Enrichment methods of N-linked glycopeptides from human serum or plasma: A mini-review.

Human diseases often correlate with changes in protein glycosylation, which can be observed in serum or plasma samples. N-glycosylation, the most common form, can provide potential biomarkers for disease prognosis and diagnosis. However, glycoproteins constitute a relatively small proportion of the total proteins in human serum and plasma compared to the non-glycosylated protein albumin, which constitutes the majority. The detection of microheterogeneity and low glycan abundance presents a challenge. Mass spectrometry facilitates glycoproteomics research, yet it faces challenges due to interference from abundant plasma proteins. Therefore, methods have emerged to enrich N-glycans and N-linked glycopeptides using glycan affinity, chemical properties, stationary phase chemical coupling, bioorthogonal techniques, and other alternatives. This review focuses on N-glycans and N-glycopeptides enrichment in human serum or plasma, emphasizing methods and applications. Although not exhaustive, it aims to elucidate principles and showcase the utility and limitations of glycoproteome characterization.

Mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via mTORC2/HK1-mediated metabolic rewiring.

Bacillus Calmette-Guérin (BCG) vaccination induces a type of immune memory known as "trained immunity", characterized by the immunometabolic and epigenetic changes in innate immune cells. However, the molecular mechanism underlying the strategies for inducing and/or boosting trained immunity in alveolar macrophages remains unknown. Here, we found that mucosal vaccination with the recombinant strain rBCGPPE27 significantly augmented the trained immune response in mice, facilitating a superior protective response against Mycobacterium tuberculosis and non-related bacterial reinfection in mice when compared to BCG. Mucosal immunization with rBCGPPE27 enhanced innate cytokine production by alveolar macrophages associated with promoted glycolytic metabolism, typical of trained immunity. Deficiency of the mammalian target of rapamycin complex 2 and hexokinase 1 abolished the immunometabolic and epigenetic rewiring in mouse alveolar macrophages after mucosal rBCGPPE27 vaccination. Most noteworthy, utilizing rBCGPPE27's higher-up trained effects: The single mucosal immunization with rBCGPPE27-adjuvanted coronavirus disease (CoV-2) vaccine raised the rapid development of virus-specific immunoglobulin G antibodies, boosted pseudovirus neutralizing antibodies, and augmented T helper type 1-biased cytokine release by vaccine-specific T cells, compared to BCG/CoV-2 vaccine. These findings revealed that mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via reprogramming mTORC2- and HK-1-mediated aerobic glycolysis, providing new vaccine strategies for improving tuberculosis (TB) or coronavirus variant vaccinations, and targeting innate immunity via mucosal surfaces.

Development and Validation of a Prediction Model for Venous Thrombus Embolism (VTE) in Patients With Colorectal Cancer.

Cancer patients are at high risk of developing venous thromboembolism (VTE). The risk of VTE could be mitigated with the administration of prophylactic anticoagulants. Therefore, risk assessment models would be a useful tool in order to identify those patients who are at higher risk and will be benefited more by prophylactic anticoagulants. This study retrospectively examined 528 newly diagnosed colorectal cancer patients from January 2019 to January 2021. Specified logistic regression models were employed to screen the factors and establish prediction tools based on nomograms according to the final included variables. Discrimination, calibration, and clinical applicability were used to assess the performance of screening tools. In addition, internal verifications were conducted through 10-fold cross-verification, leave-one-out cross-validation, and Bootstrap verification. Four risk factors, closely related to the occurrence of VTE in colorectal cancer patients, were identified after univariate and multivariate logistic regression, including age, body mass index, activated partial thromboplastin time, and D-Dimer value. Besides, the risk assessment model named ABAD was built on the basis, displaying good discriminations and calibrations. The area under the curve was 0.705 (95% confidence interval [CI], 0.644 to 0.766). According to Hosmer-Lemeshow goodness-of-fit test, a good agreement between the predicted and observed VTE events in patients with newly-diagnosed gastrointestinal cancer was observed for χ2 = 6.864, P = .551. Internal validation was applied with a C-index of 0.669 in the 10-fold cross-verification, 0.658 in the leave-one-out cross verification and 0.684 in the bootstrap verification. We developed a prediction model called ABAD for newly diagnosed colorectal cancer patients, which can be used to predict the risk of VTE. After evaluation and internal verification, we believe that ABAD exhibited high predictive performance and availability and could be recommended.

