The effects of immortalization on the N-glycome and proteome of CDK4-transformed lung cancer cells.

Biological experiments are often conducted in vitro using immortalized cells due to their accessibility and ease of propagation compared to primary cells and live animals. However, immortalized cells may present different proteomic and glycoproteomic characteristics from the primary cell source due to the introduction of genes that enhance proliferation (e.g. CDK4) or enable telomere lengthening. To demonstrate the changes in phenotype upon CDK4-transformation, we performed LC-MS/MS glycomic and proteomic characterizations of a human lung cancer primary cell line (DTW75) and a CDK4-transformed cell line (GL01) derived from DTW75. We observed that the primary and CDK4-transformed cells expressed significantly different levels of sialylated, fucosylated, and sialofucosylated N-glycans. Specifically, the primary cells expressed higher levels of hybrid- and complex-type sialylated N-glycans, while CDK4-transformed cells expressed higher levels of complex-type fucosylated and sialofucosylated N-glycans. Further, we compared the proteomic differences between the cell lines and found that CDK4-transformed cells expressed higher levels of RNA-binding and adhesion proteins. Further, we observed that the CDK4-transformed cells changed N-glycosylation after 31 days in cell culture, with a decrease in high-mannose and increase in fucosylated, sialylated, and sialofucosylated N-glycans. Identifying these changes between primary and CDK4-transformed cells will provide useful insight when adapting cell lines that more closely resemble in vivo physiological conditions.

UDP-glucose pyrophosphorylase 2, a regulator of glycogen synthesis and glycosylation, is critical for pancreatic cancer growth.

UDP-glucose pyrophosphorylase 2 (UGP2), the enzyme that synthesizes uridine diphosphate (UDP)-glucose, rests at the convergence of multiple metabolic pathways, however, the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an important role for UGP2 in the maintenance of pancreatic ductal adenocarcinoma (PDAC) growth in both in vitro and in vivo tumor models. We found that transcription of UGP2 is directly regulated by the Yes-associated protein 1 (YAP)-TEA domain transcription factor (TEAD) complex, identifying UGP2 as a bona fide YAP target gene. Loss of UGP2 leads to decreased intracellular glycogen levels and defects in N-glycosylation targets that are important for the survival of PDACs, including the epidermal growth factor receptor (EGFR). These critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer insights into therapeutic options for otherwise intractable PDACs.

Multi-glycomic analysis of spheroid glycocalyx differentiates 2- and 3-dimensional cell models.

A multi-glycomic method for characterizing the glycocalyx was employed to identify the difference between 2-dimensional (2D) and 3-dimensional (3D) culture models with two human colorectal cancer cell lines, HCT116 and HT29. 3D cell cultures are considered more representative of cancer due to their ability to mimic the microenvironment found in tumors. For this reason, they have become an important tool in cancer research. Cell-cell interactions increase in 3D models compared to 2D, indeed significant glycomic changes were observed for each cell line. Analyses included the N-glycome, O-glycome, glycolipidome, glycoproteome, and proteome providing the most extensive characterization of the glycocalyx between 3D and 2D thus far. The different glycoconjugates were affected in different ways. In the N-glycome, the 3D cells increased in high-mannose glycosylation and in core fucosylation. Glycolipids increased in sialylation. Specific glycoproteins were found to increase in the 3D cell, elucidating the pathways that are affected between the two models. The results show large structural and biological changes between the 2 models suggesting that the 2 are indeed very different potentially affecting individual outcomes in the study of diseases.

Antitumor activity of a lectibody targeting cancer-associated high-mannose glycans.

Aberrant protein glycosylation is a hallmark of cancer, but few drugs targeting cancer glycobiomarkers are currently available. Here, we showed that a lectibody consisting of the high-mannose glycan-binding lectin Avaren and human immunoglobulin G1 (IgG1) Fc (AvFc) selectively recognizes a range of cell lines derived from lung, breast, colon, and blood cancers at nanomolar concentrations. Binding of AvFc to the non-small cell lung cancer (NSCLC) cell lines A549 and H460 was characterized in detail. Co-immunoprecipitation proteomics analysis revealed that epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) are among the lectibody's common targets in these cells. AvFc blocked the activation of EGFR and IGF1R by their respective ligands in A549 cells and inhibited the migration of A549 and H460 cells upon stimulation with EGF and IGF1. Furthermore, AvFc induced potent Fc-mediated cytotoxic effects and significantly restricted A549 and H460 tumor growth in severe combined immunodeficiency (SCID) mice. Immunohistochemistry analysis of primary lung tissues from NSCLC patients demonstrated that AvFc preferentially binds to tumors over adjacent non-tumor tissues. Our findings provide evidence that increased abundance of high-mannose glycans in the glycocalyx of cancer cells can be a druggable target, and AvFc may provide a new tool to probe and target this tumor-associated glycobiomarker.

