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We developed a method to produce a soluble form of a single-chain fragment variable (scFv) targeting human epithelial growth factor receptor 2 (HER2) in Escherichia coli. By optimizing the orientations of the variable heavy (VH) and variable light (VL) domains and the His-tag, we identified the HL-His type antibody with the highest HER2-binding activity. Purification of HL-His yielded 40.7 mg from a 1 L culture, achieving >99% purity. The limit of detection was determined to be 2.9 ng, demonstrating high production yield, purity, and sensitivity. Moreover, we successfully labeled HER2+ cell lines with fluorescent dye-conjugated scFv, resulting in a significantly higher observed signal-to-background ratio, compared to that of HER2- cell lines. This highlights the potential of these fluorescent scFvs as valuable probes for HER2+ breast cancer diagnostics. Notably, the process for the complete scFv production was streamlined and required only 4-5 days. Additionally, the product maintained its activity after freeze storage, allowing for large-scale production and a wide range of practical applications.
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Escherichia coli , Receptor ErbB-2 , Proteínas Recombinantes , Anticorpos de Cadeia Única , Receptor ErbB-2/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/isolamento & purificação , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Linhagem Celular Tumoral , Neoplasias da Mama/imunologiaRESUMO
Dynamic interaction between BRCA2 and telomeric G-quadruplexes (G4) is crucial for maintaining telomere replication homeostasis. Cells lacking BRCA2 display telomeric damage with a subset of these cells bypassing senescence to initiate break-induced replication (BIR) for telomere synthesis. Here we show that the abnormal stabilization of telomeric G4 following BRCA2 depletion leads to telomeric repeat-containing RNA (TERRA)-R-loop accumulation, triggering liquid-liquid phase separation (LLPS) and the assembly of Alternative Lengthening of Telomeres (ALT)-associated promyelocytic leukemia (PML) bodies (APBs). Disruption of R-loops abolishes LLPS and impairs telomere synthesis. Artificial engineering of telomeric LLPS restores telomere synthesis, underscoring the critical role of LLPS in ALT. TERRA-R-loops also recruit Polycomb Repressive Complex 2 (PRC2), leading to tri-methylation of Lys27 on histone H3 (H3K27me3) at telomeres. Half of paraffin-embedded tissue sections from human breast cancers exhibit APBs and telomere length heterogeneity, suggesting that BRCA2 mutations can predispose individuals to ALT-type tumorigenesis. Overall, BRCA2 abrogation disrupts the dynamicity of telomeric G4, producing TERRA-R-loops, finally leading to the assembly of telomeric liquid condensates crucial for ALT. We propose that modulating the dynamicity of telomeric G4 and targeting TERRA-R-loops in telomeric LLPS maintenance may represent effective therapeutic strategies for treating ALT-like cancers with APBs, including those with BRCA2 disruptions.
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Proteína BRCA2 , Replicação do DNA , Quadruplex G , Homeostase do Telômero , Telômero , Humanos , Telômero/metabolismo , Telômero/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Homeostase do Telômero/genética , Replicação do DNA/genética , Histonas/metabolismo , Histonas/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Estruturas R-Loop , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Linhagem Celular Tumoral , Feminino , Separação de FasesRESUMO
Programmed cell death-ligand 1 (PDL1) is a transmembrane protein that is characterized as an immune regulatory molecule. We recently developed a recombinant single-chain fragment of variable domain (scFv) against PDL1, which showed high binding efficiency to purified recombinant PDL1 protein. However, at that time, proof-of-concept data for the effect of scFv using PDL1-expressing cells was lacking. In this study, we conducted two kinds of cell-based immunoassays, western blotting and enzyme-linked immunosorbent assay, using anti-PDL1 scFv. The results indicate that scFv can selectively and sensitively detect PDL1 from PDL1 positive human cancer cell lines. Our findings suggest that scFv could be used as a potential PDL1 inhibitor agent and probe for cell-based immunoassays to detect PDL1.
