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1.
PLoS Pathog ; 18(4): e1010167, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35482787

RESUMO

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the leading causes of food-borne illnesses worldwide. To colonize the gastrointestinal tract, S. Typhimurium produces multiple virulence factors that facilitate cellular invasion. Chitinases have been recently emerging as virulence factors for various pathogenic bacterial species, and the S. Typhimurium genome contains two annotated chitinases: STM0018 (chiA) and STM0233. However, the role of these chitinases during S. Typhimurium pathogenesis is unknown. The putative chitinase STM0233 has not been studied previously, and only limited data exists on ChiA. Chitinases typically hydrolyze chitin polymers, which are absent in vertebrates. However, chiA expression was detected in infection models and purified ChiA cleaved carbohydrate subunits present on mammalian surface glycoproteins, indicating a role during pathogenesis. Here, we demonstrate that expression of chiA and STM0233 is upregulated in the mouse gut and that both chitinases facilitate epithelial cell adhesion and invasion. S. Typhimurium lacking both chitinases showed a 70% reduction in invasion of small intestinal epithelial cells in vitro. In a gastroenteritis mouse model, chitinase-deficient S. Typhimurium strains were also significantly attenuated in the invasion of small intestinal tissue. This reduced invasion resulted in significantly delayed S. Typhimurium dissemination to the spleen and the liver, but chitinases were not required for systemic survival. The invasion defect of the chitinase-deficient strain was rescued by the presence of wild-type S. Typhimurium, suggesting that chitinases are secreted. By analyzing N-linked glycans of small intestinal cells, we identified specific N-acetylglucosamine-containing glycans as potential extracellular targets of S. Typhimurium chitinases. This analysis also revealed a differential abundance of Lewis X/A-containing glycans that is likely a result of host cell modulation due to the detection of S. Typhimurium chitinases. Similar glycomic changes elicited by chitinase deficient strains indicate functional redundancy of the chitinases. Overall, our results demonstrate that S. Typhimurium chitinases contribute to intestinal adhesion and invasion through modulation of the host glycome.


Assuntos
Quitinases , Salmonella enterica , Animais , Quitina , Quitinases/genética , Quitinases/metabolismo , Mamíferos , Camundongos , Salmonella enterica/metabolismo , Salmonella typhimurium , Sorogrupo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
2.
Anal Biochem ; 694: 115621, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39019205

RESUMO

Mutational study is a cornerstone methodology in biochemistry and genetics, and many mutagenesis strategies have been invented to promote the efficiency of gene engineering. In this study, we developed a simple and timesaving approach to integrate simultaneous mutagenesis at discrete sites. By using plasmid as a template and compatible oligonucleotide primers per the QuikChange strategy, our method was able to introduce multiple nucleotide insertions, deletions and replacements in one round of polymerase chain reaction. The longest insertion and deletion were achieved with 28 bp and 16 bp mismatch respectively. For minor nucleotide replacements (mismatch no more than 4 bp), mutations were achieved at up to 4 discrete locations. Usually, a successful clone with all desired mutations was found by screening 5 colonies. Clones with a subset of mutations may be stocked into the library of mutants or used as templates in the next rounds of mutagenic PCR to accomplish the entire construction project. This method can be applied to build up a combinatory library of mutants through saturation mutagenesis at multiple sites. It is promising to facilitate the research of protein biochemistry, forward genetics and synthetic biology.


