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The effects of general anesthetics on cognitive impairment are unclear and complicated. Laser laryngeal surgery (LLS) requires the administration of low levels of oxygen, which may increase the risk of desaturation and brain function impairment. This prospective randomized trial aimed to compare the effects of desflurane and propofol-based general anesthesia on the occurrence of early postoperative cognitive decline in elderly patients undergoing LLS.Seventy-three patients classified as American Society of Anesthesiologists grade I or II and at least 65 years of age were randomly allocated to receive either desflurane-based (Group D) or propofol-based (Group P) anesthesia during LLS. The standard anesthesia protocol was performed, with a bispectral index between 40 and 60 and a mean arterial pressure within 20% of baseline values. Intraoperative regional oxygen saturation values were recorded. Each patient was assessed using the mini-mental state examination (MMSE) test during the preoperative period (baseline), 30 min after extubation in the postanesthesia care unit, and 1, 3, and 24 h after surgery.MMSE scores improved slightly in both groups compared to baseline during the early postoperative period, but these increases were not statistically significant. No significant differences were identified in MMSE scores between groups. Only three patients (9.6%) in group D and one patient (3.1%) in group P developed cognitive impairment (p = 0.583).Low intraoperative inspired oxygen concentration during short-duration LLS did not reduce early postoperative cognitive function in elderly patients. Desflurane or propofol-based anesthesia had similar effects on early neurocognition after LLS.2 Laryngoscope, 2022.

Rapid and accurate prediction of drug-target affinity can accelerate and improve the drug discovery process. Recent studies show that deep learning models may have the potential to provide fast and accurate drug-target affinity prediction. However, the existing deep learning models still have their own disadvantages that make it difficult to complete the task satisfactorily. Complex-based models rely heavily on the time-consuming docking process, and complex-free models lacks interpretability. In this study, we introduced a novel knowledge-distillation insights drug-target affinity prediction model with feature fusion inputs to make fast, accurate and explainable predictions. We benchmarked the model on public affinity prediction and virtual screening dataset. The results show that it outperformed previous state-of-the-art models and achieved comparable performance to previous complex-based models. Finally, we study the interpretability of this model through visualization and find it can provide meaningful explanations for pairwise interaction. We believe this model can further improve the drug-target affinity prediction for its higher accuracy and reliable interpretability.

Bioaerosol transmission has received much attention as one of the significant ways of causing respiratory diseases. Therefore, studying bioaerosol sampling and detection is crucial. In this study, magnetic nanoparticles and cyclone sampling were combined to monitor pathogens in the form of bioaerosol at low concentrations in the environment. Magnetic nanoparticles had obvious advantages in extracting bacterial nucleic acid, which could be purified and enriched in a short time. The influencing factors on the performance of the high-flow cyclone sampler were also explored. The results showed that the high-flow cyclone sampler had good collection efficiency owing to high flow rate and small cutting size. Moreover, the sampling effect was better when the flow rate and the sampling time were set to 300 L/min and 20 min, which was probably due to desiccation effect, impact stress, embedding, and particle rebound. The experimental results indicated that the cyclone aerosol sampling technology based on magnetic nanoparticles could even detect low concentration bioaerosol, providing a reference for using high-flow cyclone samplers in different applications.

Airborne transmission is much more common than previously thought. Based on our knowledge about SARS-COV-2 (COVID-19) infection, the aerosol transmission routes for all respiratory infections must be reassessed. Thus far, the COVID-19 outbreak has caused catastrophic public health and economic crises, posing a serious threat to the lives and health of people around the world and directing public attention toward the airborne transmission of pathogens. The novel coronavirus transmission in the form of nanoaerosols in a wider range hinders prevention and early warning efforts. As a classical bioaerosol sampler, the Andersen six-stage sampler is widely used in the collection and research of aerosol particles. In this study, the physical and biological collection efficiency of the six-stage sampler was explored by qPCR and colony counting method. Results showed that the physical collection efficiency reached more than 50% when the particle size was larger than 0.75 μm. However, the overall biological collection efficiency was only 0.25%. In addition, fluorescence microscopy and flow cytometry were used to detect the microbial state after sampling, and the results showed that the proportion of the collected live bacteria was less than 15% of the total. This result is of great significance not only for the application of the Andersen six-stage sampler in collecting nanosized bioaerosols, but also provides reference for the selection of subsequent detection technologies for effective collection.

