Microwave biosensor for the detection of growth inhibition of human liver cancer cells at different concentrations of chemotherapeutic drug.

Cytotoxicity assays are crucial for assessing the efficacy of drugs in killing cancer cells and determining their potential therapeutic value. Measurement of the effect of drug concentration, which is an influence factor on cytotoxicity, is of great importance. This paper proposes a cytotoxicity assay using microwave sensors in an end-point approach based on the detection of the number of live cells for the first time. In contrast to optical methods like fluorescent labeling, this research uses a resonator-type microwave biosensor to evaluate the effects of drug concentrations on cytotoxicity by monitoring electrical parameter changes due to varying cell densities. Initially, the feasibility of treating cells with ultrapure water for cell counting by a microwave biosensor is confirmed. Subsequently, inhibition curves generated by both the CCK-8 method and the new microwave biosensor for various drug concentrations were compared and found to be congruent. This agreement supports the potential of microwave-based methods to quantify cell growth inhibition by drug concentrations.

Real-Time Detection of Yeast Growth on Solid Medium through Passive Microresonator Biosensor.

This study presents a biosensor fabricated based on integrated passive device (IPD) technology to measure microbial growth on solid media in real-time. Yeast (Pichia pastoris, strain GS115) is used as a model organism to demonstrate biosensor performance. The biosensor comprises an interdigital capacitor in the center with a helical inductive structure surrounding it. Additionally, 12 air bridges are added to the capacitor to increase the strength of the electric field radiated by the biosensor at the same height. Feasibility is verified by using a capacitive biosensor, and the change in capacitance values during the capacitance detection process with the growth of yeast indicates that the growth of yeast can induce changes in electrical parameters. The proposed IPD-based biosensor is used to measure yeast drop-added on a 3 mm medium for 100 h at an operating frequency of 1.84 GHz. The resonant amplitude of the biosensor varies continuously from 24 to 72 h due to the change in colony height during vertical growth of the yeast, with a maximum change of 0.21 dB. The overall measurement results also fit well with the Gompertz curve. The change in resonant amplitude between 24 and 72 h is then analyzed and reveals a linear relationship with time with a coefficient of determination of 0.9844, indicating that the biosensor is suitable for monitoring yeast growth. Thus, the proposed biosensor is proved to have potential in the field of microbial proliferation detection.

Highly Sensitive and Linear Resonator-Based Biosensor for White Blood Cell Counting: Feasible Measurement Method and Intrinsic Mechanism Exploration.

Since different quantities of white blood cells (WBCs) in solution possess an adaptive osmotic pressure of cells, the WBCs themselves and in solution have similar concentrations, resulting in them having similar dielectric properties. Therefore, a microwave sensor could have difficulty in sensing the quantity variation when WBCs are in solution. This paper presents a highly sensitive, linear permittivity-inspired microwave biosensor for WBCs, counting through the evaporation method. Such a measurement method is proposed to record measurements after the cell solution is dripped onto the chip and is completely evaporated naturally. The proposed biosensor consists of an air-bridged asymmetric differential inductor and a centrally located circular fork-finger capacitor fabricated on a GaAs substrate using integrated passive fabrication technology. It is optimized to feature a larger sensitive area and improved Q-factor, which increases the effective area of interaction between cells and the electromagnetic field and facilitates the detection of their changes in number. The sensing relies on the dielectric properties of the cells and the change in the dielectric constant for different concentrations, and the change in resonance properties, which mainly represents the frequency shift, corresponds to the macroscopic change in the concentration of the cells. The microwave biosensors are used to measure biological samples with concentrations ranging from 0.25 × 106 to 8 × 106 cells per mL in a temperature (26.00 ± 0.40 °C) and humidity (54.40 ± 3.90 RH%) environment. The measurement results show a high sensitivity of 25.06 Hz/cells·mL-1 with a highly linear response of r2 = 0.99748. In addition, a mathematical modeling of individual cells in suspension is performed to estimate the dielectric constant of individual cells and further explain the working mechanism of the proposed microwave biosensor.

Production of CA125 with Tn-antigens using a glycosylphosphatidylinositol anchoring system.