Three-Dimensional Gene Regulation Network in Glioblastoma Ferroptosis.

Ferroptosis is an iron-dependent form of cell death, which is reported to be associated with glioma progression and drug sensitivity. Targeting ferroptosis is a potential therapeutic approach for glioma. However, the molecular mechanism of glioma cell ferroptosis is not clear. In this study, we profile the change of 3D chromatin structure in glioblastoma ferroptosis by using HiChIP and study the 3D gene regulation network in glioblastoma ferroptosis. A combination of an analysis of HiChIP and RNA-seq data suggests that change of chromatin loops mediated by 3D chromatin structure regulates gene expressions in glioblastoma ferroptosis. Genes that are regulated by 3D chromatin structures include genes that were reported to function in ferroptosis, like HDM2 and TXNRD1. We propose a new regulatory mechanism governing glioblastoma cell ferroptosis by 3D chromatin structure.

Absolute Quantification of Dynamic Cellular Uptake of Small Extracellular Vesicles via Lanthanide Element Labeling and ICP-MS.

Small extracellular vesicles (sEVs) are increasingly reported to play important roles in numerous physiological and pathological processes. Cellular uptake of sEVs is of great significance for functional regulation in recipient cells. Although various sEV quantification, labeling, and tracking methods have been reported, it is still highly challenging to quantify the absolute amount of cellular uptake of sEVs and correlate this information with phenotypic variations in the recipient cell. Therefore, we developed a novel strategy using lanthanide element labeling and inductively coupled plasma-mass spectrometry (ICP-MS) for the absolute and sensitive quantification of sEVs. This strategy utilizes the chelation interaction between Eu3+ and the phosphate groups on the sEV membrane for specific labeling. sEVs internalized by cells can then be quantified by ICP-MS using a previously established linear relationship between the europium content and the particle numbers. High Eu labeling efficiency and stability were demonstrated by various evaluations, and no structural or functional alterations in the sEVs were discovered after Eu labeling. Application of this method revealed that 4020 ± 171 sEV particles/cell were internalized by HeLa cells at 37 °C and 61% uptake inhibition at 4 °C. Further investigation led to the quantitative differential analysis of sEV cellular uptake under the treatment of several chemical endocytosis inhibitors. A 23% strong inhibition indicated that HeLa cells uptake sEVs mainly through the macropinocytosis pathway. This facile labeling and absolute quantification strategy of sEVs with ppb-level high sensitivity is expected to become a potential tool for studying the functions of sEVs in intracellular communication and cargo transportation.

Pressure and Light Multi-Physics Fields Comodulate the Multi-Length Scales of Chemical Wave Propagation in Diffusion-Fed Gels.

During plant growth, the combined effects of multi-physics fields such as light, temperature, and material concentration create a complex multi-length scales phenomenon. However, the mechanism of multi-physical field interactions on biological multi-length scales has not been thoroughly investigated. In this paper, an open diffusion-fed system is constructed by coupling gels with a Belousov-Zhabotinsky (BZ) chemical reaction system. The multi-length scales propagation of chemical waves in the gel system under the combined effect of multi-physical fields such as light (I) and pressure (ΔP) is investigated. It is found that when 85 Pa ≤ ΔP ≤ 100 Pa or 200 µW·cm-2 ≤ I ≤ 300 µW·cm-2, the complexity of the multi-length scales periodic structure of chemical waves shows a nonlinear change with increasing light intensity or pressure. Beyond this range, the complexity of the chemical wave multi-length scales periodic structure decreases linearly on enhancing the light intensity or increasing the pressure.

Tandem enrichment of serum exosomes and exosomal RNA with titanium dioxide.