In the dual-channel supply chain, the relationship between the electronic direct selling channel and the traditional selling channel is situated in an antagonistic game. The present study used the master-slave game model to analyze the dual-channel supply chain structure, where the manufacturer is the leader of the game, and the seller is the follower of the game. Based on the game analysis theory, the game order of manufacturer and seller is analyzed in the state of demand information sharing. The decision model of each member in the dual-channel supply chain is constructed, and the optimal production decision and product price decision under the demand information sharing between the seller and the manufacturer are determined by the reverse induction method. The expected economic benefits of both are calculated, respectively. The experimental results showed that the economic benefit of the model is higher than that of the traditional demand information nonsharing model, which could enhance the economic benefit of the manufacturer and the seller at the same time and alleviate the contradiction between the electronic direct selling channel and the traditional selling channel.

Background Ovary is an important reproductive organ for poultry. MicroRNA (miRNA) is a highly conserved class of small non-coding RNA that function in a specific manner to post-transcriptionally regulate gene expression in organisms. Currently, miRNA has been studied extensively but research on duck ovarian tissue is relatively rare. Thus, in this study, we performed the first miRNA analysis of ovarian tissues in Leizhou black ducks with low and high rates of egg production. Method: Using high-throughput sequencing technology, miRNA library was constructed to obtain miRNA expression profile; differentially expressed miRNAs were screened, to predict miRNA target genes. Results A total of 29 differentially expressed miRNAs were obtained from the miRNA library, of which 7 were up-regulated and 22 were down-regulated. Verification of 12 randomly selected miRNAs, using RT-qPCR technology showed the results are consistent with the sequencing data, indicating that the sequencing results are reliable. The GO enrichment and KEGG pathway enrichment analysis of differential miRNAs showed that target genes were significantly enriched in signal pathways related to ovarian development, such as the oxytocin signaling pathway, GnRH signaling pathway, progesterone-mediated oocyte maturation, and AMPK signal pathway. Conclusion The research results enrich the data resources of duck miRNAs, and provide a theoretical basis for further research on the laying performance of poultry and molecular-assisted breeding of poultry in the future.

BackgroundEstrogen receptor 2 (ESR2) plays significant biological roles in the reproductive system and ovarian follicle development. This study, therefore, aimed to reveal the expression pattern and cell-specific localization of ESR2 in the ovarian follicles of Leizhou black ducks. MethodFour laying Leizhou black ducks at 43 weeks old were annihilated and different grade-sized follicles were collected for immunohistochemistry and expression profile study. The follicles were grouped into seven (7) as small white follicles (SWF), large white follicles (LWF), small yellow follicles (SYF), large yellow follicles (LYF), follicle 5 (F5), follicle 2 (F2), and follicle 1 (F1). ResultsThe qRT/PCR results displayed that ESR2 mRNA was expressed in all follicles with the highest (P < 0.05) level of expression found in F1 compared to other follicles. Immunohistochemistry analysis of the cell-specific localization of ESR2 protein revealed that ESR2 was distributed in both granulosa and theca cells region in all the follicles examined. There was a significantly higher localization of ESR2 protein in the granulosa cells than the theca cells of SWF, SYF, LYF, F2, and F1. Comparatively, ESR2 was highly expressed in the granulosa cells of LYF than in all the other follicles. ConclusionThese results provide theoretical knowledge for the in-depth study of the related biological functions of the ESR2 gene and its application at the cellular level.

BackgroundOur previous study on the ovarian transcriptomic analysis in Leizhou black duck revealed that the ESR2 gene was involved in hormone regulation in reproduction and the estrogen signaling pathway related to reproductive performance was enriched. This suggested that ESR2 may have a functional role in the reproductive performance of the Leizhou black duck. Thus, this study aimed at evaluating the polymorphism of the ESR2 gene and its association with egg-laying traits and the distribution pattern of ESR2 mRNA in laying and non-laying Leizhou black ducks. MethodIn this study, genomic DNA was extracted from blood samples of 101 Leizhou black ducks to identify single nucleotide polymorphisms (SNPs) of the ESR2 gene to elucidate molecular markers highly associated with egg-laying traits. Four (4) each of laying and non-laying Leizhou black ducks were selected to collect different tissues to analyze the ESR2 gene expression. ResultsA total of 23 SNPs were identified and association analysis of the single SNP sites showed that SNPs g.56805646 T>C and exon 3-20G>A were significantly (P < 0.05) associated with egg weight. Ducks with CT and AG genotypes had significantly higher (P < 0.05) egg weights than their respective other genotypes. Haplotype association analysis of g.56805646 T>C and exon 320G>A showed that the haplotypes were significantly associated with egg weight where higher egg weight was seen in individuals with H3H4 haplotypes. In the hypothalamus-pituitary-gonadal (HPG) axis, the results of qRT/PCR showed that ESR2 mRNA was significantly (P < 0.05) expressed in the ovaries of both duck groups than in the hypothalamus and pituitary. In the oviduct, ESR2 was significantly (P < 0.05) higher in the infundibulum and magnum of laying and nonlaying ducks respectively. ConclusionThis study provides molecular marker for selecting Leizhou black ducks for egg production and provides theoretical knowledge for the study of the related biological functions of the ESR2 gene at the cellular level.