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Antígeno B7-H1 , Proteínas Recombinantes , Anticorpos de Cadeia Única , Humanos , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Linhagem Celular Tumoral , Ensaio de Imunoadsorção EnzimáticaRESUMO
There is a critical need to identify new therapeutic vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). Transcriptional co-regulators C-terminal binding proteins (CtBP) 1 and 2 are highly overexpressed in human PDAC, and CRISPR-based homozygous deletion of Ctbp2 in a mouse PDAC cell line (CKP) dramatically decreased tumor growth, reduced metastasis, and prolonged survival in orthotopic mouse allografts. Transcriptomic profiling of tumors derived from CKP vs. Ctbp2-deleted CKP cells (CKP/KO) revealed significant downregulation of the EGFR-superfamily receptor Erbb3, the heterodimeric signaling partner for both EGFR and ErbB2. Compared with CKP cells, CKP/KO cells also demonstrated reduced Erbb2 expression and did not activate downstream Akt signaling after stimulation of Erbb3 by its ligand neuregulin-1. ErbB3 expression in human PDAC cell lines was similarly dependent on CtBP2 and depletion of ErbB3 in a human PDAC cell line severely attenuated growth, demonstrating the critical role of ErbB3 signaling in maintaining PDAC cell growth. Sensitivity to the ErbB2-targeted tyrosine kinase inhibitor lapatinib, but not the EGFR-targeted agent erlotinib, varied in proportion to the level of ErbB3 expression in mouse and human PDAC cells, suggesting that an ErBb2 inhibitor can effectively leverage CtBP2-driven transcriptional activation of physiologic ErbB2/3 expression and signaling in PDAC cells for therapeutic benefit.
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The number of non-accidental deaths and heat-related illnesses due to the co-occurrence of heatwaves and COVID-19 has been identified to estimate compound health impacts between two risks. We have analyzed data from historical years (2013-2019) to calculate the baseline values of the number of non-accidental deaths and heat-related illness patients from May to September using a quasi-Poisson generalized linear model and compared them to data from 2020 in Korea. We also assessed the relative risk and absolute cumulative number of non-accidental deaths and heat-related illnesses in the summer of 2020 in Seoul, Daegu, and Gyeongnam region of Korea. In the Summer of 2020, Korea experienced 0.8% of non-accidental excess deaths, with the highest in August, and 46% of reduction was observed in heat-related throughout the study period, except in Daegu, where excess of heat-related illness occurred in August. The relative risk (RR) of non-accidental deaths at 33.1 °C, was 1.00 (CI 0.99-1.01) and 1.04 (CI 1.02-1.07) in 2013-2019 and 2020, respectively. The RR of heat-related illness at 33.1 °C, was 1.44 (CI 1.42-1.45) and 1.59 (CI 1.54-1.64) in 2013-2019 and 2020, respectively. The absolute cumulative trends of non-accidental deaths and heat-related illnesses were similar in the three regions, indicating increased non-accidental deaths and decreased heat-related illnesses at similar temperatures in 2020. During the COVID-19 pandemic, the fear of infection by the virus and the limited access to healthcare services led to changes in health-seeking behaviors. These results indicate social distancing could have had adverse impacts on other health conditions. A comprehensive health risk assessment is important when facing simultaneous risks, such as heatwaves and pandemics, in the implementation of effective countermeasures.
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COVID-19 , Humanos , Animais , COVID-19/epidemiologia , Pandemias , Raios Infravermelhos , Estro , SeulRESUMO
Biomimetic silica deposition is an in-situ immobilization method for bioactive molecules under biocompatible conditions. The osteoinductive P4 peptide derived from the knuckle epitope of bone morphogenetic protein (BMP), which binds to BMP receptor-II (BMPRII), has been newly found to contain silica formation ability. We found that the two lysine residues at the N-terminus of P4 played a vital role in silica deposition. The P4 peptide co-precipitated with silica during P4-mediated silicification, yielding P4/silica hybrid particles (P4@Si) with a high loading efficiency of 87%. P4 was released from P4@Si at a constant rate for over 250 h, representing a zero-order kinetic model. In flow cytometric analysis, P4@Si showed a 1.5-fold increase in the delivery capacity to MC3T3 E1 cells than the free form of P4. Furthermore, P4 was found anchored to hydroxyapatite (HA) through a hexa-glutamate tag, followed by P4-mediated silicification, yielding P4@Si coated HA. This suggested a superior osteoinductive potential compared to silica or P4 alone coated HA in the in vitro study. In conclusion, the co-delivery of the osteoinductive P4 peptide and silica by P4-mediated silica deposition is an efficient method for capturing and delivering its molecules and inducing synergistic osteogenesis.