Exosomes have great potential as biomarker carriers for disease diagnosis and prognosis. In recent years, exosomal RNA (exoRNA) has become a promising candidate for the early diagnosis and prognosis of cancers, and its pathophysiological roles in various diseases have been revealed. For example, exosome-derived mRNAs, miRNAs, circRNAs, and lncRNAs function as signalling molecules to regulate tumour growth, angiogenesis, invasion, metastasis, and the response to chemotherapy. However, the isolation of exosomes and exoRNA with high quality and purity remains challenging due to the relatively small size of exosomes and the limited amount of RNA in exosomes. In this work, we developed a novel tandem enrichment method to isolate exoRNA from serum based on the specific interaction between titanium dioxide (TiO2) and the phosphate groups on the lipid bilayer of exosomes and of the exoRNA. TiO2-based RNA isolation was first demonstrated and optimized in HeLa cells. A total of 130.9 ± 8.34 µg of RNA was rapidly enriched from approximately 5 × 106 HeLa cells within 10 min. This was a 41.5% higher yield than that using a commercial Ultrapure RNA Kit. TiO2-based tandem enrichment of exoRNA was then performed using human serum, obtaining 64.53±3.41 ng of exoRNA from 500 µL of human serum within 30 min. A total of 2,137,902 reads, including seven types of exoRNAs, were identified from the exosomes. This method is compatible with various downstream RNA processing techniques and does not use toxic or irritating reagents, such as phenol or chloroform, providing a simple, economical, rapid, and safe approach for exoRNA extraction from biological samples.

hMSH2 coordinated with the expression of E2F1 promotes platinum response in epithelial ovarian cancer.

OBJECTIVE: To explore underlying mechanisms that regulate hMSH2 expression and drug susceptibility in epithelial ovarian cancer (EOC). METHODS: Using data from the Cancer Genome Atlas (TCGA) we used bioinformatical analysis to predict transcription factors (TFs) that potentially regulate hMSH2. RT-qPCR, Western blot, and luciferase assays were undertaken using ovarian cancer cell lines to verify the identified TF. Expressions of the TF were modulated using overexpression or knockdown, and the corresponding cellular responses to cisplatin were examined. RESULTS: The TF, E2F1, was found to regulate the hMSH2 gene. The expression level of E2F1 correlated with cisplatin susceptibility in vitro. Kaplan-Meier analysis of 77 patients with EOC showed that low E2F1 expression was associated with worse survival. CONCLUSIONS: To our knowledge, this is the first report of E2F1 regulated MSH2 expression playing a role in drug resistance of platinum-based treatments for patients with EOC. Further work is need to confirm our results.

Hsa_circ_0119412 Contributes to Development of Retinoblastoma by Targeting miR-186-5p/ELK4 Axis.

Increasing evidences indicate the crucial role of circRNAs in tumorigenesis, but little is understood about their molecular mechanism in retinoblastoma (RB). This paper was designed for exploring the circ_0119412 function in cases with RB and the potential mechanism. RT-qPCR was utilized to ascertain circ_0119412 and miR-186-5p levels in RB tissues and cells, and western blotting was used to quantify ELK4 in RB cells. In addition, CCK-8 and scratch assays were carried out for evaluation of cell proliferation and migration, respectively. Apoptosis-related proteins levels (Bax and Bcl-2) were measure by western blotting. Tumor growth in vivo was detected utilizing xenograft tumor experiment. The targeting relationship between circ_0119412, miR-186-5p, and ELK4 was validated using a dual-luciferase reporter assay and an RNA immunoprecipitation (RIP) assay. In RB tissues and cells, Circ_0119412 and ELK4 expression were upregulated, while miR-186-5p expression was downregulated. In vitro assay revealed that downregulating circ_0119412 accelerated the cell apoptosis of RB cells and slowed down their migration and proliferation, and the in vivo assay indicated that circ_0119412 downregulation reduced the weight and volume of tumor in nude mice. In addition, miR-186-5p interference promoted the malignant behavior of RB cells, while ELK4 silencing showed an opposite trend. Mechanically, circ_0119412 can promote RB malignant phenotypes via miR-186-5p/ELK4 axis. Circ_0119412 was found to be upregulated in RB, and could accelerate the progression of RB via the miR-186-5p/ELK4 axis, indicating circ_0119412 may serve a promising clinical therapeutic target of RB.