Urban sports space is the basis for the fitness activities of urban residents. The service radiation range and intensity reflect the supply problem of urban resources. Based on point of interest (POI) data of Gaode map crawled by Python, this study systematically discussed the accessibility and equity of Nanjing stadiums through further analysis of kernel density, buffer zone, accessibility, and spatial autocorrelation. It is found that the centralized and dispersed layout of sports venues in Nanjing is not reasonable, the accessibility level of different types of stadiums is different, and the number of stadiums does not determine the accessibility. From the further investigation of the local residents and students, this paper again confirmed that there is no necessary relationship between accessibility, equity, and demand in Nanjing. At present, residents, especially college students, still have a low level of cognition of physical exercise. In terms of sports services, in addition to improving the construction of stadiums and facilities, the government and education departments should also try to change citizens’ attitudes towards fitness.

A novel protective roasting strategy was applied to prepare mesoporous hollow CeO2 microspheres (mh-CeO2) using silica as both a hard template and a roasting protectant. Through detailed characterization techniques such as SEM, TEM, EDX, FT-IR, XRD, XPS, and N2 adsorption–desorption, it could be determined that hollow CeO2 microspheres with good dispersibility and crystallinity, uniform spherical morphology and suitable pore width as heterogeneous catalysts had been successfully prepared. mh-CeO2 showed much higher catalytic activity than the as-prepared SiO2@CeO2 and reported CeO2 catalysts for one-pot synthesis of imines from anilines and benzyl alcohols, which is attributed to its super-high specific surface area and mesoporous hollow structure. In addition, this protective roasting strategy can also superiorly stabilize the hollow structure during the reaction, recovery and multiple calcination processes.

Our previous study on the ovarian transcriptomic analysis in Leizhou black duck revealed that the ESR2 gene was involved in hormone regulation in reproduction and the estrogen signaling pathway related to reproductive performance was enriched. This suggested that ESR2 may have a functional role in the reproductive performance of the Leizhou black duck. Thus, this study aimed at evaluating the polymorphism of the ESR2 gene and its association with egg-laying traits and the distribution pattern of ESR2 mRNA in laying and non-laying Leizhou black ducks. In this study, genomic DNA was extracted from blood samples of 101 Leizhou black ducks to identify single nucleotide polymorphisms (SNPs) of the ESR2 gene to elucidate molecular markers highly associated with egg-laying traits. Four each of laying and non-laying Leizhou black ducks were selected to collect different tissues to analyze the ESR2 gene expression. A total of 23 SNPs were identified and association analysis of the single SNP sites showed that SNPs g.56805646 T>C and exon 3-20G>A were significantly (P < 0.05) associated with egg weight. Ducks with CT and AG genotypes had significantly higher (P < 0.05) egg weights than their respective other genotypes. Haplotype association analysis of g.56805646 T>C and exon 3-20G>A showed that the haplotypes were significantly associated with egg weight. Higher egg weight was seen in individuals with H3H4 haplotypes. In the hypothalamus-pituitary-gonadal (HPG) axis, the results of qRT/PCR showed that ESR2 mRNA was significantly (P < 0.05) expressed in the ovaries of both duck groups than in the hypothalamus and pituitary. In the oviduct, ESR2 was significantly (P < 0.05) higher in the infundibulum and magnum of laying and non-laying ducks respectively. This study provides a molecular marker for selecting Leizhou black ducks for egg production. In addition, it offers theoretical knowledge for studying the related biological functions of the ESR2 gene at the cellular level.

Background: To evaluate the efficacy of controlled hypertension on hemostasis before closure of the operative incision in vertical partial laryngectomy (VPL).Methods: Seventy males scheduled for VPL were randomly assigned to Group H who received either ephedrine or phenylephrine to maintain a mean arterial pressure (MAP) of 90–110 mmHg 10 min before the operative incision closure or to Group N who received normal saline. The primary endpoint was the volume of drainage after surgery. Heart rate, MAP, surgical field bleeding score before and after drug administration, the amount of liquid administered, operation and recovery durations were recorded. Heart rate, MAP, incisional bleeding score, coughing score, nausea and vomiting score, and frequency of bloody sputum aspiration were also recorded when the patient left the postanesthesia care unit and at 6 and 24 hours postoperatively.Results: The intraoperative bleeding score of the surgical field was significantly higher in patients receiving vasopressor. The volume of drainage in control group was more than that in Group H at different time points after surgery. Compared with Group N, Group H had a significantly lower incisional bleeding score and frequency of bloody sputum aspiration at all time-points after surgery, and a significantly lower nausea and vomiting score at 6 and 24 hours postoperatively. Conclusion: Controlled hypertension (MAP 90–110 mmHg) before wound closure may be used as an alternative way to detect intraoperative bleeding points in patients undergoing VPL, which also decreased the postoperative volume of drainage and the incidences of nausea and vomiting.Trial registration: The China Clinical Research Information Service (registration number, ChiCTR1900026097). Registered 21 September 2019, http://www.chictr.org.cn/showproj.aspx?proj=37850

A general-applicable two-phase ligand exchange method is developed to prepare a series of monodisperse water-soluble AuAg nanoclusters. This method relies on the ligand exchange of the water-solubl...