Cancer antigen 125 (CA125) is a serum marker associated with ovarian cancer. Despite its widespread use, CA125 levels can also be elevated in benign conditions. Recent reports suggest that detecting serum CA125 that carries the Tn-antigen, a truncated O-glycan containing only N-acetylgalactosamine on serine or threonine residues, can improve the specificity of ovarian cancer diagnosis. In this study, we engineered cells to express CA125 with a Tn-antigen. To achieve this, we knocked out C1GALT1 and SLC35A1, genes encoding Core1 synthase and a transporter for cytidine-5'-monophospho-sialic acid respectively, in human embryonic kidney 293 (HEK293) cells. In ClGALT1-SLC35A1-knockout (KO) cells, the expression of the Tn-antigen showed a significant increase, whereas the expression of the T-antigen (galactose-ß1,3-N-acetylgalactosamine on serine or threonine residues) was decreased. Due to the inefficient secretion of soluble CA125, we employed a glycosylphosphatidylinositol (GPI) anchoring system. This allowed for the expression of GPI-anchored CA125 on the cell surface of ClGALT1-SLC35A1-KO cells. Cells expressing high levels of GPI-anchored CA125 were then enriched through cell sorting. By knocking out the PGAP2 gene, the GPI-anchored form of CA125 was converted to a secretory form. Through the engineering of O-glycans and the use of a GPI-anchoring system, we successfully produced CA125 with Tn-antigen modification.

Protein Signatures for Distinguishing Colorectal Cancer Liver Metastases from Primary Liver Cancer Using Tissue Slide Proteomics.

BACKGROUND: Colorectal cancer liver metastasis (CRLM) and hepatocellular carcinoma (HCC) are both high incidence tumors in China. In certain poorly differentiated cases they can exhibit comparable imaging and pathological characteristics, which impedes accurate clinical diagnosis. The use of protein-based techniques with tissue slides offers a more precise means to assess pathological changes and has the potential to assist with tumor diagnosis. METHODS: A simple in situ protein digestion protocol was established for protein fingerprint analysis of paraffin-embedded tissue slide samples. Additionally, machine learning techniques were employed to construct predictive models for CRLM and HCC. The accuracy of these models was validated using tissue slides and a clinical database. RESULTS: Analysis of differential protein expression between CRLM and HCC groups reliably identified 977 proteins. Among these, 53 were highly abundant in CRLM samples and 57 were highly abundant in HCC samples. A prediction model based on the expression of six proteins (CD9, GSTA1, KRT20, COL1A2, AKR1C3, and HIST2H2BD) had an area under curve (AUC) of 0.9667. This was further refined to three proteins (CD9, ALDH1A1, and GSTA1) with an AUC of 0.9333. CONCLUSIONS: Tissue slide proteomics can facilitate accurate differentiation between CRLM and HCC. This methodology holds great promise for improving clinical tumor diagnosis and for identifying novel markers for challenging pathological specimens.

Site- and Structure-Specific Glycosylation Signatures of Bovine, Caprine, Porcine, and Human Milk-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are membrane-bound vesicles released by living cells. As vesicles for macromolecule transmission and intercellular communication, EVs are broadly applied in clinical diagnosis and biomimetic drug delivery. Milk-derived EVs (MEVs) are an ideal choice for scale-up applications because they exhibit biocompatibility and are easily obtained. Herein, intact glycopeptides in MEVs from bovines, caprines, porcines, and humans were comprehensively analyzed by high-resolution mass spectrometry using the sceHCD, followed by the EThcD fragment method, revealing that protein glycosylation is abundant and heterogeneous in MEVs. The dominant glycans in all MEVs were sialic acid-modified N-linked glycans (over 50%). A couple of species-specific glycans were also characterized, which are potentially markers of different original EVs. Interestingly, the Neu5Gc-modified glycans were enriched in caprine milk-derived EVs (58 ± 2%). Heterogeneity of MEV protein glycosylation was observed for glycosites and glycan compositions, and the structural heterogeneity of protein glycosylation was also identified and validated. The glycosignatures of EV biogenesis- and endocytosis-related proteins (CD63 and MFGE8) were significantly different in these four species. Overall, we comprehensively characterized the glycosylation signature of MEVs from four different species and provided insight into protein glycosylation related to drug target delivery.

