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1.
J Sep Sci ; 45(16): 3150-3168, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35770343

RESUMO

The standard bottom-up proteomic workflow is comprised of sample preparation, data acquisition, and data analysis. While the latter two parts have made considerable advances in the last decade, sample preparation has remained an important challenge within the workflow due to the multi-step nature of complex biological samples, and still requires much development. Several sample preparation methods have been developed and used in the last two decades, including in-gel, in-solution, on-bead, filter-aided sample preparation, and suspension trapping, to improve reproducibility, efficiency, scalability, and reduce the handling time of this process. One of the most recent methods developed and applied in proteomics studies in recent years is suspension trapping, which combines rapid detergent removal, reactor-type protein digestion, and peptide clean-up in a tip or spin column. Suspension trapping is a simple, rapid, and reproducible digestion method that can effectively handle proteins in low microgram or sub-microgram amounts. This review discusses the benefits of the suspension trapping digestion method in relation to its development and application in bottom-up proteomics studies. We also discuss recent applications of suspension trapping digestion to different sample types and the features of the suspension trapping digestion method compared with other sample preparation methods.


Assuntos
Proteínas , Proteômica , Digestão , Peptídeos , Reprodutibilidade dos Testes
2.
Molecules ; 26(7)2021 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-33918234

RESUMO

Emiliania huxleyi is a cosmopolitan coccolithophore that plays an essential role in global carbon and sulfur cycling, and contributes to marine cloud formation and climate regulation. Previously, the proteomic profile of Emiliania huxleyi was investigated using a three-dimensional separation strategy combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current study reuses the MS/MS spectra obtained, for the global discovery of post-translational modifications (PTMs) in this species without specific enrichment methods. Twenty-five different PTM types were examined using Trans-Proteomic Pipeline (Comet and PeptideProphet). Overall, 13,483 PTMs were identified in 7421 proteins. Methylation was the most frequent PTM with more than 2800 modified sites, and lysine was the most frequently modified amino acid with more than 4000 PTMs. The number of proteins identified increased by 22.5% to 18,780 after performing the PTM search. Compared to intact peptides, the intensities of some modified peptides were superior or equivalent. The intensities of some proteins increased dramatically after the PTM search. Gene ontology analysis revealed that protein persulfidation was related to photosynthesis in Emiliania huxleyi. Additionally, various membrane proteins were found to be phosphorylated. Thus, our global PTM discovery platform provides an overview of PTMs in the species and prompts further studies to uncover their biological functions. The combination of a three-dimensional separation method with global PTM search is a promising approach for the identification and discovery of PTMs in other species.


Assuntos
Haptófitas/química , Processamento de Proteína Pós-Traducional , Ontologia Genética , Metilação , Peptídeos/química , Fosforilação , Proteínas/química , Espectrometria de Massas em Tandem
3.
Int J Mol Sci ; 21(4)2020 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-32102244

RESUMO

Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are typically highly complex and large in abundance range. To fulfill high accuracy and sensitivity of proteome analysis, the hybrid platforms of multidimensional (MD) separations and mass spectrometry have provided the most powerful solution. Multidimensional separations provide enhanced peak capacity and reduce sample complexity, which enables mass spectrometry to analyze more proteins with high sensitivity. Although two-dimensional (2D) separations have been widely used since the early period of proteomics, three-dimensional (3D) separation was barely used by low reproducibility of separation, increased analysis time in mass spectrometry. With developments of novel microscale techniques such as nano-UPLC and improvements of mass spectrometry, the 3D separation becomes a reliable and practical selection. This review summarizes existing offline and online 3D-LC platforms developed for proteomics and their applications. In detail, setups and implementation of those systems as well as their advances are outlined. The performance of those platforms is also discussed and compared with the state-of-the-art 2D-LC. In addition, we provide some perspectives on the future developments and applications of 3D-LC in proteomics.


Assuntos
Cromatografia Líquida/métodos , Proteoma/análise , Proteômica/métodos , Animais , Cromatografia Líquida/instrumentação , Humanos , Fígado/metabolismo , Espectrometria de Massas/métodos , Sistemas On-Line , Proteômica/instrumentação , Reprodutibilidade dos Testes
4.
Molecules ; 25(13)2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32630776