Assuntos
Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da Polimerase , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Mutagênese Sítio-Dirigida/métodos , DNA/genética
3.
Anal Bioanal Chem ; 416(18): 4071-4082, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38958703

RESUMO

The study of glycoproteomics presents a set of unique challenges, primarily due to the low abundance of glycopeptides and their intricate heterogeneity, which is specific to each site. Glycoproteins play a crucial role in numerous biological functions, including cell signaling, adhesion, and intercellular communication, and are increasingly recognized as vital markers in the diagnosis and study of various diseases. Consequently, a quantitative approach to glycopeptide research is essential. One effective strategy to address this need is the use of multiplex glycopeptide labeling. By harnessing the synergies of 15N metabolic labeling via the isotopic detection of amino sugars with glutamine (IDAWG) technique for glycan parts and tandem mass tag (TMT)pro labeling for peptide backbones, we have developed a method that allows for the accurate quantification and comparison of multiple samples simultaneously. The adoption of the liquid chromatography-synchronous precursor selection (LC-SPS-MS3) technique minimizes fragmentation interference, enhancing data reliability, as shown by a 97% TMT labeling efficiency. This method allows for detailed, high-throughput analysis of 32 diverse samples from 231BR cell lines, using both 14N and 15N glycopeptides at a 1:1 ratio. A key component of our methodology was the precise correction for isotope and TMTpro distortions, significantly improving quantification accuracy to less than 5% distortion. This breakthrough enhances the efficiency and accuracy of glycoproteomic studies, increasing our understanding of glycoproteins in health and disease. Its applicability to various cancer cell types sets a new standard in quantitative glycoproteomics, enabling deeper investigation into glycopeptide profiles.


Assuntos
Glicopeptídeos , Marcação por Isótopo , Isótopos de Nitrogênio , Espectrometria de Massas em Tandem , Glicopeptídeos/análise , Glicopeptídeos/metabolismo , Humanos , Isótopos de Nitrogênio/análise , Espectrometria de Massas em Tandem/métodos , Marcação por Isótopo/métodos , Proteômica/métodos , Linhagem Celular Tumoral , Cromatografia Líquida/métodos
4.
Anal Chem ; 95(50): 18388-18397, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38069741

RESUMO

Determination of the relative expression levels of the α2,3/α2,6-sialic acid linkage isomers on glycoproteins is critical to the analysis of various human diseases such as cancer, inflammation, and viral infection. However, it remains a challenge to separate and differentiate site-specific linkage isomers at the glycopeptide level. Some derivatization methods on the carboxyl group of sialic acid have been developed to generate mass differences between linkage isomers. In this study, we utilized chemical derivatization that occurred on the vicinal diol of sialic acid to separate linkage isomers on a reverse-phase column using a relatively short time. 2-Aminobenzamide (2AB) labeling derivatization, including periodate oxidation and reductive amination, took only ∼3 h and achieved high labeling efficiency (>90%). Within a 66 min gradient, the sialic acid linkage isomers of 2AB-labeled glycopeptides from model glycoproteins can be efficiently resolved compared to native glycopeptides. Two different methods, neuraminidase digestion and higher-energy collision dissociation tandem mass spectrometry (HCD-MS2) fragmentation, were utilized to differentiate those isomeric peaks. By calculating the diagnostic oxonium ion ratio of Gal2ABNeuAc and 2ABNeuAc fragments, significant differences in chromatographic retention times and in mass spectral peak abundances were observed between linkage isomers. Their corresponding MS2 PCA plots also helped to elucidate the linkage information. This method was successfully applied to human blood serum. A total of 514 2AB-labeled glycopeptide structures, including 152 sets of isomers, were identified, proving the applicability of this method in linkage-specific structural characterization and relative quantification of sialic acid isomers.


Assuntos
Ácido N-Acetilneuramínico , Espectrometria de Massas em Tandem , Humanos , Ácido N-Acetilneuramínico/química , Espectrometria de Massas em Tandem/métodos , Sialoglicoproteínas , Espectrometria de Massa com Cromatografia Líquida , Cromatografia Líquida , Glicoproteínas , Glicopeptídeos/análise , Polissacarídeos/química
5.
Anal Chem ; 95(44): 16059-16069, 2023 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-37843510