Sialylation: A Cloak for Tumors to Trick the Immune System in the Microenvironment.

The tumor microenvironment (TME), where the tumor cells incite the surrounding normal cells to create an immune suppressive environment, reduces the effectiveness of immune responses during cancer development. Sialylation, a type of glycosylation that occurs on cell surface proteins, lipids, and glycoRNAs, is known to accumulate in tumors and acts as a "cloak" to help tumor cells evade immunological surveillance. In the last few years, the role of sialylation in tumor proliferation and metastasis has become increasingly evident. With the advent of single-cell and spatial sequencing technologies, more research is being conducted to understand the effects of sialylation on immunity regulation. This review provides updated insights into recent research on the function of sialylation in tumor biology and summarizes the latest developments in sialylation-targeted tumor therapeutics, including antibody-mediated and metabolic-based sialylation inhibition, as well as interference with sialic acid-Siglec interaction.

Comprehensive proteome, phosphoproteome and kinome characterization of luminal A breast cancer.

Background: Breast cancer is one of the most frequently occurring malignant cancers worldwide. Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the two most common histological subtypes of breast cancer. In this study, we aimed to deeply explore molecular characteristics and the relationship between IDC and ILC subtypes in luminal A subgroup of breast cancer using comprehensive proteomics and phosphoproteomics analysis. Methods: Cancer tissues and noncancerous adjacent tissues (NATs) with the luminal A subtype (ER- and PR-positive, HER2-negative) were obtained from paired IDC and ILC patients respectively. Label-free quantitative proteomics and phosphoproteomics methods were used to detect differential proteins and the phosphorylation status between 10 paired breast cancer and NATs. Then, the difference in protein expression and its phosphorylation between IDC and ILC subtypes were explored. Meanwhile, the activation of kinases and their substrates was also revealed by Kinase-Substrate Enrichment Analysis (KSEA). Results: In the luminal A breast cancer, a total of 5,044 high-confidence proteins and 3,808 phosphoproteins were identified from 10 paired tissues. The protein phosphorylation level in ILC tissues was higher than that in IDC tissues. Histone H1.10 was significantly increased in IDC but decreased in ILC, Conversely, complement C4-B and Crk-like protein were significantly decreased in IDC but increased in ILC. Moreover, the increased protein expression of Septin-2, Septin-9, Heterogeneous nuclear ribonucleoprotein A1 and Kinectin but reduce of their phosphorylation could clearly distinguish IDC from ILC. In addition, IDC was primarily related to energy metabolism and MAPK pathway, while ILC was more closely involved in the AMPK and p53/p21 pathways. Furthermore, the kinomes in IDC were primarily significantly activated in the CMGC groups. Conclusions: Our research provides insights into the molecular characterization of IDC and ILC and contributes to discovering novel targets for further drug development and targeted treatment.

Studies on the Proteinaceous Structure Present on the Surface of the Saccharomyces cerevisiae Spore Wall.

The surface of the Saccharomyces cerevisiae spore wall exhibits a ridged appearance. The outermost layer of the spore wall is believed to be a dityrosine layer, which is primarily composed of a crosslinked dipeptide bisformyl dityrosine. The dityrosine layer is impervious to protease digestion; indeed, most of bisformyl dityrosine molecules remain in the spore after protease treatment. However, we find that the ridged structure is removed by protease treatment. Thus, a ridged structure is distinct from the dityrosine layer. By proteomic analysis of the spore wall-bound proteins, we found that hydrophilin proteins, including Sip18, its paralog Gre1, and Hsp12, are present in the spore wall. Mutant spores with defective hydrophilin genes exhibit functional and morphological defects in their spore wall, indicating that hydrophilin proteins are required for the proper organization of the ridged and proteinaceous structure. Previously, we found that RNA fragments were attached to the spore wall in a manner dependent on spore wall-bound proteins. Thus, the ridged structure also accommodates RNA fragments. Spore wall-bound RNA molecules function to protect spores from environmental stresses.

Expression of a Siglec-Fc Protein and Its Characterization.