A novel Au36-xCux(m-MBT)24 (x = 1-3, m-MBT = 3-methylbenzenethiolate) nanocluster has been prepared. According to the X-ray single crystal diffractometer, the structure of Au36-xCux(m-MBT)24 is similar to that of Au36(SPhtBu)24. The Au36-xCux(m-MBT)24 nanocluster contains a face-centered cubic (FCC) M28 core, which is protected by 4 M2S3 (M = Au/Cu) staple motifs and 12 bridging SR ligands. The Cu dopants could possibly occupy 14 sites (six in the sub-surface and eight in the staple motifs). Spectral monitoring indicates that the number of Cu dopants sequentially increased on increasing the amount of Cu precursors (relative to a Au control). Meanwhile, DFT calculations imply that the maximum doping number of Cu is 3, and doping occurs preferentially at the staple sites and sub-surface sites (instead of the centre of the core). Because the atomic orbital of the peripheral locations hardly contributes to the frontier molecular orbitals, the UV-vis of the AuCu alloy is almost the same as that of its homometallic Au counterpart.

Skeletal muscle is one of the most important economic traits in the poultry industry whose development goes through several processes influenced by several candidate genes. This study explored the regulatory role of DCN on MSTN and the influence of these genes on the proliferation and differentiation of embryonic myoblasts in Leizhou black ducks. Embryonic myoblasts were transfected with over-expressing DCN, Si-DCN, and empty vector and cultured for 24 h, 48 h, and 72 h of proliferation and the comparative expression of DCN and MSTN were measured. The results showed that cells transfected with the over-expression DCN had a significantly (P 0.05) higher expression of DCN mRNA than the normal group and the expression of MSTN mRNA showed a downward trend during the proliferation of myoblasts. DCN mRNA expression was lower in cells transfected with Si-DCN than the normal group in all stages of proliferation. While the expression of MSTN in the Si-DCN transfected group was higher than the normal group with a significant (P 0.05) difference at the 72 h stage. DCN mRNA increased at the early stage of differentiation but decreased (P 0.05) from the 6th day to the 8th day of differentiation. The level of MSTN increased gradually during the differentiation process of myoblasts until it decreased significantly on the 8th day. These results show that DCN enhances the proliferation and differentiation of Leizhou black duck myoblasts and suppresses MSTN activity.

Protein N-glycosylation is one of the most important post-translational modifications, participating in many key biological and pathological processes. Large-scale and precise identification of N-glycosylated proteins and peptides is especially beneficial for understanding their biological functions and for discovery of new clinical biomarkers and therapeutic drug targets. However, protein N-glycosylation is microheterogeneous and low abundant in living organisms, therefore specific enrichment of N-glycosylated proteins/peptides before mass spectrometry analysis is a prerequisite. In this work, we developed a new type of polymer hybrid graphene oxide (GO) by in situ growth of hydrazide-functionalized hydrophilic polymer chains on the GO surface (GO-PAAH) for selective N-glycopeptide enrichment and identification by mass spectrometry. The densely attached and low steric hindrance hydrazide groups as well as the highly hydrophilic nature of GO-PAAH facilitate N-glycopeptide enrichment by the combination of hydrazide capturing and HILIC interaction. Taking advantage of the unique features of GO-PAAH, all of the three N-glycopeptides of bovine fetuin were successfully enriched and identified with significantly enhanced signal intensities from a digest mixture of bovine fetuin and bovine serum albumin at a mass ratio of 1:100, demonstrating the excellent enrichment selectivity of GO-PAAH. Furthermore, a total of 507 N-glycosylation sites and 480 N-glycopeptides in 232 N-glycoproteins were enriched and identified from 10μL of human serum by three replicates using this novel enrichment material, which is nearly two times higher than the commercial hydrazide resin based method (280 N-glycosylation sites, 261 N-glycopeptides and 144 N-glycoproteins in three experiments). Among the identified, 95 N-glycosylation sites were not reported in the Uniprot database, and 106 N-glycoproteins were disease related in the Nextprot database, indicating the potential of this new enrichment material in global mapping of protein N-glycosylation.