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Different proteoform products of the same gene can exhibit differing associations with health and disease, and their patterns of modifications may offer more precise markers of phenotypic differences between individuals. However, currently employed protein-biomarker discovery and quantification tools, such as bottom-up proteomics and ELISAs, are mostly proteoform-unaware. Moreover, the current throughput for proteoform-level analyses by liquid chromatography mass spectrometry (LCMS) for quantitative top-down proteomics is incompatible with population-level biomarker surveys requiring robust, faster proteoform analysis. To this end, we developed immunoprecipitation coupled to SampleStream mass spectrometry (IP-SampleStream-MS) as a high-throughput, automated technique for the targeted quantification of proteoforms. We applied IP-SampleStream-MS to serum samples of 25 individuals to assess the proteoform abundances of apolipoproteins A-I (ApoA-I) and C-III (ApoC-III). The results for ApoA-I were compared to those of LCMS for these individuals, with IP-SampleStream-MS showing a >7-fold higher throughput with >50% better analytical variation. Proteoform abundances measured by IP-SampleStream-MS correlated strongly to LCMS-based values (R2 = 0.6-0.9) and produced convergent proteoform-to-phenotype associations, namely, the abundance of canonical ApoA-I was associated with lower HDL-C (R = 0.5) and glycated ApoA-I with higher fasting glucose (R = 0.6). We also observed proteoform-to-phenotype associations for ApoC-III, 22 glycoproteoforms of which were characterized in this study. The abundance of ApoC-III modified by a single N-acetyl hexosamine (HexNAc) was associated with indices of obesity, such as BMI, weight, and waist circumference (R â¼ 0.7). These data show IP-SampleStream-MS to be a robust, scalable workflow for high-throughput associations of proteoforms to phenotypes.
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Imunoprecipitação , Espectrometria de Massas , Proteômica , Adulto , Apolipoproteína A-I/análise , Apolipoproteína A-I/química , Cromatografia Líquida , Desenho de Equipamento , Feminino , Humanos , Imunoprecipitação/instrumentação , Imunoprecipitação/métodos , Masculino , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Proteínas/análise , Proteínas/química , Proteômica/instrumentação , Proteômica/métodosRESUMO
BACKGROUND: Mobile health (mHealth) tools may be useful platforms for dietary monitoring and assessment. OBJECTIVE: This study aims to evaluate the effectiveness of a mobile dietary self-monitoring app for weight loss versus a paper-based diary among adults with a BMI of 23 kg/m2 or above. METHODS: A total of 33 men and 17 women aged 18-39 years participated in a 6-week randomized controlled trial. We randomly assigned participants to one of two groups: (1) a smartphone app group (n=25) or (2) a paper-based diary group (n=25). The smartphone app group recorded foods and dietary supplements that they consumed and received immediate dietary feedback using Well-D, a dietary self-monitoring app developed by our team. The paper-based diary group was instructed to record foods or supplements that they consumed using a self-recorded diary. The primary outcomes were weight, BMI, waist circumference, body fat mass, and skeletal muscle mass. We also examined changes in nutrient intake, including energy, carbohydrate, protein, fat, dietary fiber, vitamins, and minerals, using 3-day 24-hour recalls. Differences in changes between the two groups were analyzed using independent t tests or Wilcoxon Mann-Whitney tests. All of the data were analyzed using intent-to-treat analysis. RESULTS: The mean number of days recorded was 18.5 (SD 14.1) for the app group and 15.5 (SD 10.1) for the paper-based diary group. The differences in changes in weight, BMI, and waist circumference were not significantly different between the app group and paper-based diary group (P=.33, .34, and .70, respectively). Similarly, changes in body fat mass or skeletal muscle mass did not differ between the two groups (P=.71 and .054, respectively). Although energy intake was reduced in both groups, there was no significant difference in changes in energy intake between the two groups (P=.98). CONCLUSIONS: There were no differences in changes in anthropometric measures and nutrient intake between the app group and the paper-based diary group. Both mobile dietary self-monitoring app and paper-based diary may be useful for improving anthropometric measures. TRIAL REGISTRATION: Clinical Research Information Service KCT0003170; https://cris.nih.go.kr/cris/search/search_result_st01_en.jsp?seq=11642<ype=&rtype=.