KLF4 inhibits early neural differentiation of ESCs by coordinating specific 3D chromatin structure.

Neural differentiation of embryonic stem cells (ESCs) requires precisely orchestrated gene regulation, a process governed in part by changes in 3D chromatin structure. How these changes regulate gene expression in this context remains unclear. In this study, we observed enrichment of the transcription factor KLF4 at some poised or closed enhancers at TSS-linked regions of genes associated with neural differentiation. Combination analysis of ChIP, HiChIP and RNA-seq data indicated that KLF4 loss in ESCs induced changes in 3D chromatin structure, including increased chromatin interaction loops between neural differentiation-associated genes and active enhancers, leading to upregulated expression of neural differentiation-associated genes and therefore early neural differentiation. This study suggests KLF4 inhibits early neural differentiation by regulation of 3D chromatin structure, which is a new mechanism of early neural differentiation.

A Novel Purine and Uric Metabolism Signature Predicting the Prognosis of Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is regarded as one of the most common cancers in the world with a poor prognosis. Patients with HCC often have abnormal purine and uric acid metabolism, but their relationship with prognosis is unclear. Methods: Here, we collected the data of peripheral blood uric acid and clinical data in 50 patients with HCC and analyzed the relationship with prognosis. At the same time, the transcriptome sequencing data of TCGA and GEO databases were collected to analyze the changes in purine metabolic pathway activity and construct a prognosis prediction model. Based on the prognosis prediction model related to purine metabolism, we further looked for the differences in the immune microenvironment and molecular level and provided possible drug targets. Results: We found that the level of serum uric acid was positively correlated with the prognosis of HCC. At the same time, purine metabolism and purine biosynthesis pathway activities were significantly activated in patients with a poor prognosis of HCC. The prognosis prediction model of HCC based on purine metabolism and purine biosynthesis pathway can accurately evaluate the prognosis of patients with HCC. Meanwhile, we found that there were significant changes in tumor immune infiltration microenvironment and biological function at the molecular level in patients with over-activation of purine metabolism and purine biosynthesis pathway. In addition, we found that uric acid level was positively correlated with peripheral blood leukocytes in HCC patients. Conclusion: In this study, we found that the level of peripheral blood uric acid in patients with HCC is correlated with their prognosis. The prognosis of patients with HCC can be accurately predicted through the metabolic process of uric acid and purine.

Preliminary Study of Pulsed Ultraviolet Technology for Low-Temperature Disinfection.

Introduction: To explore the feasibility of pulsed ultraviolet (UV) light technology for low-temperature disinfection, a series of experiments were conducted. Methods: Pulsed UV technology's effectiveness in disinfecting Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli on different carriers were studied under varying temperatures. Results: Under different temperatures and constant radiation illumination (i.e., distance), the disinfection effect was correlated with irradiation time; among the three carriers, the disinfection effect of cloth sheets was the best, followed by stainless steel sheets, and corrugated paper sheet. The disinfection effect on Gram-negative bacteria Escherichia coli was better than that on Gram-positive bacteria Staphylococcus aureus overall. Discussion: Temperature has a limited effect on pulsed UV disinfection. Irradiation times and carrier types are influencing factors.

Prognostic implications of PPL expression in ovarian cancer.