The emerging importance of the Siglec-sialic acid axis in human disease, especially cancer, has necessitated the identification of ligands for Siglecs. Recombinant Siglec-Fc fusion proteins have been widely used as ligand detectors, and also as sialic acid-targeted antibody-like proteins for cancer treatment. However, the heterogenetic properties of the Siglec-Fc fusion proteins prepared from various expression systems have not been fully elucidated. In this study, we selected HEK293 and CHO cells for producing Siglec9-Fc and further evaluated the properties of the products. The protein yield in CHO (8.23 mg/L) was slightly higher than that in HEK293 (7.46 mg/L). The Siglec9-Fc possesses five N-glycosylation sites and one of them is located in its Fc domain, which is important for the quality control of protein production and also the immunogenicity of Siglec-Fc. Our glycol-analysis confirmed that the recombinant protein from HEK293 received more fucosylation, while CHO showed more sialylation. Both products revealed a high dimerization ratio and sialic acid binding activity, which was confirmed by the staining of cancer cell lines and bladder cancer tissue. Finally, our Siglec9-Fc product was used to analyze the potential ligands on cancer cell lines.

Accumulated precursors of specific GPI-anchored proteins upregulate GPI biosynthesis with ARV1.

We previously reported that glycosylphosphatidylinositol (GPI) biosynthesis is upregulated when endoplasmic reticulum-associated degradation (ERAD) is defective; however, the underlying mechanistic basis remains unclear. Based on a genome-wide CRISPR-Cas9 screen, we show that a widely expressed GPI-anchored protein CD55 precursor and ER-resident ARV1 are involved in upregulation of GPI biosynthesis under ERAD-deficient conditions. In cells defective in GPI transamidase, GPI-anchored protein precursors fail to obtain GPI, with the remaining uncleaved GPI-attachment signal at the C-termini. We show that ERAD deficiency causes accumulation of the CD55 precursor, which in turn upregulates GPI biosynthesis, where the GPI-attachment signal peptide is the active element. Among the 31 GPI-anchored proteins tested, only the GPI-attachment signal peptides of CD55, CD48, and PLET1 enhance GPI biosynthesis. ARV1 is prerequisite for the GPI upregulation by CD55 precursor. Our data indicate that GPI biosynthesis is balanced to need by ARV1 and precursors of specific GPI-anchored proteins.

Nucleotide binding as an allosteric regulatory mechanism for Akkermansia muciniphila ß-N-acetylhexosaminidase Am2136.

ß-N-acetylhexosaminidases (EC3.2.1.52), which belong to the glycosyl hydrolase family GH20, are important enzymes for oligosaccharides modification. Numerous microbial ß-N-acetylhexosaminidases have been investigated for applications in biology, biomedicine and biotechnology. Akkermansia muciniphila is an anaerobic intestinal commensal bacterium which possesses specific ß-N-acetylhexosaminidases for gut mucosal layer colonization and mucin degradation. In this study, we assessed the in vitro mucin glycan cleavage activity of the A. muciniphila ß-N-acetylhexosaminidase Am2136 and demonstrated its ability that hydrolyzing the ß-linkages joining N-acetylglucosamine to a wide variety of aglycone residues, which indicated that Am2136 may be a generalist ß-N-acetylhexosaminidase. Structural and enzyme activity assay experiments allowed us to probe the essential function of the inter-domain interactions in ß23-ß33. Importantly, we revealed that the hydrolysis activity of Am2136 was enhanced by nucleotides. We further speculated that this activation mechanism might be associated with the conformational motions between domain III and IV. To our knowledge, this is the first report of nucleotide effector regulated ß-N-acetylhexosaminidase, to reveal its novel biological functions. These findings contribute to understanding the distinct properties within the GH20 family and lay a certain foundation to develop controllable glycan hydrolyzing catalysts.Abbreviations: OD600 - optical cell densities at 600 nm; LB - Luria-Bertani; IPTG - isopropyl ß-D-1-thiogalactopyranoside; PMSF - phenylmethanesulfonyl fluoride; rmsd - root mean square deviation; GlcNAc - N-acetyl-ß-D-glucosamine; GalNAc - N-acetyl-ß-D-galactosamine; Gal - galactose.

Proteome and Glycoproteome Analyses Reveal the Protein N-Linked Glycosylation Specificity of STT3A and STT3B.