RESUMO

Emiliania huxleyi is one of the most abundant marine planktons, and it has a crucial feature in the carbon cycle. However, proteomic analyses of Emiliania huxleyi have not been done extensively. In this study, a three-dimensional liquid chromatography (3D-LC) system consisting of strong cation exchange, high- and low-pH reversed-phase liquid chromatography was established for in-depth proteomic profiling of Emiliania huxleyi. From tryptic proteome digest, 70 fractions were generated and analyzed using liquid chromatography-tandem mass spectrometry. In total, more than 84,000 unique peptides and 10,000 proteins groups were identified with a false discovery rate of ≤0.01. The physicochemical properties of the identified peptides were evaluated. Using ClueGO, approximately 700 gene ontology terms and 15 pathways were defined from the identified protein groups with p-value ≤0.05, covering a wide range of biological processes, cellular components, and molecular functions. Many biological processes associated with CO2 fixation, photosynthesis, biosynthesis, and metabolic process were identified. Various molecular functions relating to protein binding and enzyme activities were also found. The 3D-LC strategy is a powerful approach for comparative proteomic studies on Emiliania huxleyi to reveal changes in its protein level and related mechanism.


Assuntos
Haptófitas/química , Proteínas/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia de Fase Reversa/métodos , Ontologia Genética , Peptídeos/análise , Peptídeos/isolamento & purificação , Proteínas/química , Proteínas/isolamento & purificação , Proteoma/análise , Proteoma/genética , Proteoma/isolamento & purificação , Fluxo de Trabalho
5.
Int Ophthalmol ; 40(2): 447-457, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31720953

RESUMO

PURPOSE: To investigate the changes in corneal topography including parameters such as corneal curvature and corneal aberrations, along with anterior chamber angle (ACA) after laser iridotomy (LI) combined with peripheral iridoplasty (PI) using dual Scheimpflug analyzer. METHODS: In this prospective observational study, dual Scheimpflug analyzer images were acquired before and 1 week after LI plus PI. Corneal curvature of both axial and instantaneous maps from anterior and posterior surface, respectively, and total corneal power (TCP) were acquired. These corneal parameters from three zones (central, middle, and peripheral) and total corneal wavefront aberration, trefoil, and coma were obtained. The ACA from four quadrants, anterior chamber depth (ACD), anterior chamber volume (ACV), and intraocular pressure (IOP) were also inspected. RESULTS: ACD increased significantly from 2.15 ± 0.25 to 2.18 ± 0.24 mm (P = 0.002). ACV and ACA from all four quadrants increased significantly after the laser treatment (all P < 0.05). IOP decreased significantly from 16.9 ± 3.1 to 14.7 ± 2.9 mmHg following LI plus PI (P = 0.000). No significant changes were detected in corneal axial and instantaneous curvature from three zones on the anterior and posterior corneal surface after LI plus PI (all P > 0.05). The TCP, total corneal wavefront aberration, trefoil, and coma also revealed no significant changes after the laser procedure (all P > 0.05). CONCLUSIONS: Treatment with LI combined with PI did not affect the corneal topographic parameters from both anterior and posterior surfaces. However, LI plus PI improved ACA parameters significantly and effectively.


Assuntos
Córnea/patologia , Topografia da Córnea/métodos , Glaucoma de Ângulo Fechado/cirurgia , Pressão Intraocular/fisiologia , Iridectomia/métodos , Terapia a Laser/métodos , Lasers de Estado Sólido/uso terapêutico , Córnea/cirurgia , Feminino , Glaucoma de Ângulo Fechado/diagnóstico , Glaucoma de Ângulo Fechado/fisiopatologia , Gonioscopia , Humanos , Iris/cirurgia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Tonometria Ocular
6.
J Periodontal Res ; 54(1): 53-62, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30298515

RESUMO

BACKGROUND AND OBJECTIVES: Proteome analysis of periodontal ligament stem cells (PDLSCs) could be used to study the function of PDL tissue. We used a label-free quantitative proteomic technique to investigate differentially expressed proteins (DEPs) in human PDLSCs (hPDLSCs) compared to human bone marrow mesenchymal stem cells (hBMSCs) and identify proteins specific to hPDLSCs. MATERIAL AND METHODS: hPDLSCs (n = 3) and hBMSCs (n = 3) were cultured and harvested for protein extraction and trypsin digestion. The proteomes of both cell types were analyzed by nano-liquid chromatography/tandem mass spectrometry. DEPs in hPDLSCs compared to hBMSCs were detected by label-free quantification and evaluated through signal transduction pathway and gene ontology (GO) analysis. RESULTS: In total, 690 and 771 proteins were identified from hPDLSCs and hBMSCs, of which 561 proteins were in common and 124 DEPs were found between hPDLSCs and hBMSCs. Fifty-eight proteins were expressed at significantly higher levels in hPDLSCs, whereas 66 proteins were expressed at lower levels compared to hBMSCs. The more highly expressed proteins were associated with translation and initiating protein synthesis, and lower expressed proteins were related to cell aging and metabolic processes. Proteins unique to hPDLSCs and hBMSCs were associated with translation and metabolic processes, respectively. CONCLUSION: Our results demonstrate evidence of distinct differences in protein expression between hPDLSCs and hBMSCs by using label-free quantitative proteomic analysis which was the first attempt in this field. DEPs included previously reported hPDLSC marker proteins and novel marker candidates, such as microtubule-associated protein, CTP synthase 1 and stathmin, which could be the markers for developing periodontal disease diagnostics and therapies.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células-Tronco/metabolismo , Espectrometria de Massas em Tandem , Proteínas Reguladoras de Apoptose , Biomarcadores/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Células Cultivadas , Cromatografia Líquida , Humanos , Células-Tronco Mesenquimais/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Doenças Periodontais/diagnóstico , Estatmina/metabolismo , Proteínas Supressoras de Tumor/metabolismo
7.
J Cell Mol Med ; 22(10): 5165-5169, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30019838