Comparative proteomics reveals anticancer compounds from Lansium domesticum against NSCLC cells target mitochondrial processes.

Lansium domesticum is identified as a potential source of anticancer compounds. However, there are minimal studies on its anti-lung cancer properties as well as its mechanism of action. Here, we show the specificity of lanzones hexane (LH) leaf extracts to non-small cell lung cancer cells (A549) compared to normal lung fibroblast cells (CCD19-Lu) and normal epithelial prostate cells (PNT2). Subsequent bioassay-guided fractionation of the hexane leaf extracts identified two bioactive fractions with IC50 values of 2.694 µg/ml (LH6-6) and 2.883 µg/ml (LH7-6). LH 6-6 treatment (1 µg/ml concentration) also showed a significantly reduced migration potential of A549 relative to the control. Thirty-one phytocompounds were isolated and identified using gas chromatography-mass spectrometric (MS) analysis and were then subjected to network pharmacology analysis to assess its effects on lung cancer target proteins. Using liquid chromatography-tandem mass spectrometry proteomics experiments, we were able to show that these compounds cause cytotoxic effects through targeting mitochondrial processes in A549 lung cancer cells.

Surface Reconstruction of an FeNi Foam Substrate for Efficient Oxygen Evolution.

Designing earth-abundant electrocatalysts that are highly active, low-cost, and stable for the oxygen evolution reaction (OER) is crucial for electrochemical water splitting. However, in conventional electrode fabrication strategies, NiFe layered double hydroxide (NiFe LDH) catalysts are usually coated onto substrates as external components, which suffers from poor conductivity, easily detaches from the substrate, and hinders their long-term utilization. Herein, the surface-reconstruction strategy is used to synthesize in situ autologous NiFe LDH to increase the surficial active sites numbers. The FeNi foam (FNF) serves as both the metal source and substrate, and the obtained NiFe LDH nanosheets (NSs) are firmly anchored in the monolithic FNF. What needs to be emphasized is that the strategy does not involve any high-temperature or high-pressure processes, apart from a cost-effective etching and a specified drying treatment. The nanostructure of NiFe LDH and the synergistic effect between Fe and Ni simultaneously lead to an enhanced catalytic effect for the OER. Remarkably, the sr-FNF46 requires only an ultralow overpotential of 283 mV to achieve a current density of 100 mA cm-2 for the OER in 1 M KOH electrolyte, and exhibits excellent stability. Thus, the obtained electrode holds promise for electrocatalytic applications. Finally, the formation mechanism of NiFe LDH NSs due to surface reconstruction is investigated and discussed in detail.

An Integrated Mass Spectrometry-Based Glycomics-Driven Glycoproteomics Analytical Platform to Functionally Characterize Glycosylation Inhibitors.

Cancer progression is linked to aberrant protein glycosylation due to the overexpression of several glycosylation enzymes. These enzymes are underexploited as potential anticancer drug targets and the development of rapid-screening methods and identification of glycosylation inhibitors are highly sought. An integrated bioinformatics and mass spectrometry-based glycomics-driven glycoproteomics analysis pipeline was performed to identify an N-glycan inhibitor against lung cancer cells. Combined network pharmacology and in silico screening approaches were used to identify a potential inhibitor, pictilisib, against several glycosylation-related proteins, such as Alpha1-6FucT, GlcNAcT-V, and Alpha2,6-ST-I. A glycomics assay of lung cancer cells treated with pictilisib showed a significant reduction in the fucosylation and sialylation of N-glycans, with an increase in high mannose-type glycans. Proteomics analysis and in vitro assays also showed significant upregulation of the proteins involved in apoptosis and cell adhesion, and the downregulation of proteins involved in cell cycle regulation, mRNA processing, and protein translation. Site-specific glycoproteomics analysis further showed that glycoproteins with reduced fucosylation and sialylation were involved in apoptosis, cell adhesion, DNA damage repair, and chemical response processes. To determine how the alterations in N-glycosylation impact glycoprotein dynamics, modeling of changes in glycan interactions of the ITGA5-ITGB1 (Integrin alpha 5-Integrin beta-1) complex revealed specific glycosites at the interface of these proteins that, when highly fucosylated and sialylated, such as in untreated A549 cells, form greater hydrogen bonding interactions compared to the high mannose-types in pictilisib-treated A549 cells. This study highlights the use of mass spectrometry to identify a potential glycosylation inhibitor and assessment of its impact on cell surface glycoprotein abundance and protein-protein interaction.