STT3A and STT3B are the main catalytic subunits of the oligosaccharyltransferase complex (OST-A and OST-B in mammalian cells), which primarily mediate cotranslational and post-translocational N-linked glycosylation, respectively. To determine the specificity of STT3A and STT3B, we performed proteomic and glycoproteomic analyses in the gene knock-out (KO) and wild-type HEK293 cells. In total, 3961 proteins, 4265 unique N-linked intact glycopeptides and 629 glycosites representing 349 glycoproteins were identified from all these cells. Deletion of the STT3A gene had a greater impact on the protein expression than deletion of STT3B, especially on glycoproteins. In addition, total mannosylated N-glycans were reduced and fucosylated N-glycans were increased in STT3A-KO cells, which were caused by the differential expression of glycan-related enzymes. Interestingly, hyperglycosylated proteins were identified in KO cells, and the hyperglycosylation of ENPL was caused by the endoplasmic reticulum (ER) stress due to the STT3A deletion. Furthermore, the increased expression of the ATF6 and PERK indicated that the unfolded protein response also happened in STT3A-KO cells. Overall, the specificity of STT3A and STT3B revealed that defects in the OST subunit not only broadly affect N-linked glycosylation of the protein but also affect protein expression.

Comprehensive N- and O-Glycoproteomic Analysis of Multiple Chinese Hamster Ovary Host Cell Lines.

Glycoproteomic analysis of three Chinese hamster ovary (CHO) suspension host cell lines (CHO-K1, CHO-S, and CHO-Pro5) commonly utilized in biopharmaceutical settings for recombinant protein production is reported. Intracellular and secreted glycoproteins were examined. We utilized an immobilization and chemoenzymatic strategy in our analysis. Glycoproteins or glycopeptides were first immobilized through reductive amination, and the sialyl moieties were amidated for protection. The desired N- or O-glycans and glycopeptides were released from the immobilization resin by enzymatic or chemical digestion. Glycopeptides were studied by Orbitrap Liquid chromatography-mass spectrometry (LC/MS), and the released glycans were analyzed by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Differences were detected in the relative abundances of N- and O-glycopeptide types, their resident and released glycans, and their glycoprotein complexity. Ontogeny analysis revealed key differences in features, such as general metabolic and biosynthetic pathways, including glycosylation systems, as well as distributions in cellular compartments. Host cell lines and subfraction differences were observed in both N- and O-glycan and glycoprotein pools. Differences were observed in sialyl and fucosyl glycan distributions. Key differences were also observed among glycoproteins that are problematic contaminants in recombinant antibody production. The differences revealed in this study should inform the choice of cell lines best suited for a particular bioproduction application.

Triosephosphate Isomerase and Its Product Glyceraldehyde-3-Phosphate Are Involved in the Regulatory Mechanism That Suppresses Exit from the Quiescent State in Yeast Cells.

Cells of the budding yeast Saccharomyces cerevisiae form spores or stationary cells upon nutrient starvation. These quiescent cells are known to resume mitotic growth in response to nutrient signals, but the mechanism remains elusive. Here, we report that quiescent yeast cells are equipped with a negative regulatory mechanism which suppresses the commencement of mitotic growth. The regulatory process involves a glycolytic enzyme, triosephosphate isomerase (Tpi1), and its product, glyceraldehyde-3-phosphate (GAP). GAP serves as an inhibitory signaling molecule; indeed, the return to growth of spores or stationary cells is suppressed by the addition of GAP even in nutrient-rich growth media, though mitotic cells are not affected. Reciprocally, dormancy is abolished by heat treatment because of the heat sensitivity of Tpi1. For example, spores commence germination merely upon heat treatment, which indicates that the negative regulatory mechanism is actively required for spores to prevent premature germination. Stationary cells of Candida glabrata are also manipulated by heat and GAP, suggesting that the regulatory process is conserved in the pathogenic yeast. IMPORTANCE Our results suggest that, in quiescent cells, nutrient signals do not merely provoke a positive regulatory process to commence mitotic growth. Exit from the quiescent state in yeast cells is regulated by balancing between the positive and negative signaling pathways. Identifying the negative regulatory pathway would provide new insight into the regulation of the transition from the quiescent to the mitotic state. Clinically, quiescent cells are problematic because they are resistant to environmental stresses and antibiotics. Given that the quiescent state is modulated by manipulation of the negative regulatory mechanism, understanding this process is important not only for its biological interest but also as a potential target for antifungal treatment.