Periplakin (PPL) is a main member in plakin family, which plays important role in cellular adhesion complexes supporting and cytoskeletal integrity supplying. PPL was reported to be a potential biomarker candidate for several types of cancers. However, the biological functions and underlying mechanisms of PPL in ovarian cancer (OV) remain unclear. In the present study, we used GEPIA 2, Human Protein Atlas, Oncomine, LinkedOmics, Kaplan-Meier Plotter, STRING, CytoHubba plug-in and TIMER to determine the associations among PPL expression, prognosis, and immune cell infiltration in OV. RT-qPCR and IHC analysis were conducted to validated the role of PPL in an independent OV cohort. Compared with the normal ovary tissues, the levels of PPL mRNA and protein expression were both obviously higher in OV tumors from multiple datasets (P < 0.05), and a poor survival was observed to be strongly correlated with high PPL expression (P < 0.05). Moreover, the results were further validated by RT-qPCR and IHC analysis in an independent OV cohort. A gene-clinical nomogram was constructed, including PPL mRNA expression and clinical factors in TCGA. Functional network analysis suggested that PPL participates in the important pathways like Wnt signaling pathway, MAPK signaling pathway. Ten hub genes (LAMC2, PXN, LAMA3, LAMB3, LAMA5, ITGA3, TLN1, ACTN4, ACTN1, and ITGB4) were identified to be positively associated with PPL. Furthermore, PPL expression was negatively correlated with infiltrating levels of CD4+ T cell, macrophages, neutrophils, and dendritic cells. In conclusion, PPL may be an unfavorable prognostic biomarker candidate in OV, which was also correlated with immune infiltrating and function in immunotherapy response.

Research progress in self-oscillating polymer brushes.

Polymer brushes possess unique changes in physical and chemical properties when they are exposed to external stimuli and have a wide range of applications. Self-oscillating polymers are anchored on surfaces of certain materials and are coupled with some self-oscillating reactions (with the Belousov-Zhabotinsky (BZ) reaction as an example) to form self-oscillating polymer brushes. As an independent field of stimulus response functional surface research, the development of new intelligent bionic materials has good potential. This article reviews the oscillation mechanisms of self-oscillating polymer brushes and their classifications. First, the oscillation mechanisms of self-oscillating polymer brushes are introduced. Second, the research progress in self-oscillating polymers is discussed in terms of the type of self-oscillation reactions. Finally, possible future developments of self-oscillating polymer brushes are prospected.

Synthesis of zwitterionic polymer modified graphene oxide for hydrophilic enrichment of N-glycopeptides from urine of healthy subjects and patients with lung adenocarcinoma.

As one of the most common and important post-translational modifications, protein N-glycosylation plays essential roles in many biological processes and have long been considered closely correlated with the occurrence and progression of multiple diseases. Systematic characterization of these disease-related protein N-glycosylation is one of the most convenient ways for new diagnostic biomarker and therapeutic drug target discovering. However, the biological samples are extremely complex and the abundance of N-glycoproteins are especially low, which make highly efficient N-glycoprotein/glycopeptide enrichment before mass spectrometry analysis a prerequisite. In this work, a new type of hydrophilic material (GO-pDMAPS) was prepared by in situ growth of linear zwitterionic polymer chains on the surface of GO and it was successfully applied for N-glycopeptide enrichment from human urine. Due to the excellent hydrophilicity and the facilitate interactions between this GO-pDMAPS and the targets, a total of 1426 N-glycosylated sites corresponding to 766 N-glycoproteins as well as 790 N-glycosylation sites corresponding to 470 N-glycoproteins were enriched and identified from urine of healthy subjects and patients with lung adenocarcinoma, respectively. Among which, 27 N-glycoproteins were expressed exclusively and 4 N-glycoproteins were upregulated at least 3 times comparing with the healthy group, demonstrating the tremendous potential of this new hydrophilic material for large scale and in depth N-glycoproteome research.

Glutathione synthesis primes monocytes metabolic and epigenetic pathway for ß-glucan-trained immunity.