RESUMO

Mesenchymal stem cells (MSCs) have been investigated to treat liver diseases, but the efficiency of MSCs to treat chronic liver diseases is conflicting. FGF21 can reduce inflammation and fibrosis. We established FGF21-secreting adipose derived stem cells (FGF21_ADSCs) to enhance the effects of ADSCs and transplanted them into thioacetamide (TAA)-induced liver fibrosis mice via the tail vein. Transplantation of FGF21_ADSCs significantly improved liver fibrosis by decreasing serum hyaluronic acid and reducing the expression of fibrosis-related factors such as α-smooth muscle actin (α-SMA), collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) compared with the Empty_ADSCs by inhibition of p-JNK, NF-κB and p-Smad2/3 signalling. α-lactoalbumin (LA) and lactotransferrin (LTF), secretory factors produced from FGF21_ADSCs inhibited TGF-ß1-induced expression of α-SMA and collagen in LX-2 cells. These results suggest that transplantation of FGF21_ADSCs inhibited liver fibrosis more effectively than Empty_ADSCs, possibly via secretion of α-LA and LTF.


Assuntos
Fatores de Crescimento de Fibroblastos/genética , Cirrose Hepática/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Actinas/genética , Animais , Colágeno/genética , Fatores de Crescimento de Fibroblastos/uso terapêutico , Células Estreladas do Fígado , Humanos , Lactalbumina/genética , Lactoferrina/genética , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Camundongos , Transdução de Sinais/genética , Tioacetamida/toxicidade , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Crescimento Transformador beta1/genética
8.
Nutr Cancer ; 69(4): 663-673, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28353361

RESUMO

Methionine (Met) is involved in one-carbon de novo nucleotide synthesis and is an essential amino acid for cell survival. The impact of lactate calcium salt (CaLa) on the Met metabolism was investigated to evaluate the enhanced antitumor effect of methotrexate (MTX) on colorectal cancer (CRC) cells. Met dependency relating to homocysteine (Hcy) and betaine was investigated in human CRC cells (HCT-116 and HT-29) using a viability assay and liquid chromatography-mass spectrometry. Expression of betaine transporter-1 (BGT-1) following treatment with MTX alone or with CaLa was determined by Western blot. Enhanced antitumor effect due to malfunction of Met synthesis was confirmed. CRC cell viability decreased in Met-restricted medium, but was maintained after Hcy and betaine treatment while overcoming Met restriction. BGT-1 expression was downregulated following the treatment of dose-increased CaLa, whereas there was no effect on BGT-1 expression after MTX treatment. CaLa in combination with MTX induced reduced Met synthesis when CRC cell viability was reduced. The results indicated that CaLa-mediated BGT-1 downregulation inhibits Met synthesis by disrupting betaine homeostasis. CaLa raised the antitumor effect of MTX via secondary role in the inhibition of the de novo nucleotide synthesis. Combination therapy of MTX and CaLa could maximize the effectiveness of CRC treatment.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Metionina/metabolismo , Betaína/administração & dosagem , Betaína/metabolismo , Betaína/farmacologia , Compostos de Cálcio/administração & dosagem , Compostos de Cálcio/farmacologia , Proteínas de Transporte/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas da Membrana Plasmática de Transporte de GABA , Células HCT116/efeitos dos fármacos , Células HT29/efeitos dos fármacos , Humanos , Lactatos/administração & dosagem , Lactatos/farmacologia , Metotrexato/administração & dosagem , Metotrexato/farmacologia , Terapia de Alvo Molecular
9.
BMC Infect Dis ; 16(1): 706, 2016 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-27887598