Herein, we report the synthesis and atomic structures of the cluster-assembled CuxAu25–x(PPh3)10(PhCH2CH2S)5Cl22+ and Cu3Au34(PPh3)13(tBuPhCH2S)6S23+ nanoclusters (NCs). The atomic structures of both NCs were precisely determined by single-crystal X-ray crystallography. The CuxAu25–x(PPh3)10(PhC2H4S)5Cl22+ NC was assembled by two icosahedral M13 via a vertex-sharing mode. The Cu atom partially occupies the top and waist sites and is monocoordinated with chlorine or thiol ligands. Meanwhile, the Cu3Au34(PPh3)13(tBuPhCH2S)6S23+ NC can be described as three 13-atom icosahedra sharing three vertexes in a cyclic fashion. The three Cu atoms all occupy the internal positions of the cluster core. What is more important is that all three Cu atoms in Cu3Au34 are monocoordinated by the bare S atoms. The absorption spectra of the as-synthesized bimetallic NCs reveal that the additional metal doping and different cluster assemblies affect the electronic structure of the monometallic NCs.

In recent years, lead-free metal-halide perovskite photovoltaics has attracted ever-growing attention, in view of its potential to replicate the outstanding properties of lead-halide perovskite photovoltaics, but without the toxicity burden of the latter. Despite a research effort much smaller in scale than that pursued with lead-based perovskites, considerable progress has been achieved in lead-free perovskite photovoltaics, with the highest power conversion efficiencies now being in the region of 13%. In this Perspective, we first discuss the state of the art of lead-free perovskite photovoltaics and additionally highlight promising directions and strategies that could lead to further progress in material exploration and understanding as well as in photovoltaic efficiency. Furthermore, we point out the widespread lack of experimental data on the fundamental optoelectronic properties of lead-free halide perovskite absorbers (e.g., charge carrier mobility, defect parameters, Urbach energy, and the impact of dimensionality). All of this currently hampers a rational approach to further improving their performance and points to the need for a concerted effort that could bridge this knowledge gap. Additionally, this Perspective brings to the fore the manifold photovoltaic opportunities—thus far largely unexplored with lead-free perovskite absorbers—beyond single-junction outdoor photovoltaics, which may potentially enable the realization of their full potential. The exploration of these opportunities (tandem photovoltaics, indoor photovoltaics, and building-integrated and transparent photovoltaics) could energize the investigation of existing and new classes of lead-free perovskite absorbers beyond current paradigms and toward high photovoltaic performance.

Chemically-triggered drug delivery systems (DDSs) have been extensively studied as they do not require specialized equipment to deliver the drug and can deeply penetrate human tissue. However, their syntheses are complicated and they tend to be cytotoxic, which restricts their clinical utility. In this work, the self-regulated drug loading and release capabilities of peptide-protected gold nanoclusters (Pep-Au NCs) are investigated using vancomycin (Van) as the model drug. Gold nanoclusters (Au NCs) coated with a custom-designed pentapeptide are synthesized as drug delivery nanocarriers and loaded with Van - a spontaneous process reliant on the specific binding between Van and the custom-designed peptide. The Van-loaded Au NCs show comparable antimicrobial activity with Van on its own, and the number of Van released by the Pep-Au NCs is found to be proportional to the amount of bacteria present. The controlled nature of the Van release is very encouraging, and predominantly due to the stronger binding affinity of Van with bacteria than that with Au NCs. In addition, these fluorescent Au NCs could also be used to construct temperature sensors, which enable the in vitro and in vivo bioimaging.

As one of the most common post-translational modifications, protein N-glycosylation precipitates in many important biological processes and has closely correlations with the occurrence and progression of multiple diseases. Plasma exosomes secreted by cells contain various bioactive N-glycoproteins which may serve as potential biomarkers for early disease diagnosis and treatment. However, the protein N-glycosylation profile in human plasma exosome is largely unknown, due to the technical challenges in glycoprotein identification. Signals of the rare N-glycoproteins/N-glycopeptides are severely suppressed by the abundant coexisting non-glycosylated counterparts in mass spectrometry analysis. Therefore, specific enrichment of N-glycoprotein/glycopeptide is a prerequisite for large scale N-glycosylation profiling. In this work, we developed a hydrazide functionalized thermosensitive polymer for efficient enrichment and in-depth identification of protein N-glycosylation in human plasma exosome by mass spectrometry. The polymer chains completely dissolve in the enrichment system to form a homogeneous solution. Therefore, efficient covalent coupling between the N-glycoprotein/glycopeptide and the polymer chain is achieved, due to the reduced interfacial mass transfer resistance and the densely packed accessible functional groups on the polymer chains. Furthermore, the thermosensitive polymer can be easily precipitated and recovered by simply rising the system temperature to above 34 °C. As a result, 329 N-glycosylation sites corresponding to 180 N-glycoproteins were enriched and identified from plasma exosomes of glioma patients and healthy subjects using the thermosensitive polymer. By quantitative comparison, we found 26 N-glycoproteins significantly changed between the glioma patients and the healthy subjects, demonstrating the potential of this new strategy for N-glycoproteome research of plasma exosome and biomarker discovery.