Mesenchymal Stromal Cells Regulate Sialylations of N-Glycans, Affecting Cell Migration and Survival.

N-Glycosylations are an important post-translational modification of proteins that can significantly impact cell function. Terminal sialic acid in hybrid or complex N-glycans has been shown to be relevant in various types of cancer, but its role in non-malignant cells remains poorly understood. We have previously shown that the motility of human bone marrow derived mesenchymal stromal cells (MSCs) can be modified by altering N-glycoforms. The goal of this study was to determine the role of sialylated N-glycans in MSCs. Here, we show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model. We also show that pre-treatment or continuous treatment with 3F-Neu5Ac inhibits both osteogenic and adipogenic differentiation of MSCs. Finally, secretion of key trophic factors by MSCs is variably affected upon exposure to 3F-Neu5Ac. Altogether, our experiments suggest that sialylation of N-glycans is tightly regulated in response to environmental cues and that glycoengineering MSCs to reduce sialylated N-glycans could be beneficial to increase both cell migration and survival, which may positively impact the therapeutic potential of the cells.

Dss1 Regulates Association of Brh2 with Rad51.

Brh2, the BRCA2 ortholog in the fungus Ustilago maydis, mediates delivery of Rad51 to DNA during the course of homology-directed DNA repair. Rad51 interacts with Brh2 through the highly conserved BRC element and through a second region termed CRE located at the extreme carboxy terminus. Dss1, a small intrinsically unstructured protein that interacts with Brh2, is crucial for its activity in DNA repair, but the mechanism of this regulation is uncertain. In previous studies, we found that interaction of Brh2 with DNA was strongly modulated by association with Dss1. Here we report that CRE influences interaction of Dss1 with Brh2 and that Dss1 status markedly alters interaction of Brh2 with Rad51. While it appears that a single Rad51 protomer associates with Brh2 in complex with Dss1, loss of Dss1 is accompanied by a large increase in the number of Rad51 protomers that can associate with Brh2. Concomitant with this buildup of Rad51, Brh2 loses its ability to bind DNA. These observations suggest a feedback circuit in which release of Dss1 from Brh2 as it binds DNA triggers nucleation of a short Rad51 oligomer on Brh2, which in turn promotes dissociation of Brh2 from the DNA.

Novel Co2 VO4 Anodes Using Ultralight 3D Metallic Current Collector and Carbon Sandwiched Structures for High-Performance Li-Ion Batteries.

A novel spinel Co2 VO4 is studied as the Li-ion battery anode material and it is sandwiched with a 3D ultralight porous current collector (PCC) and amorphous carbon. Co2 VO4 demonstrates the high capacity and excellent cyclability because of the mixed lithium storage mechanisms. The 3D composite structure requires no binders and replaces the conventional current collector (Cu foil) with a 3D ultralight porous metal scaffold, yielding the high electrode-based capacity. Such a novel composite anode also enables the close adhesion of Co2 VO4 to the PCC scaffold. The resulting monolithic electrode has the rapid electron pathway and stable mechanical properties, which lead to the excellent rate capabilities and cycling properties. At a current density of 1 A g-1 , the PCC and carbon sandwiched Co2 VO4 anode is able to deliver a stable reversible capacity of about 706.8 mAh g-1 after 1000 cycles. Generally, this study not only develops a new Co2 VO4 anode with high capacity and good cyclability, but also demonstrates an alternative approach to improve the electrochemical properties of high capacity anode materials by using ultralight porous metallic current collector instead of heavy copper foil.

Carbon doping enhances the fluoride removal performance of aluminum-based adsorbents.

Excessive fluoride presence in water poses significant environmental and public health risks, necessitating the development of effective remediation techniques. Conventional aluminum-based adsorbents face inherent limitations such as limited pH range and low adsorption capacity. To overcome these challenges, we present a facile solvent-thermal method for synthesizing a carbon-doped aluminum-based adsorbent (CDAA). Extensive characterization of CDAA reveals remarkable features including substantial carbon-containing groups, unsaturated aluminum sites, and a high pH at point of zero charge (pHpzc). CDAA demonstrates superior efficiency and selectivity in removing fluoride contaminants, surpassing other adsorbents. It exhibits exceptional adaptability across a broad pH spectrum from 3 to 12, with a maximum adsorption capacity of 637.4 mg/g, more than 110 times higher than alumina. The applicability of the Langmuir isotherm and pseudo-second-order models effectively supports these findings. Notably, CDAA exhibits rapid kinetics, achieving near-equilibrium within just 5 min. Comprehensive analyses utilizing Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) offer detailed insights into the mechanisms involving electrostatic attraction, ion exchange, and ligand exchange. Carbon-based groups play a role in ligand exchange processes, synergistically interacting with the unsaturated aluminum structure to provide a multitude of adsorption sites. The exceptional attributes of CDAA establish its immense potential as a transformative solution for the pressing challenge of fluoride removal from water sources.