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Dieta , Aplicativos Móveis , Sobrepeso/dietoterapia , Redução de Peso , Adolescente , Adulto , Feminino , Humanos , Masculino , Smartphone , Adulto JovemRESUMO
AIM: To investigate the chemical and mechanical properties of teeth affected by a 1-bp deletion (c.2688delT) in the DSPP gene. METHODS AND MATERIALS: Maxillary first premolars were extracted from the affected individual at age 9 years due to the orthodontic reason for crowding. A sample was imbedded in epoxy resin and sectioned buccolingually, after micro-computerized tomography (µCT) images were taken. Scanning Electron Microscopy (SEM), Energy Dispersive Spectrometry (EDS) and Vickers microhardness testing were also performed. RESULTS: µCT reconstruction and analysis showed an irregularly obliterated pulp chamber and an extremely small pulpal volume in the DGI-II sample. The mineral density and microhardness scores were smaller in the dentin of the DGI-II sample compared to the wild-type. Mg content was lower in the dentin of the DGI-II sample compared to the wild-type. CONCLUSION: This study shows that dentin affected by a 1-bp deletion in DSPP has a reduced mineral density, diminished microhardness and reduced Mg content.
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Dentinogênese Imperfeita , Proteínas da Matriz Extracelular/genética , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Criança , Dentina/patologia , Dentinogênese Imperfeita/diagnóstico por imagem , Dentinogênese Imperfeita/genética , Humanos , Mutação , Linhagem , Deleção de SequênciaRESUMO
BACKGROUND: With the recent interest in personalized medicine for cancer patients and immune therapy, the field of cancer vaccines has been resurrected. Previous autologous, whole cell tumour vaccine trials have not produced convincing results due, in part to poor patient selection and inactivation methos that are harsh on the cells. These methods can alter protein structure and antigenic profiles making vaccine candidates ineffective in stimulating immune response to autochthonous tumour cells. MATERIALS AND METHODS: We investigated a novel method for inactivating tumour cells that uses UVA/UVB light and riboflavin (vitamin B2) (RF + UV). RF + UV inactivates the tumour cells' ability to replicate, yet preserves tumour cell integrity and antigenicity. RESULTS: Our results demonstrate that proteins are preserved on the surface of RF + UV-inactivated tumour cells and that they are immunogenic via induction of dendritic cell maturation, increase in IFNγ production and generation of tumour cell-specific IgG. Moreover, when formulated with an adjuvant ('Innocell vaccine') and tested in different murine tumour primary and metastatic disease models, decreased tumour growth, decreased metastatic disease and prolonged survival were observed. In addition, immune cells obtained from tumour tissue following vaccination had decreased exhausted and regulatory T cells, suggesting that activation of intra-tumoural T cells may be playing a role leading to reduced tumour growth. CONCLUSIONS: These data suggest that the RF + UV inactivation of tumour cells may provide an efficacious method for generating autologous whole tumour cell vaccines for use in cancer patients.
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Vacinas Anticâncer/imunologia , Imunoterapia/métodos , Neoplasias Experimentais/terapia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Imunogenicidade da Vacina , Camundongos , Riboflavina/toxicidade , Raios UltravioletaRESUMO
With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform. Here, we describe a new analytical platform that enables direct and high-throughput coupling to electrospray ionization mass spectrometry. The SampleStream platform is compatible with both native and denaturing electrospray, operates with a throughput of up to 15 s/sample, provides extensive concentration of dilute samples, and affords similar sensitivity to comparable liquid chromatographic methods.