Benefiting from the excellent photostability and biocompatibility, fluorescent nanoclusters have recently emerged as a highly attractive bio-sensing and imaging material, especially in early diagnosis of cancer. However, their clinic applications were limited by the unsatisfactory specificity and the complex synthesis. In this study, novel methionine coated gold nanoclusters (Met-AuNCs) have been prepared via an easily-achievable one-pot synthetic method. The prepared Met-AuNCs showed high imaging-specificity: after incubating with Met-AuNCs for 1 h, cancer cells (including A549, Hela, MCF-7, HepG2) were fluorescent, while the normal cells (WI-38 and CHO) showed no fluorescence. According to a series of controlled experiments, the reason for the high imaging-selectivity was proposed to originate from the specific recognition of L-type amino acid transporter overexpressed in cancer cells.

A novel Au

Liquid phase homogeneous reactions using soluble polymer supports have found numerous applications in homogeneous catalysis and organic synthesis because of their advantages of no interface mass transfer limitation and a high conversion rate. However, their application in analytical separation is limited by the inefficient/inconvenient recovery of the target molecules from the extremely complex biological samples. Here, we report a stimuli-responsive polymer system for facile and efficient enrichment of trace amounts of biomolecules from complex biological samples. The soluble polymer supports provide a homogeneous reaction system with fast mass transfer and facilitate interactions between the supports and the target molecules. More importantly, the stimuli-responsive polymers exhibit reversible self-assembly and phase separation under pH variations, which leads to facial sample recovery with a high yield of the target biomolecules. The stimuli-responsive polymer is successfully applied to the enrichment of low abundant N-glycoproteins/glycopeptides, which play crucial roles in various key biological processes in mammals and are closely correlated with the occurrence, progression and metastasis of cancer. N-Glycoprotein is coupled to the stimuli-responsive polymer using the reported hydrazide chemistry with pre-oxidation of the oligosaccharide structure. Highly efficient enrichment of N-glycoproteins/N-glycopeptides with >95% conversion rate is achieved within 1 h, which is eight times faster than using solid/insoluble hydrazide enrichment materials. Mass spectrometry analysis achieves low femtomolar identification sensitivity and obtained 1317 N-glycopeptides corresponding to 458 N-glycoproteins in mouse brain, which is more than twice the amount obtained after enrichment using commercial solid/insoluble materials. These results demonstrate the capability of this “smart” polymer system to combine stimuli-responsive and target-enrichment moieties to achieve improved identification of key biological and disease related biomolecules.

Herein, we report the synthesis and atomic structures of the cluster-assembled Cu

Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy. Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and decreasing missed cleavages, which results in more peptides that are compatible with mass-spectrometry analysis. Compared to conventional solution digestion, immobilized protease digestion has the obvious advantages of short digestion time, no self-proteolysis, and reusability. We proposed a multiple-immobilized proteases-digestion strategy that combines the advantages of the two digestion strategies mentioned above. Graphene-oxide (GO)-based immobilized trypsin and endoproteinase Glu-C were prepared by covalently attaching them onto the GO surface. The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoanaerobacter tengcongensis. Compared to 12-hour solution digestion using trypsin or Glu-C, 14% and 7% improvement were obtained, respectively, in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-Glu-C. Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3% and 48.7% enhancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone. The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO, which leads to fewer missed cleavages and more complete digestion. As a result, improved protein identification and sequence coverage can be expected.

In an age of whole-genome analysis, the mass spectrometry-based bottom-up strategy is now considered to be the most powerful method for in-depth proteomics analysis. As part of this strategy, highly efficient and complete proteolytic digestion of proteins into peptides is crucial for successful proteome profiling with deep coverage. To achieve this goal, prolonged digestion time and the use of multiple proteases have been adopted. The long digestion time required and tedious sample treatment steps severely limit the sample processing throughput. Though utilization of immobilized protease greatly reduces the digestion time, highly efficient proteolysis of extremely complex proteomic samples remains a challenging task. Here, we propose a dual matrix-based complementary digestion method using two types of immobilized trypsin with opposite matrix hydrophobicity prepared by attaching trypsin on hydrophobic or hydrophilic polymer-brush-modified nanoparticles. The polymer brushes on the nanoparticles serve as three-dimensional supports for a large amount of trypsin immobilization and lead to ultrafast and highly efficient protein digestion. More importantly, the two types of immobilized trypsin show high complementarity in protein digestion with only ∼60% overlap in peptide identification for yeast and membrane protein of mouse liver. Complementary digestion by applying these two types of immobilized trypsin together leads to obviously enhanced protein and peptide identification. Furthermore, the dual matrix-based complementary digestion shows particular advantage in the digestion of membrane proteins, as twice the number of identified peptides is obtained compared with solution digestion using free proteases, demonstrating its potential as a promising alternative to promote proteomics analysis with higher protein sequence coverage.