Trained monocytes and macrophages produce reactive oxygen species (ROS), which trigger antioxidative glutathione (GSH) response to buffer the rising ROS. However, whether and how the trained immunity is shaped by GSH synthesis remains unknown. Here, we report that ß-glucan-trained macrophages from mice harboring a myeloid-specific deletion of the catalytic subunit of glutamate-cysteine ligase (Gclc) showed impaired GSH synthesis and decreased proinflammatory cytokine production in response to lipopolysaccharide challenge. Gclc deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of c-Myc transcription factors, abrogating the energy utilization and the metabolic reprogramming that allows ß-glucan-trained macrophages to switch to glycolysis and glutaminolysis. Furthermore, Gclc deletion repressed effective H3K27me3 demethylation in the promoters of immunometabolic genes, such as Gls, Hk2, and Glut1, in ß-glucan-trained macrophages by promoting the methyltransferase enhancer of zeste homolog 2 (EZH2). In vivo, myeloid-specific ablation of Gclc decreased the secretion of proinflammatory cytokines upon rechallenge with Candida albicans and these animals were less protected against the infection, compared with control littermates. Moreover, pharmacological inhibition of EZH2 enhanced the trained immunity response against Candida infection in Gclc-deficient mouse and human peripheral blood mononuclear cells treated with GCLC inhibitor buthionine sulfoximine (BSO). Thus, antioxidative GSH synthesis supports an environment conducive to ß-glucan-induced metabolic and epigenetic reprogramming in trained immunity, allowing exploration of its functional consequences in autoimmune or inflammatory disease.

Monitoring the Effective Sterilization of Low-Temperature Hydrogen Peroxide Gas Plasma Sterilizers in 58 Hospitals - 22 PLADs, China, June 2015-December 2019.

WHAT IS ALREADY KNOWN ON THIS TOPIC?: Hydrogen peroxide sterilizeation is widely used for luminal devices. However, the low penetrability of the sterilant is of major concern. WHAT IS ADDED BY THIS REPORT?: This report investigated the effective sterilization of low-temperature hydrogen peroxide gas plasma sterilizers and compared the applicability of different biological monitoring methods based on medical luminal devices. WHAT ARE THE IMPLICATIONS FOR PUBLIC HEALTH PRACTICE?: It is recommended to use a biological process challenge device for monitoring the sterilization of luminal devices with low-temperature hydrogen peroxide gas plasma sterilizers.

Electrooxidation of Formamidine Disulfide Simultaneously Investigated by On-Line High Performance Liquid Chromatography and Cyclic Voltammetry.

The electro-oxidation of formamidine disulfide, an important sulfur-containing compound, was simultaneously investigated with on-line high-performance liquid chromatography and cyclic voltammetry. Using a home-made microporous sampler located at the electrode interface, the solution on the electrode surface was in situ sampled and analyzed. The electrochemical scanning was synchronously performed, which allowed the electro-oxidation products to be detected at a given potential. The main products on the surface of platinum electrode were found to be thiourea, formamidine sulfinic acid, cyanamide, and elemental sulfur. Forced convection arising from the sampling played an important role in the electrochemical oxidation. The extraction of electrode surface solution promoted the renewal of reactant and its intermediates, which induced the change of cyclic voltammetry curve. The forced convection also contributed to the redox peak current of the species on the cyclic voltammetry curves through the change of concentration of reactant and its intermediates. This technique can help to explore the reaction mechanism of complex electrochemical reactions.

Prevention and control of COVID-19 in public transportation: Experience from China.

Due to continuous spread of coronavirus disease 2019 (COVID-19) worldwide, long-term effective prevention and control measures should be adopted for public transport facilities, as they are increasing in popularity and serve as the principal modes for travel of many people. The human infection risk could be extremely high due to length of exposure time window, transmission routes and structural characteristics during travel or work. This can result in the rapid spread of the infection. Based on the transmission characteristics of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and the nature of public transport sites, we identified comprehensive countermeasures toward the prevention and control of COVID-19, including the strengthening of personnel management, personal protection, environmental cleaning and disinfection, and health education. Multi-pronged strategies can enhance safety of public transportation. The prevention and control of the disease during the use of public transportation will be particularly important when all countries in the world resume production. The aim of this study is to introduce experience of the prevention and control measures for public transportation in China to promote the global response to COVID-19.