Facile method to activate substrate for oxygen evolution by a galvanic-cell reaction.

NiFe layered double hydroxide (NiFe LDH) is a promising material with multiple functions. In this communication, a novel method is used to prepare NiFe LDH. This synthesis method is achieved via galvanic-cell corrosion between nickel and iron substrates in aqueous solutions containing a halogen group anion (e.g., Cl) at ambient temperature. The as-prepared NiFe LDH electrodes are developed as electrocatalysts for the oxygen evolution reaction (OER) and exhibit excellent catalytic activities and durability. This work provides an energy-efficient, cost-effective, and scaled-up corrosion engineering approach for manufacturing NiFe LDH materials.

Enhancing fluoride removal from wastewater using Al/Y amended sludge biochar.

This study explored the potential of utilizing aluminum and yttrium amended (Al/Y amended) sewage sludge biochar (Al/Y-CSBC) for efficient fluoride removal from wastewater. The adsorption kinetics of fluoride on bimetallic modified Al/Y-CSBC followed the pseudo-second-order model, while the adsorption isotherm conformed to the Freundlich equation. Remarkably, the material exhibited excellent fluoride removal performance over a wide pH range, achieving a maximum adsorption capacity of 62.44 mg·g-1. Moreover, Al/Y-CSBC demonstrated exceptional reusability, maintaining 95% removal efficiency even after six regeneration cycles. The fluoride adsorption mechanism involved ion exchange, surface complexation, and electrostatic adsorption interactions. The activation and modification processes significantly increased the specific surface area of Al/Y-CSBC, leading to a high isoelectric point (pHpzc = 9.14). The incorporation of aluminum and yttrium metals exhibited a novel approach, enhancing the adsorption capacity for fluoride ions due to their strong affinity. Furthermore, the dispersing effect of biochar played a crucial role in improving defluoridation efficiency by enhancing accessibility to active sites. These findings substantiate the significant potential of Al/Y-CSBC for enhanced fluoride removal from wastewater.

Acid etching followed by water soaking: a top-down strategy to induce highly reactive substrates for electrocatalysis.

A top-down strategy using acid etching followed by water soaking is utilized to in situ synthesize autologous NiFe LDH nanosheets on NiFe foam without other metal ions, oxidizing agents or heating steps. The NiFe foam serves as both the metal source and substrate, and the obtained nanosheets are firmly anchored on the foam. The obtained ultrathin nanosheet arrays could greatly increase the electrocatalytic active sites. This factor together with the synergistic effect between Fe and Ni simultaneously leads to an enhanced catalytic effect for water splitting and urea oxidation. This strategy could be scaled up to pave a viable way for low-cost fabrication of highly efficient electrodes for electrocatalysis.

Unlocking the potential of a NiMo foam substrate for water splitting using an ultrafast two-step dipping strategy.

In this paper, a facile and ultrafast two-step dipping process was developed to in situ form an electrocatalyst on a NiMo foam substrate without consuming extra energy. The obtained electrode showed a porous coral-like structure decorated with nanosheets and exhibited excellent overall water splitting properties in alkaline solution. This study provides a feasible strategy for developing an environmentally friendly and energy-efficient non-noble metal electrode for hydrogen production from water splitting.

Plasma-spray-enabled microcosmic explosion to construct Ni mesh-based electrodes for water splitting.

Currently, the fabrication of low cost and high efficiency electrocatalysts is a hotspot in the study of water splitting. Herein, plasma spray (PS) was used to induce a microcosmic explosion (me) on Ni mesh to modify the nanoscale Ni for the preparation of me-PS-NM electrodes with excellent hydrogen evolution. We also demonstrated that oxygen evolution could be significantly enhanced after the me-PS-NM electrodes were doped with Fe3+. Both electrodes formed a system exhibiting superior activity and stability for overall water splitting without noble metals.

Water activating fresh NiMo foam for enhanced urea electrolysis.