RESUMO

The complexity and heterogeneity of protein glycosylation present an analytical challenge to the studies of characterization and quantitation. Various LC-MS-based quantitation strategies have emerged in recent decades. Metabolic stable isotope labeling has been developed to enhance the accurate LC/MS-based quantitation between different cell lines. Stable isotope labeling by amino acids in a cell culture (SILAC) is the most widely used metabolic labeling method in proteomic analysis. However, it can only label the peptide backbone and is thus limited in glycomic studies. Here, we present a metabolic isotope labeling strategy, named GlyProSILC (Glycan Protein Stable Isotope Labeling in Cell Culture), that can label both the glycan motif and peptide backbone from the same batch of cells. It was performed by feeding cells with a heavy medium containing amide-15N-glutamine, 13C6-arginine (Arg6), and 13C6-15N2-lysine (Lys8). No significant change of cell line metabolism after GlyProSILC labeling was observed based on transcriptomic, glycomic, and proteomic data. The labeling conditions, labeling efficiency, and quantitation accuracy were investigated. After quantitation correction, we simultaneously quantified 62 N-glycans, 574 proteins, and 344 glycopeptides using the same batch of mixed 231BR/231 cell lines. So far, GlyProSILC provides an accurate and effective quantitation approach for glycomics, proteomics, and glycoproteomics in a cell culture system.


Assuntos
Glicômica , Proteômica , Marcação por Isótopo/métodos , Glicômica/métodos , Proteômica/métodos , Proteínas , Técnicas de Cultura de Células , Glicopeptídeos/metabolismo , Polissacarídeos/química
6.
Electrophoresis ; 43(1-2): 370-387, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34614238

RESUMO

Protein glycosylation is one of the most common posttranslational modifications, and plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, host-pathogen interaction, and protein stability. Glycoproteomics is a proteomics subfield dedicated to identifying and characterizing the glycans and glycoproteins in a given cell or tissue. Aberrant glycosylation has been associated with various diseases such as Alzheimer's disease, viral infections, inflammation, immune deficiencies, congenital disorders, and cancers. However, glycoproteomic analysis remains challenging because of the low abundance, site-specific heterogeneity, and poor ionization efficiency of glycopeptides during LC-MS analyses. Therefore, the development of sensitive and accurate approaches to efficiently characterize protein glycosylation is crucial. Methods such as metabolic labeling, enrichment, and derivatization of glycopeptides, coupled with different mass spectrometry techniques and bioinformatics tools, have been developed to achieve sophisticated levels of quantitative and qualitative analyses of glycoproteins. This review attempts to update the recent developments in the field of glycoproteomics reported between 2017 and 2021.


Assuntos
Glicopeptídeos , Proteômica , Cromatografia Líquida , Glicopeptídeos/química , Glicosilação , Espectrometria de Massas/métodos , Proteômica/métodos
7.
Analyst ; 146(13): 4374-4383, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34132263

RESUMO

Glycosylation is a complex and common post-translational modification of proteins. To study glycosylation, liquid chromatography-mass spectrometry (LC-MS) is often used to profile and structurally characterize the glycans in biological systems. While bed packed reverse phase columns are frequently utilized for the separation of permethylated glycans, the use of newly commercialized micro array pillar nanoLC columns (µPAC) have not been demonstrated previously. Owing to its advantages such as low back pressure, reproducibility, and durability, we have investigated the viability of the µPAC for the analysis of permethylated glycans. In this work, we demonstrate the online purification ability of µPAC trapping column compared against PepMap trapping column. We also found that the 50 cm µPAC can be used for the analysis of both permethylated N- and O-glycans. The use of 50 cm µPAC was compared against the previous method. The use of 200 cm µPAC was also investigated for the permethylated glycan analysis. 200 cm µPAC demonstrated efficient separation of oligomannose glycan isomers as well as other complex glycans.