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Anticorpos Monoclonais/análise , Ensaios de Triagem em Larga Escala , Imunoconjugados/análise , Ensaios de Triagem em Larga Escala/instrumentação , Software , Espectrometria de Massas por Ionização por Electrospray/instrumentaçãoRESUMO
This article describes the key features of the Well-D, a mobile dietary self-monitoring application developed to assess and track dietary intake. To test the acceptability of the app, 102 adults aged 18 years or older were asked to use Well-D for 3 days or more. After using the app, they recorded their likes/dislikes and recommendations regarding ways to improve Well-D. A mobile application for dietary assessment and monitoring may have the potential to help individuals and groups to engage in healthy behaviors.
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Ctbp2 is a uniquely targetable oncogenic transcriptional coregulator, exhibiting overexpression in most common solid tumors, and critical to the tumor-initiating cell (TIC) transcriptional program. In the "CKP" mouse pancreatic ductal adenocarcinoma (PDAC) model driven by mutant K-Ras, Ctbp2 haploinsufficiency prolonged survival, abrogated peritoneal metastasis, and caused dramatic downregulation of c-Myc, a known critical dependency for TIC activity and tumor progression in PDAC. A small-molecule inhibitor of CtBP2, 4-chloro-hydroxyimino phenylpyruvate (4-Cl-HIPP) phenocopied Ctbp2 deletion, decreasing tumor burden similarly to gemcitabine, and the combination of 4-Cl-HIPP and gemcitabine further synergistically suppressed tumor growth. Pharmacodynamic monitoring revealed that the 4-Cl-HIPP/gemcitabine combination induced robust and synergistic tumor apoptosis and marked downregulation of the TIC marker CD133 in CKP PDAC tumors. Collectively, our data demonstrate that targeting CtBP represents a fruitful avenue for development of highly active agents in PDAC that cooperate with standard therapy to limit both primary and metastatic tumor burden.
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Cocaine addiction afflicts nearly 1 million adults in the United States, and to date, there are no known treatments approved for this psychiatric condition. Women are particularly vulnerable to developing a cocaine use disorder and suffer from more serious cardiac consequences than men when using cocaine. Estrogen is one biological factor contributing to the increased risk for females to develop problematic cocaine use. Animal studies have demonstrated that estrogen (17ß-estradiol or E2) enhances the rewarding properties of cocaine. Although E2 affects the dopamine system, the molecular and cellular mechanisms of E2-enhanced cocaine reward have not been characterized. In this study, quantitative top-down proteomics was used to measure intact proteins in specific regions of the female mouse brain after mice were trained for cocaine-conditioned place preference, a behavioral test of cocaine reward. Several proteoform changes occurred in the ventral tegmental area after combined cocaine and E2 treatments, with the most numerous proteoform alterations on myelin basic protein, indicating possible changes in white matter structure. There were also changes in histone H4, protein phosphatase inhibitors, cholecystokinin, and calmodulin proteoforms. These observations provide insight into estrogen signaling in the brain and may guide new approaches to treating women with cocaine use disorder.
