewater treatment process; iii) AI-aided soft-sensors for precisely on-line/off-line estimation of hard-to-measure parameters in wastewater treatment plants; iv) Single and hybrid AI methods applied to estimate pollutants concentrations and design monitoring and early-warning systems for both aquatic and atmospheric environments; v) AI modelings of short-term, mid-term and long-term solid waste generations, and various ANNs for solid waste recycling and reduction. Finally, the future challenges of AI-based models employed in the environmental fields are discussed and proposed.

BP-ANN Model Coupled with Particle Swarm Optimization for the Efficient Prediction of 2-Chlorophenol Removal in an Electro-Oxidation System.

Electro-oxidation is an effective approach for the removal of 2-chlorophenol from wastewater. The modeling of the electrochemical process plays an important role in improving the efficiency of electrochemical treatment and increasing our understanding of electrochemical treatment without increasing the cost. The backpropagation artificial neural network (BP-ANN) model was applied to predict chemical oxygen demand (COD) removal efficiency and total energy consumption (TEC). Current density, pH, supporting electrolyte concentration, and oxidation-reduction potential (ORP) were used as input parameters in the 2-chlorophenol synthetic wastewater model. Prediction accuracy was increased by using particle swarm optimization coupled with BP-ANN to optimize weight and threshold values. The particle swarm optimization BP-ANN (PSO-BP-ANN) for the efficient prediction of COD removal efficiency and TEC for testing data showed high correlation coefficient of 0.99 and 0.9944 and a mean square error of 0.0015526 and 0.0023456. The weight matrix analysis indicated that the correlation of the five input parameters was a current density of 18.85%, an initial pH 21.11%, an electrolyte concentration 19.69%, an oxidation time of 21.30%, and an ORP of 19.05%. The analysis of removal kinetics indicated that oxidation-reduction potential (ORP) is closely correlated with the chemical oxygen demand (COD) and total energy consumption (TEC) of the electro-oxidation degradation of 2-chlorophenol in wastewater.

Discovery of Butyrylcholinesterase-Activated Near-Infrared Fluorogenic Probe for Live-Cell and In Vivo Imaging.

Butyrylcholinesterase (BChE) is widely distributed in various tissues and highly implicated in several important human diseases, especially Alzheimer's disease (AD). However, the role of BChE in AD is still controversial, which may be partially attributed to the lack of a direct tool for real-time and noninvasive monitoring of BChE in in vivo. Here, we report three rationally designed near-infrared fluorogenic probes that possess excellent discrimination for butyrylcholinesterase (BChE) over the related enzyme acetylcholinesterase (AChE). The refined probe, BChE-NIRFP, not only functions as an exquisite substrate for BChE in in vitro assays but also represents a superb "signal-on" imaging tool to real-time track BChE levels in human cells, zebrafish, and a mouse model of AD. A further application of BChE-NIRFP to identify the cellular mechanism reveals that Aß fibrils and insulin resistance may be important contributors to the abnormally elevated BChE levels observed during AD progression. Based on the results from the present study, this new probe is a valuable tool for basic and clinical research designed to obtain a complete understanding of the physiological roles of BChE in diverse human diseases, particularly AD.

Characterization of the interactions between inhibitor-1 and recombinant PP1 by NMR spectroscopy.

Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35 is phosphorylated by cAMP-dependent protein kinase (PKA). However, PKA-phosphorylated form of inhibitor-1 displayed a weak activity in inhibition of recombinant PP1. The mechanism for the impaired activity of PKA-phosphorylated inhibitor-1 toward inhibition of recombinant PP1 remained elusive. By using NMR spectroscopy in combination with site-directed mutagenesis and inhibitory assay, we found that the interaction between recombinant PP1 and the consensus PP1-binding motif of PKA-thiophosphorylated form of inhibitor-1 was unexpectedly weak. Unlike binding to native PP1, the subdomains 1 (residues around and including the phosphorylated Thr35) and 2 (the consensus PP1-binding motif) of PKA-thiophosphorylated form of inhibitor-1 do not exhibit a synergistic effect in inhibition of recombinant PP1. This finding implied that a slight structural discrepancy exists between native and recombinant PP1, resulting in PKA-thiophosphorylated form of inhibitor-1 displaying a different affinity to native and recombinant enzyme.