Assuntos
Polissacarídeos , Espectrometria de Massas em Tandem , Cromatografia Líquida , Glicosilação , Reprodutibilidade dos Testes
8.
J Sep Sci ; 44(1): 403-425, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33090644

RESUMO

Changes in the glycome of human proteins and cells are associated with the progression of multiple diseases such as Alzheimer's, diabetes mellitus, many types of cancer, and those caused by viruses. Consequently, several studies have shown essential modifications to the isomeric glycan moieties for diseases in different stages. However, the elucidation of extensive isomeric glycan profiles remains challenging because of the lack of analytical techniques with sufficient resolution power to separate all glycan and glycopeptide iso-forms. Therefore, the development of sensitive and accurate approaches for the characterization of all the isomeric forms of glycans and glycopeptides is essential to tracking the progression of pathology in glycoprotein-related diseases. This review describes the isomeric separation achievements reported in glycomics and glycoproteomics in the last decade. It focuses on the mass spectrometry-based analytical strategies, stationary phases, and derivatization techniques that have been developed to enhance the separation mechanisms in liquid chromatography systems and the detection capabilities of mass spectrometry systems.


Assuntos
Glicômica , Glicopeptídeos/isolamento & purificação , Polissacarídeos/isolamento & purificação , Proteômica , Cromatografia Líquida , Glicopeptídeos/química , Humanos , Espectrometria de Massas , Polissacarídeos/química
9.
Sensors (Basel) ; 20(18)2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32906657

RESUMO

Pose estimation is a typical problem in the field of image processing, the purpose of which is to compare or fuse images acquired under different conditions. In recent years, many studies have focused on pose estimation algorithms, but so far there are still many challenges, such as efficiency, complexity and accuracy for various targets and conditions, in the field of algorithm research and practical applications. In this paper, a multi-view-based pose estimation method is proposed. This method can solve the pose estimation problem effectively for large-scale targets and achieve good performance accuracy and stability. Compared with existing methods, this method uses different views (positions and angles), each of which only observes some features of large-size parts, to estimate the six-degree-of-freedom pose of the entire large-size parts. Experimental results demonstrate that the accurate six-degree-of-freedom pose for different targets can be obtained by the proposed method which plays an important role in many actual production lines. What is more, a new visual guidance system, applied into intelligent manufacturing, is presented based on this method. The new visual guidance system has been widely used in automobile manufacturing with high accuracy and efficiency but low cost.

10.
Methods Mol Biol ; 2762: 267-280, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38315371

RESUMO

Glycosylation of proteins is an important post-translational modification that plays a role in a wide range of biological processes, including immune response, intercellular signaling, inflammation, and host-pathogen interaction. Abnormal protein glycosylation has been correlated with various diseases. However, the study of protein glycosylation remains challenging due to its low abundance, microheterogeneity of glycosylation sites, and low ionization efficiency. During the past decade, several methods for enrichment and for isolation of glycopeptides from biological samples have been developed and successfully employed in glycoproteomics research. In this chapter, we discuss the sample preparation protocol and the strategies for effectively isolating and enriching glycopeptides from biological samples, using PolyHYDROXYETHYL A as a hydrophilic interaction liquid chromatography (HILIC) enrichment technique.


Assuntos
Glicopeptídeos , Processamento de Proteína Pós-Traducional , Glicopeptídeos/análise , Cromatografia Líquida/métodos , Glicosilação , Interações Hidrofóbicas e Hidrofílicas
11.
J Proteomics ; : 105333, 2024 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-39426592