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Encéfalo/efeitos dos fármacos , Cocaína/farmacologia , Estradiol/farmacologia , Proteoma/metabolismo , Proteômica/métodos , Animais , Encéfalo/metabolismo , Condicionamento Clássico/efeitos dos fármacos , Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Estrogênios/farmacologia , Feminino , Camundongos Endogâmicos C57BL , Ovariectomia , Recompensa , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/metabolismoRESUMO
The hypoxia of high-altitude (HA) residence increases the risk of intrauterine growth restriction (IUGR) and preeclampsia 3-fold, augmenting perinatal morbidity and mortality and the risk for childhood and adult disease. Currently, no effective therapies exist to prevent these vascular disorders of pregnancy. The peroxisome proliferator-activated receptor γ (PPAR-γ) is an important regulator of uteroplacental vascular development and function and has been implicated in the pathogenesis of IUGR and preeclampsia. Here, we used a model of HA pregnancy in mice to determine whether hypoxia-induced fetal growth restriction reduces placental PPAR-γ protein expression and placental vascularization and, if so, to evaluate the effectiveness of the selective PPAR-γ agonist pioglitazone (PIO) for preventing hypoxia-induced IUGR. Hypoxia resulted in asymmetric IUGR, placental insufficiency, and reduced placental PPAR-γ expression; PIO prevented approximately half of the fetal growth restriction and attenuated placental insufficiency. PIO did not affect fetal growth under normoxia. Although PIO was beneficial for fetal growth, PIO treatment reduced placental vascular density of the labrynthine zone in normoxic and hypoxic (Hx) conditions, and mean vascular area was reduced in the Hx group. Our results suggest that pharmacological PPAR-γ activation is a potential strategy for preventing IUGR in pregnancies complicated by hypoxia, although further studies are needed to identify its likely metabolic or vascular mechanisms.-Lane, S. L., Dodson, R. B., Doyle, A. S., Park, H., Rathi, H., Matarrazo, C. J., Moore, L. G., Lorca, R. A., Wolfson, G. H., Julian, C. G. Pharmacological activation of peroxisome proliferator-activated receptor γ (PPAR-γ) protects against hypoxia-associated fetal growth restriction.
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Retardo do Crescimento Fetal/prevenção & controle , Hipóxia Fetal/complicações , PPAR gama/agonistas , Pioglitazona/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Doença da Altitude/complicações , Animais , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/etiologia , Retardo do Crescimento Fetal/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Placenta/irrigação sanguínea , Placenta/efeitos dos fármacos , Placenta/metabolismo , Insuficiência Placentária/etiologia , Insuficiência Placentária/metabolismo , Insuficiência Placentária/prevenção & controle , Pré-Eclâmpsia/etiologia , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/prevenção & controle , GravidezRESUMO
OBJECTIVES: To identify the molecular genetic etiology of the families with non-syndromic multiple missing permanent teeth (oligodontia). MATERIALS AND METHODS: Genomic DNA was isolated and measured, and whole-exome sequencing was performed. The obtained sequencing reads were aligned to the human reference genome and subsequently processed by a series of bioinformatics programs. Finally, short insertions/deletions and single nucleotide variations were annotated with dbSNP build 138. RESULTS: The proband of family 1 was missing 14 permanent teeth, and the mutational analysis revealed compound heterozygousWNT10A mutations (c.364A > T and c.511C > T). Two affected individuals in family 2 were missing 20 and 12 permanent teeth, respectively, and compound heterozygous WNT10A mutations (c.364A > T and c.637G > A) were also identified. CONCLUSIONS: This study reveals compound heterozygousWNT10A missense mutations in two families with non-syndromic oligodontia which will improve the understanding of odontogenesis and the pathogenesis related to WNT10A mutations.
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Anodontia , Proteínas Wnt/genética , Anodontia/genética , Análise Mutacional de DNA , Dentição Permanente , Humanos , Mutação , Linhagem , Sequenciamento do ExomaRESUMO
Serum is one of the most commonly used samples in many studies to identify protein biomarkers to diagnose cancer. Although conventional enzyme-linked immunosorbent assay (ELISA) or liquid chromatography-mass spectrometry (LC-MS)-based methods have been applied as clinical tools for diagnosing cancer, there have been troublesome problems, such as inferior multiplexing capabilities, high development costs and long turnaround times, which are inappropriate for high-throughput analytical platforms. Here, we developed a simple and robust cancer diagnostic method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based total serum protein fingerprinting. First, serum samples were simply diluted with distilled water and subsequently spotted onto a MALDI plate without prior chromatographic purification or separation. The sample preparation method was enough to collect reproducible total serum protein fingerprints and would be highly advantageous for high-throughput assay. Each of the integrated main spectrum profiles (MSPs), which are representative of liver cancer patients (n = 40) or healthy controls (n = 80), was automatically generated by the MALDI Biotyper 3 software. The reliability of the integrated MSPs was successfully evaluated in comparison with a blind test set (n = 31), which consisted of 13 liver cancer patients and 18 healthy controls. Additionally, our partial least squares discriminant analysis (PLS-DA) demonstrated a statistically significant difference in MALDI-TOF MS-based total serum protein fingerprints between liver cancer patients and healthy controls. Taken together, this work suggests that this method may be an effective high-throughput platform technology for various cancer diagnoses and disease evaluations.