Protein glycosylation is involved in the control of many important biological processes and structural alterations of the N-linked glycans are correlated with various kinds of disease. High-throughput N-glycan profiling is a key technique for elucidating the functions of glycans in biological process and disease development as well as discovering new diagnostic biomarkers. However, the low abundance of glycans existing in living organism, the competition/suppression effect of other highly abundant biological molecules and the inherent lack of alkalinity and hydrophobicity of glycans leads to particularly poor detection sensitivity in MS analysis. Here, we demonstrated the first “one-step” approach for highly efficient glycan enrichment and derivatization using reduced graphene oxide as nanoreactors and 1-pyrenebutyric hydrazide for glycan capture and derivatization, which resulted in a 33-fold increase in the glycan detection sensitivity in MALDI-TOF-MS and the identification of 48N-glycoforms from human plasma.

Gold nanoparticles (GNPs) and graphene oxide (GO) sheets are excellent nano carriers in many analytical methods. In this study, a modified enzyme-linked immunosorbent assay (ELISA) strategy was developed using antibody-functionalized GO sheets and GNPs. This modification significantly reduced the limit of detection (LOD) and cost greatly of this assay. The applicability of the method was demonstrated by detecting HSP70 in a human serum sample. This result suggests that the 3G-ELISA method is feasible to detect an antigen in a complex mixture, and the LOD is up to 64-fold and the cost is as low as one-tenth of the conventional ELISA method.

As one of the most important subproteomes in eukaryote cells, N-glycoproteins play crucial roles in various of biological processes and have long been considered closely correlated with the occurrence, progression, and metastasis of cancer. Comprehensive characterization of protein N-glycosylation and association of their aberrant patterns to the corresponding cancer stage may provide a unique way to discover new diagnostic biomarkers and therapeutic drug targets. However, the extremely complex nature of biological samples and relatively low abundance of N-glycosylated proteins makes the enrichment of glycoprotein/glycopeptide a prerequisite for large scale N-glycosylation identification. In this work, we prepared sequence controlled triblock copolymer grafted silica-microparticles (TCP-SMs) by sequential atom transfer radical polymerization (sequential-ATRP) of monosaccharides and zwitterionic-ion monomers for highly efficient and selective glycopeptides enrichment. The triblock copolymer is composed of sequence defined poly zwitterionic-ion, poly-N-acetylglucosamine and poly mannose blocks. The glycopolymer blocks carrying densely packed pendent sugars are excellent mimics of the natural carbohydrate clusters and may induce multivalent carbohydrate-carbohydrate interaction (CCI) with the target glycopeptides. Therefore, increased retention of glycopeptides can be expected by the combination of CCI and zwitterionic-HILIC interaction. As a result, 1244 glycopeptides were identified after TCP-SMs enrichment from mouse liver, which are 65-120% higher than that obtained by homoglycopolymer or random-copolymer grafted silica microparticles prepared using the conventional free radical polymerization. These results demonstrate the critical role of sequence-defined block copolymer of TCP-SMs for obtaining enhanced affinity toward glycopeptides and the potential of this sequential-ATRP strategy to integrate different affinity moieties into one enrichment material to achieve deeper coverage in protein PTM mapping.

N-glycosylation of proteins is one of the most important post-translational modifications (PTM). Many diagnostic biomarkers and therapeutic targets are glycosylated proteins. However, it is still a big challenge to identify glycoproteins in human plasma, because of the high dynamic range and microheterogeneity of glycosylation. In this work, hydrophilic interaction liquid chromatography (HILIC) enrichment, high-pH reversed-phase prefractionation and high accurate mass spectrometry (MS) analysis were combined to profile the N-glycoproteins in human plasma. In total, 637 N-glycosites from 299 glycoproteins (protein groups) were identified. There were also 31 glycoforms recognized after HILIC enrichment and MS analysis. Among the results, 107 glycosylation sites (16.8%) are newly found. This study provided a simple and reliable strategy for exploring potential N-glycoprotein biomarkers from complicated biological systems.