RESUMO

Alterations in glycoprotein abundance and glycan structures are closely linked to numerous diseases. The quantitative exploration of glycoproteomics is pivotal for biomarker discovery, but comprehensive analysis within biological samples remains challenging due to low abundance, complexity, and lack of universal technology. We developed a multiplex glycoproteomic approach using an LC-ESI-MS platform for direct comparison of glycoproteomic quantitation. Glycopeptides were isotopically labeled during cell culture, achieving high labeling efficiency (≥ 95 %) for both glycans and peptides. Quantitation was validated by mixing the same cell line in a 1:1:1:1 ratio, with mathematical correction applied to deconvolute the ratios. This method proved reliable and was applied to a comparative glycoproteomic study of three breast cancer cell lines (HTB22, MDA-MB-231, MDA-MB-231BR) and one brain cancer cell line (CRL-1620), quantifying glycopeptides from three replicates. The expression of glycopeptides was relatively quantified, and up/down-regulation between cell lines was investigated. This approach provided insights into glycosylation microheterogeneity, crucial for breast cancer brain metastasis research. Benefits include eliminating fluctuations from nano electrospray ionization and reducing analysis time, enabling up to 4-plex profiling in a single injection. Metabolic labeling introduced mass differences at the MS1 level, ensuring increased sensitivity and higher resolution for accurate quantitation. SIGNIFICANCE: Alternations in glycoprotein abundance, changes in glycosylation levels, and variations in glycan structures are closely linked to numerous diseases. The quantitative exploration of glycoproteomics has emerged as a popular area of research for biomarker discovery. However, conducting a comprehensive quantitative analysis of the glycoproteome within biological samples remains challenging due to low abundance, inherent complexities, and the absence of universal quantitative technology. Here, we developed a multiplex glycoproteomic approach using an LC-ESI-MS platform to facilitate direct comparison of glycoproteomic quantitation and enhance throughput. This approach offers benefits such as eliminating quantitative fluctuations arising from nano electrospray ionization (ESI) and reducing analysis time, enabling up to 4-plex glycoproteomic profiling in a single injection. Glycopeptides were stable isotopic labeled during cell culture procedure, attaching to monosaccharides, amino acids, or both. We achieved a high labeling efficiency (≥ 95 %) for both glycans and peptides. Quantitation validation was tested on glycopeptides by mixing the same cell line with 1:1:1:1 ratio. Due to the overlapped isotopes, a mathematical correction was applied to deconvolute the ratio of 4-plex glycopeptides. This method demonstrated quantitative reliability and was successfully applied to a comparative glycoproteomic study of three breast cancer cells (HTB22, MDA-MB-231, and MDA-MB-231BR) and one brain cancer cell (CRL-1620), identifying a total of 264 glycopeptides from three replicates. The expression of glycopeptides among these four cells was relatively quantified and up/down-regulation between two cell lines was investigated. The exploration of glycosylation microheterogeneity through glycopeptide quantification may offer valuable insights for further investigation into breast cancer brain metastasis. Conclusion: The primary advantage of our presented work lies in the multiplexing offered by combining two established labeling techniques, SILAC and IDAWG, both of which have been effectively used and widely cited in the scientific community. This combination enhances the applicability and accuracy of our method, as demonstrated by the extensive citations and successful use of these techniques independently. We believe that this multiplexing approach significantly advances the field, despite the method's current limitation to cell systems.

12.
Technol Health Care ; 31(S1): 347-355, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37066935

RESUMO

BACKGROUND: Coronary artery disease (CAD) manifests with a blockage the coronary arteries, usually due to plaque buildup, and has a serious impact on the human life. Atherosclerotic plaques, including fibrous plaques, lipid plaques, and calcified plaques can lead to occurrence of CAD. Optical coherence tomography (OCT) is employed in the clinical practice as it clearly provides a detailed display of the lesion plaques, thereby assessing the patient's condition. Analyzing the OCT images manually is a very tedious and time-consuming task for the clinicians. Therefore, automatic segmentation of the coronary OCT images is necessary. OBJECTIVE: In view of the good utility of Unet network in the segmentation of medical images, the present study proposed the development of a Unet network based on Sk-Conv and spatial pyramid pooling modules to segment the coronary OCT images. METHODS: In order to extract multi-scale features, these two modules were added at the bottom of UNet. Meanwhile, ablation experiments are designed to verify each module is effective. RESULTS: After testing, our model achieves 0.8935 on f1 score and 0.7497 on mIOU. Compared to the current advanced models, our model performs better. CONCLUSION: Our model achieves good results on OCT sequences.