Recently, production of hydrogen (H2) through the urea oxidation reaction (UOR) and hydrogen evolution reaction (HER) has acquired great attention because it is more environmentally friendly and energy-saving. Herein, an approach of water activation was developed for in situ growth of NiMo LDH nanosheet arrays on NiMo foam without using any binder or pressurizing or heating steps. The obtained NiMo foam electrodes showed exceptional catalytic activity and durability for both the UOR and HER. This work offers a new standpoint on designing electrodes with high activation for efficient and sustainable hydrogen production coupled with urea organic oxidation.

Fermented Chinese Herbs Improve the Growth and Immunity of Growing Pigs through Regulating Colon Microbiota and Metabolites.

(1) Background: the development of new antibiotic substitutes to promote pig growth and health has become an important way to solve the current dilemma and promote the pig industry. (2) Methods: to assess the effects of a fermented Chinese herbal (FCH) formula on the growth and immunity of growing pigs, 100 Duroc × Landrace × Yorshire three-way crossed growing pigs were randomly divided into control and treatment groups that were fed a basal diet, and a basal diet with 1% (group A), 2% (group B), and 3% (group C) FCH formulas, respectively. A sixty-day formal experiment was conducted, and their growth and serum indices, colonic microbiota, and metabolites were analyzed. (3) Results: the daily gain of growing pigs in groups A, B, and C increased by 7.93%, 17.68%, and 19.61%, respectively, and the feed-to-gain ratios decreased by 8.33%, 15.00%, and 14.58%, respectively. Serum immunity and antioxidant activities were significantly increased in all treatment groups. Particularly, adding a 2% FCH formula significantly changed the colon's microbial structure; the Proteobacteria significantly increased and Firmicutes significantly decreased, and the metabolite composition in the colon's contents significantly changed. (4) Conclusions: these results indicate that the FCH formula is a good feed additive for growing pigs, and the recommended addition ratio was 3%.

Glycomic, Glycoproteomic, and Proteomic Profiling of Philippine Lung Cancer and Peritumoral Tissues: Case Series Study of Patients Stages I-III.

Lung cancer is the leading cause of cancer death and non-small cell lung carcinoma (NSCLC) accounting for majority of lung cancers. Thus, it is important to find potential biomarkers, such as glycans and glycoproteins, which can be used as diagnostic tools against NSCLC. Here, the N-glycome, proteome, and N-glycosylation distribution maps of tumor and peritumoral tissues of Filipino lung cancer patients (n = 5) were characterized. We present several case studies with varying stages of cancer development (I-III), mutation status (EGFR, ALK), and biomarker expression based on a three-gene panel (CD133, KRT19, and MUC1). Although the profiles of each patient were unique, specific trends arose that correlated with the role of aberrant glycosylation in cancer progression. Specifically, we observed a general increase in the relative abundance of high-mannose and sialofucosylated N-glycans in tumor samples. Analysis of the glycan distribution per glycosite revealed that these sialofucosylated N-glycans were specifically attached to glycoproteins involved in key cellular processes, including metabolism, cell adhesion, and regulatory pathways. Protein expression profiles showed significant enrichment of dysregulated proteins involved in metabolism, adhesion, cell-ECM interactions, and N-linked glycosylation, supporting the protein glycosylation results. The present case series study provides the first demonstration of a multi-platform mass-spectrometric analysis specifically for Filipino lung cancer patients.

Dss1 release activates DNA binding potential in Brh2.

Dss1 is an intrinsically unstructured polypeptide that partners with the much larger Brh2 protein, the BRCA2 ortholog in Ustilago maydis, to form a tight complex. Mutants lacking Dss1 have essentially the same phenotype as mutants defective in Brh2, implying that through physical interaction Dss1 serves as a positive activator of Brh2. Dss1 associates with Brh2 through an interaction surface in the carboxy-terminal region. Certain derivatives of Brh2 lacking this interaction surface remain highly competent in DNA repair as long as a DNA-binding domain is present. However, the Dss1-independent activity raises the question of what function might be met in the native protein by having Brh2 under Dss1 control. Using a set of Brh2 fusions and truncated derivatives, we show here that Dss1 is capable of exerting control when there is a cognate Dss1-interacting surface present. We find that association of Dss1 attenuates the DNA binding potential of Brh2 and that the amino-terminal domain of Brh2 helps evict Dss1 from its carboxy-terminal interaction surface. The findings presented here add to the notion that Dss1 serves in a regulatory capacity to dictate order in association of Brh2's amino-terminal and carboxy-terminal domains with DNA.