Protein glycosylation regulates numerous important biological processes and plays key roles in many diseases including cancer, diabetes and inflammation. The ability to efficiently profile variation of protein glycosylation in biological samples is very useful for identifying new diagnostic biomarkers or developing new therapeutic approaches. Due to the low availability of glycoprotein/glycopeptide from natural sources, enrichment before mass spectrometry (MS) analysis is usually a prerequisite. Affinity enrichment using lectins is currently one of the most widely adopted approaches. Conventionally, lectins are immobilized on solid supporting materials for sample recovery. However, the limited specific surface area, high steric hindrance and rigid nature of such supporting materials restricts lectin loading amount and results in low flexibility as well as accessibility of the immobilized lectins. Therefore, we proposed using core–shell microparticles composed of silica core and brush-like polymer chains shell for improved lectin immobilization. The surface bound brush-like polymer are synthesized by in situ growth of polymer chains from microparticle surface using surface initiated atom transfer radical polymerization (SI-ATRP). The flexible non-crosslinked polymer chains not only provide numerous binding sites, but also work as three-dimensional support for lectin immobilization, which leads to high loading amount and good accessibility of the immobilized lectin. Successful enrichment which facilitated glycoprotein/glycopeptide identification is demonstrated.

Protein N-glycosylation is one of the most important post-translational modifications closely correlated with many important biological and pathological processes. The structural alterations of N-linked glycans in glycoproteins are always associated with many diseases, such as diabetes, heart failure and malignant tumors. Therefore, it is very important to establish sensitive methods for high-throughput N-glycan profiling. However, the low abundance of the N-glycoproteins and the heterogeneity of the N-glycans make it a challenge to analyse the protein glycosylation sensitively. In this work, we had synthesized core-shell hydrophilic polymer-silica hybrid materials (pGMAG-SiO2) for the efficient enrichment of protein N-glycans. Firstly, pGMAG-SiO2 was prepared by in situ growth of glucose polymer on the surface of silica microparticles using surface-initiated atom transfer radical polymerization (SI-ATRP) technique. The strong hydrophilicity of the material makes it suitable for the enrichment of N-glycans released from complex samples. Secondly, maltoheptaose and the N-glycans from chicken egg albumin were used as standard samples to optimize the enrichment conditions and evaluate the enrichment efficiency of pGMAG-SiO2. Finally, pGMAG-SiO2 was applied to the enrichment of N-linked glycans from human plasma proteins and 47 glycoforms were successfully identified after enrichment. These results demonstrated the high enrichment efficiency and significant application value of pGMAG-SiO2 in the analysis of N-glycans.

Protein N-glycosylation plays an important role in a variety of biological processes, and unusual changes in glycan structures are often correlated with various pathological states. Therefore, N-glycan profiling not only plays a vital role in the mechanism study of biological processes but also contributes to the discovery of biomarkers for early disease diagnosis. However, the conventional in-solution release of N-glycans by free endoglycosidases, such as peptide N-glycosidase F (PNGase F), suffers from several drawbacks, including prolonged incubation time, possible incomplete digestion and high cost due to the non-reusability of the enzyme. Herein, we report a novel graphene oxide (GO)-based immobilized PNGase F reagent for fast and highly efficient N-glycan release and glycoform profiling of glycoproteins. To create this reagent, PNGase F was attached to the GO surface via carbodiimide-activated amidation. The ultra-high surface area of GO allows for high loading with PNGase F and significantly facilitates the deglycosylation process. Highly efficient N-glycan release of ribonuclease B and asialofetuin was achieved with this reagent within 2 min. Furthermore, the successful application of our GO-based immobilized PNGase F in the N-glycan release of the human plasma glycoproteome was demonstrated by reproducible N-glycan identification using MALDI-TOF MS.

A novel type of glycopolymer brushes grafted open-tubular capillary column (OTCC) was developed for the polar compound separation. Briefly, the glycerol methacrylate (GMA) polymer brushes were grafted on the inner wall of OTCC by the surface initiated atom transfer radical polymerization (SI-ATRP). Next, glucose was coupled to the side chains of the GMA polymer brushes to obtain the hydrophilic stationary phase (PGMA-N-Glucose). The optimized SI-ATRP reaction conditions were GMA/CuCl/CuCl2/cyclohexanol system grafting at 25 degrees C for 1 h. After the glucose coupling, the column back pressure was about 3,585 kPa. The structure of the glycopolymer brushes on the inner surface of OTCC was characterized by scanning electron microscopy (SEM). The glycopolymer grafting resulted in the formation of three-dimensional wave-like polymer structure on the inner surface of OTCC and largely increased the interior surface area. Therefore, improved column efficiency and loading capacity can be achieved. Under the optimized conditions, the electro osmotic flow (EOF) strength of the glycopolymer brushes grafted OTCC was obviously less than that of the bare column when the pH value of the mobile phase ranging from 3 to 11. Using the glycopolymer brushes grafted OTCC, the baseline separations of polar molecules and proteins were obtained without peak tailing. The future work will focus on the further development of the glycopolymer brushes for the highly polar compound separation, such as glycans and glycoproteins.