Assuntos
Doença da Artéria Coronariana , Placa Aterosclerótica , Humanos , Tomografia de Coerência Óptica/métodos , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/patologia , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/patologia , Coração , Vasos Coronários/diagnóstico por imagem , Vasos Coronários/patologia
13.
Mar Environ Res ; 188: 106005, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37156673

RESUMO

Riverine microplastic (MP) discharge into the ocean contributes greatly to global MP contamination, yet our understanding of this process remains primitive. To deepen our interpretation of the dynamic MP variation throughout the estuarine water columns, we sampled at Xuliujing, the saltwater intrusion node of the Yangtze River Estuary, over the course of ebb and flood tides in four seasons (July and October 2017, January and May 2018 respectively). We observed that the collision of downstream and upstream currents contributed to the high MP concentration and that the mean MP abundance fluctuated with the tide. A model of microplastics residual net flux (MPRF-MODEL), taking the seasonal abundance and vertical distribution of MP along with current velocity into consideration, was developed to predict the net flux of MP throughout the full water columns. 2154 ± 359.7 t/year of MP was estimated to flow into the East China Sea via the River in 2017-2018. Our study suggests that riverine MP flux can be overestimated due to reciprocating current carried MP from the estuary. Using the tidal and seasonal variation in MP distribution, we calculated the tide impact factor index (TIFI) for the Yangtze River Estuary to be between 38.11% and 58.05%. In summary, this study provides a baseline of MP flux research in the Yangtze River for similar tidal-controlled rivers and a contextual understanding of how to appropriately sample and accurately estimate in a dynamic estuary system. The redistribution of microplastics may be impacted by complex tide processes. Although not observed in this study, it may merit investigation.


Assuntos
Estuários , Poluentes Químicos da Água , Água , Microplásticos , Plásticos , Monitoramento Ambiental , Poluentes Químicos da Água/análise , China
14.
Biomolecules ; 13(9)2023 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-37759821

RESUMO

The SARS-CoV-2 virus rapidly spread worldwide, threatening public health. Since it emerged, the scientific community has been engaged in the development of effective therapeutics and vaccines. The subunit S1 in the spike protein of SARS-CoV-2 mediates the viral entry into the host and is therefore one of the major research targets. The S1 protein is extensively glycosylated, and there is compelling evidence that glycans protect the virus' active site from the human defense system. Therefore, investigation of the S1 protein glycome alterations in the different virus variants will provide a view of the glycan evolution and its relationship with the virus pathogenesis. In this study, we explored the N-glycosylation expression of the S1 protein for eleven SARS-CoV-2 variants: five variants of concern (VOC), including alpha, beta, gamma, delta, and omicron, and six variants of interest (VOI), including epsilon, eta, iota, lambda, kappa, and mu. The results showed significant differences in the N-glycome abundance of all variants. The N-glycome of the VOC showed a large increase in the abundance of sialofucosylated glycans, with the greatest abundance in the omicron variant. In contrast, the results showed a large abundance of fucosylated glycans for most of the VOI. Two glycan compositions, GlcNAc4,Hex5,Fuc,NeuAc (4-5-1-1) and GlcNAc6,Hex8,Fuc,NeuAc (6-8-1-1), were the most abundant structures across all variants. We believe that our data will contribute to understanding the S1 protein's structural differences between SARS-CoV-2 mutations.