N-glycosylation of GDF15 abolishes its inhibitory effect on EGFR in AR inhibitor-resistant prostate cancer cells.

Castration-resistance of prostate cancer is one of the most challenging clinical problems. In the present study, we have performed proteomics and glycomics using LNCaP model. Growth differentiation factor-15 (GDF15) level is increased in androgen receptor (AR) inhibitor-resistant cells and the inhibitory effect of GDF15 on epithelial growth factor receptor (EGFR) pathway is relieved by GDF15 N70 glycosylation. Interference of GDF15 (siRNA or N70Q dominant negative) or EGFR pathway (inhibitor or siRNA for EGFR, SRC or ERK) decreases the resistant-cell survival in culture and tumor growth in mice. Our study reveals a novel regulatory mechanism of prostate cancer AR inhibitor resistance, raises the possibility of AR/SRC dual-targeting of castration-resistance of prostate cancer, and lays foundation for the future development of selective inhibitors of GDF15 glycosylation.

Glycoproteomic Characterization of FUT8 Knock-Out CHO Cells Reveals Roles of FUT8 in the Glycosylation.

The α1,6-fucosyltransferase (encoded by FUT8 gene) is the key enzyme transferring fucose to the innermost GlcNAc residue on an N-glycan through an α-1,6 linkage in the mammalian cells. The presence of core fucose on antibody Fc region can inhibit antibody-dependent cellular cytotoxicity (ADCC) and reduce antibody therapeutic efficiency in vivo. Chinese hamster ovary (CHO) cells are the predominant production platform in biopharmaceutical manufacturing. Therefore, the generation of FUT8 knock-out (FUT8KO) CHO cell line is favorable and can be applied to produce completely non-fucosylated antibodies. The characterization of monoclonal antibodies as well as host cell glycoprotein impurities are required for quality control purposes under regulation rules. To understand the role of FUT8 in the glycosylation of CHO cells, we generated a FUT8 knock-out CHO cell line and performed a large-scale glycoproteomics to characterize the FUT8KO and wild-type (WT) CHO cells. The glycopeptides were enriched by hydrophilic chromatography and fractionated 25 fractions by bRPLC followed by analysis using high-resolution liquid chromatography mass spectrometry (LC-MS). A total of 7,127 unique N-linked glycosite-containing intact glycopeptides (IGPs), 928 glycosites, and 442 glycoproteins were identified from FUT8KO and WT CHO cells. Moreover, 28.62% in 442 identified glycoproteins and 26.69% in 928 identified glycosites were significantly changed in the FUT8KO CHO compared to wild-type CHO cells. The relative abundance of all the three N-glycan types (high-mannose, hybrid, and complex) was determined in FUT8KO comparing to wild-type CHO cells. Furthermore, a decrease in fucosylation content was observed in FUT8KO cells, in which core-fucosylated glycans almost disappeared as an effect of FUT8 gene knockout. Meantime, a total of 51 glycosylation-related enzymes were also quantified in these two cell types and 16 of them were significantly altered in the FUT8KO cells, in which sialyltransferases and glucosyltransferases were sharply decreased. These glycoproteomic results revealed that the knock-out of FUT8 not only influenced the core-fucosylation of proteins but also altered other glycosylation synthesis processes and changed the relative abundance of protein glycosylation.

A knockout cell library of GPI biosynthetic genes for functional studies of GPI-anchored proteins.

Over 100 kinds of proteins are expressed as glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on the cell surface in mammalian cells. GPI-APs possess unique properties in terms of their intracellular trafficking and association with lipid rafts. Although it is clear that GPI-APs play critical roles in various biological phenomena, it is poorly understood how the GPI moiety contributes to these mechanisms. More than 30 genes are involved in the correct biosynthesis of GPI-APs. We here constructed a cell library in which 32 genes involved in GPI biosynthesis were knocked out in human embryonic kidney 293 cells. Using the cell library, the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C of GPI-APs were analyzed. Furthermore, we identified structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. The cell-based GPI-knockout library could be applied not only to basic researches, but also to applications and methodologies related to GPI-APs.