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Proteínas Sanguíneas/análise , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estudos de Casos e Controles , HumanosRESUMO
Although several biomarkers can be used to distinguish cholangiocarcinoma (CCA) from healthy controls, differentiating the disease from benign biliary disease (BBD) or pancreatic cancer (PC) is a challenge. CCA biomarkers are associated with low specificity or have not been validated in relation to the biological effects of CCA. In this study, we quantitatively analyzed 15 biliary bile acids in CCA (n = 30), BBD (n = 57) and PC (n = 17) patients and discovered glycocholic acid (GCA) and taurochenodeoxycholic acid (TCDCA) as specific CCA biomarkers. Firstly, we showed that the average concentration of total biliary bile acids in CCA patients was quantitatively less than in other patient groups. In addition, the average composition ratio of primary bile acids and conjugated bile acids in CCA patients was the highest in all patient groups. The average composition ratio of GCA (35.6%) in CCA patients was significantly higher than in other patient groups. Conversely, the average composition ratio of TCDCA (13.8%) in CCA patients was significantly lower in all patient groups. To verify the biological effects of GCA and TCDCA, we analyzed the gene expression of bile acid receptors associated with the development of CCA in a CCA cell line. The gene expression of transmembrane G protein coupled receptor (TGR5) and sphingosine 1-phosphate receptor 2 (S1PR2) in CCA cells treated with GCA was 8.6-fold and 3.4-fold higher compared with control (untreated with bile acids), respectively. Gene expression of TGR5 and S1PR2 in TCDCA-treated cells was not significantly different from the control. Taken together, our study identified GCA and TCDCA as phenotype-specific biomarkers for CCA.
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Neoplasias dos Ductos Biliares/metabolismo , Biomarcadores Tumorais/metabolismo , Colangiocarcinoma/metabolismo , Ácido Glicocólico/metabolismo , Ácido Tauroquenodesoxicólico/metabolismo , Neoplasias dos Ductos Biliares/genética , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Regulação Neoplásica da Expressão Gênica , Humanos , FenótipoRESUMO
Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.
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Química Encefálica , Espectrometria de Massas/métodos , Camundongos Endogâmicos , Proteoma/análise , Proteômica/métodos , Animais , Encéfalo/metabolismo , Cromatografia Líquida/métodos , Feminino , Camundongos Endogâmicos BALB C/metabolismo , Camundongos Endogâmicos C57BL/metabolismo , Camundongos Endogâmicos DBA/metabolismo , Camundongos Endogâmicos/metabolismo , Proteoma/metabolismo , SoftwareRESUMO
Permethylation is one of the most practical modifications for mass spectrometric analysis of carbohydrates. In this study, we showed a facile platform for the on-line permethylation and analysis of acidic N-glycans using capillary liquid chromatography-electrospray ionization-tandem mass spectrometry to elucidate the N-glycan structures derived from glycoprotein samples. Two capillaries, individually packed with NaOH and C18 powder, were coupled to electrospray ion trap mass spectrometer. Prior to mass spectrometry, oligosaccharides were permethylated in NaOH-packed capillary by methyl iodide, and the permethylated glycans were consequently extracted from residual reagents, such as NaOH and methyl iodide, in C18-capillary, instead of using conventional chloroform extraction. After permethylation and purification, the permethylated glycans were released from C18 capillary by acetonitrile, and sprayed into mass spectrometer. Using this on-line strategy, a picomolar range of sialylated N-glycans from bovine fetuin were analyzed by tandem mass spectrometry.