Assuntos
COVID-19 , Humanos , COVID-19/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
15.
Cell Rep ; 42(4): 112294, 2023 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-36947544

RESUMO

Stroke is a leading cause of adult disability worldwide, and better drugs are needed to promote functional recovery after stroke. Growing evidence suggests the critical role of network excitability during the repair phase for stroke recovery. Here, we show that ß-hydroxybutyrate (ß-HB), an essential ketone body (KB) component, is positively correlated with improved outcomes in patients with stroke and promotes functional recovery in rodents with stroke during the repair phase. These beneficial effects of ß-HB depend on HDAC2/HDAC3-GABA transporter 1 (GAT-1) signaling-mediated enhancement of excitability and phasic GABA inhibition in the peri-infarct cortex and structural and functional plasticity in the ipsilateral cortex, the contralateral cortex, and the corticospinal tract. Together with available clinical approaches to elevate KB levels, our results offer a clinically translatable means to promote stroke recovery. Furthermore, GAT-1 can serve as a pharmacological target for developing drugs to promote functional recovery after stroke.


Assuntos
Corpos Cetônicos , Acidente Vascular Cerebral , Humanos , Proteínas da Membrana Plasmática de Transporte de GABA
16.
Anal Chim Acta ; 1233: 340492, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36283781

RESUMO

Glycosylation is one of the most important post-translational modifications. However, the characterizations of glycopeptides, especially the negatively charged sialoglycopeptides that are associated with various diseases, remain challenging, due to the co-existence with high abundant peptides and the low ionization efficiency of sialoglycopeptides resulting from the carboxyl groups. Therefore, it is essential to develop an efficient enrichment method for sialoglycopeptides. Here, we present a novel derivatization-based enrichment method that can (i) identify linkage isomers of sialic acids by generating mass difference, (ii) unify the net charge of peptides into zero, and (iii) introduce positive charges to sialoglycopeptides by conjugating quaternary ammonium with sialic acid. The derivatization, termed derivatization of sialylated glycopeptides plus (DOSG+), enables efficient enrichment through electrostatic interaction using weak cation exchange (WCX) media. DOSG+ -based WCX enrichment was validated and optimized with samples derived from bovine fetuin. Peptides were removed efficiently (recovery rate <1%). The signal intensity of a selected model sialoglycopeptide was increased by ∼30% (suggesting recovery rate >100%). The method was employed on human alpha-1 acid glycoprotein (AGP), and recombinant human erythropoietin (EPO), demonstrating the application of DOSG+ -based WCX enrichment on complexed N-linked and O-linked sialoglycopeptides. The method is simple, efficient, and targets small-scale sialoglycopeptide enrichment.


Assuntos
Compostos de Amônio , Eritropoetina , Bovinos , Animais , Humanos , Glicopeptídeos/química , Sialoglicoproteínas/química , Ácido N-Acetilneuramínico , Ácidos Siálicos , Peptídeos , Cátions , Fetuínas
17.
Mar Pollut Bull ; 171: 112914, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34488149

RESUMO

The environmental pollution caused by microplastics has received increasing attention recently. In this paper, we present the results of research into the bacterium attached to microplastics in four coastal mariculture zones in southeast China during winter and summer. Polyethene and polypropylene are the main microplastics in the surface water of mariculture area. The differences between the bacteria species composition found on the surface of microplastics in winter and summer were less than that found in the planktonic bacteria, indicating that biofilms protect the bacterium that live inside. Potentially pathogenic Vibrio and Pseudomonas spp. were more abundant in samples from ShanTou and QuanZhou during the summer. Bacteria related to the degradation of microplastics were found extensively on the surface of microplastics at all of the sampling sites. More attention should be paid to the risks resulting from the accumulation of harmful bacteria on microplastic surfaces during the summer.


Assuntos
Microplásticos , Poluentes Químicos da Água , Biofilmes , China , Monitoramento Ambiental , Plásticos , Estações do Ano , Poluentes Químicos da Água/análise
18.
Int J Nanomedicine ; 14: 963-976, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30787610

RESUMO

INTRODUCTION: Combined therapies utilizing inhibitors to remove pathogens are needed to suppress lipopolysaccharide (LPS)-induced periodontal disease. We prepared a novel, multi-agent delivery scaffold for periodontal treatment. METHODS: In this study, we synthesized SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) drug-loaded poly(ethylene glycol)-block-caprolactone copolymer via dialysis method. The physical property of micelles was characterized through dynamic light scattering and transmission electron microscopy. The cell growth and LPS-induced MMP-2 and MMP-13 expression were evaluated through CCK-8, real-time PCR and Western blot assay. The release of SP600125 and SB203580 from different scaffolds was estimated. Microcomputed tomography and histology were used for evaluating the effect of the micelles-loaded nanofibers on the treatment of class II furcation defects in dogs. RESULTS: The drug was then successfully incorporated into gelatin fibers during electrospinning process. We confirmed that the micelles had spherical structure and an average particle size of 160 nm for SP600125-micelles (SP-Ms) and 150 nm for SB203580-micelles (SB-Ms). The nanofiber scaffold showed excellent encapsulation capability, in vitro drug-release behavior, and cell compatibility. Real-time PCR and Western blot assay further indicated that LPS-induced MMP-2 and MMP-13 expression was significantly inhibited by the scaffold. CONCLUSION: The results suggested that the dual drug-loaded system developed in this study might become a highly effective therapy for periodontal disease.


Assuntos
Gelatina/química , Micelas , Nanofibras/química , Doenças Periodontais/terapia , Adulto , Animais , Cães , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Lipopolissacarídeos , Masculino , Metaloproteinases da Matriz/metabolismo , Nanofibras/ultraestrutura , Tamanho da Partícula , Doenças Periodontais/diagnóstico por imagem , Ligamento Periodontal/patologia , Poliésteres/química , Polietilenoglicóis/química , Testes de Toxicidade , Microtomografia por Raio-X
19.
Sci Total Environ ; 624: 48-54, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29247904

RESUMO

Plastic trash is common in oceans. Terrestrial and marine ecosystem interactions occur in the intertidal zone where accumulation of plastic frequently occurs. However, knowledge of the plastic-associated microbial community (the plastisphere) in the intertidal zone is scanty. We used high-throughput sequencing to profile the bacterial communities attached to microplastic samples from intertidal locations around the Yangtze estuary in China. The structure and composition of plastisphere communities varied significantly among the locations. We found the taxonomic composition on microplastic samples was related to their sedimentary and aquatic origins. Correlation network analysis was used to identify keystone bacterial genera (e.g. Rhodobacterales, Sphingomonadales and Rhizobiales), which represented important microbial associations within the plastisphere community. Other species (i.e. potential pathogens) were considered as hitchhikers in the plastic attached microbial communities. Metabolic pathway analysis suggested adaptations of these bacterial assemblages to the plastic surface-colonization lifestyle. These adaptations included reduced "cell motility" and greater "xenobiotics biodegradation and metabolism." The findings illustrate the diverse microbial assemblages that occur on microplastic and increase our understanding of plastisphere ecology.


Assuntos
Bactérias/classificação , Estuários , Plásticos , Microbiologia da Água , Poluentes Químicos da Água , Bactérias/isolamento & purificação , China , Oceanos e Mares
20.
ACS Macro Lett ; 7(10): 1192-1197, 2018 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-35651271

RESUMO

This study reports the scalable fabrication of a poly(butylene terephthalate) fiber assembly featured with controllable pore size and gradient wettability. Pore size is controlled via adjusting the throughput of melt blown process, while gradient wettability is achieved through single-sided plasma exposure and subsequent chitosan coating. When used in cell culture, the fiber assembly takes much less time in reaching a high cell collecting/releasing rate up to ≥99.5%, which is similar to that of the conventional centrifugal method. Other advantages of the fiber assembly, such as improved cell viability, reduced risk of contamination, and excellent reusability are also proved, leading us to believe its great potential in making the current cell culture procedure simpler and faster.

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