RESUMO

BACKGROUND: To compare the related factors or manifestations of the two most common isolates of post-operative endophthalmitis, which were Enterococcus spp. and coagulase-negative staphylococci (CNS) in South Korea. METHODS: Medical records were reviewed for cases of post-operative endophthalmitis caused by Enterococcus spp. and CNS at eight institutions between January 2004 and July 2010. Various factors including age, sex, residence, systemic diseases, smoking and drinking history, and best corrected visual acuity, and length of time between causative intraocular surgery and symptom development were compared between the two groups. RESULTS: The total number of post-operative endophthalmitis cases was 128 and in 116 cases, microbiological culture tests from the aqueous humor or vitreous were performed. Among these cases, 67 (57.8%) were culture proven. Among these 67 cases, 19 (28.4%) were caused by Enterococcus spp., 14 (20.9%) were caused by Staphylococcus epidermidis endophthalmitis, and 5 (7.5%) were caused by other CNS spp. Age, sex, causative procedure, past medical history, social history, and laterality were not different in the two groups. Mean initial and final visual acuity were significantly worse in the Enterococcus spp. endophthalmitis group than in the CNS group (p = 0.049, 0.042, respectively). Length of time between the causative procedure and symptom development was significantly shorter in cases of Enterococcus spp. endophthalmitis (p = 0.004). CONCLUSIONS: Enterococcus spp. induce more severe and rapid-onset postoperative endophthalmitis than CNS. Infectious endophthalmitis developed within 2 days after cataract operation could be caused by Enterococcus spp. and have chance to be poor prognosis in South Korea.


Assuntos
Coagulase/metabolismo , Endoftalmite/microbiologia , Enterococcus faecalis/isolamento & purificação , Infecções por Bactérias Gram-Positivas/diagnóstico , Complicações Pós-Operatórias/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Endoftalmite/diagnóstico , Endoftalmite/etiologia , Infecções Oculares Bacterianas , Feminino , Infecções por Bactérias Gram-Positivas/etiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico , Prognóstico , República da Coreia , Estudos Retrospectivos , Índice de Gravidade de Doença , Infecções Estafilocócicas/etiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus/isolamento & purificação
10.
Opt Lett ; 40(18): 4241-4, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26371906

RESUMO

We present a novel, electromagnetically induced transparency system based on guided-mode resonances and numerically demonstrate its transmission characteristics through finite-difference time-domain simulations. The system is composed of two planar dielectric waveguides and a subwavelength grating. It is shown that by coupling the two resonant guide modes with a low- and high-quality factor, a narrow transparency window is generated inside a broad background transmission dip produced by the guided-mode resonance. Our work could provide another efficient way toward the realization of electromagnetically induced transparency.

11.
BMC Infect Dis ; 15: 177, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25885441

RESUMO

BACKGROUND: To investigate clinical features of infectious endophthalmitis over five years in a South Korean population. METHODS: Medical records of consecutive patients diagnosed with infectious endophthalmitis at eight institutions located in Gyeongsangnam-do and Pusan city between January 1, 2004 and July 31, 2010 were reviewed. RESULTS: A total of 197 patients were diagnosed and treated. An average of 30.0 infectious endophthalmitis per year was developed. The annual incidence rate of postoperative endophthalmitis during 2006~2009 was 0.037%. The ratios of male to female and right to left were 50.2%: 49.8 % and 54.8%: 43.2%, respectively. Eighth decade and spring were the peak age (36.6%) and season (32.0%) to develop the infectious endophthalmitis. The most common past history in systemic disease was hypertension (40.4%), followed by diabetes (23.4%). Cataract operation (60.4%) was the most common cause, among which most of them was uneventful phacoemulsification (95.9%). Corneal laceration (51.6%) and liver abscess (42.9%) were the most common causes of traumatic and endogenous endophthalmitis, respectively. The percentages of patients with initial and final visual acuity less than counting fingers were 62.6% and 35.2%, respectively. Treatment with vitrectomy with or without intravitreal antibiotics injection was administered to 72.6% of patients, while 17.3% received intravitreal antibiotics only. CONCLUSIONS: Our study revealed that the development of infectious endophthalmitis was related with seasonal variation and increased during our study period. Pars plana vitrectomy was preferred for the treatment of infectious endophthalmitis in South Korea.


Assuntos
Extração de Catarata , Lesões da Córnea/epidemiologia , Endoftalmite/epidemiologia , Infecções Oculares Bacterianas/epidemiologia , Abscesso Hepático/epidemiologia , Infecção da Ferida Cirúrgica/epidemiologia , Adulto , Distribuição por Idade , Idoso , Antibacterianos/uso terapêutico , Estudos de Coortes , Comorbidade , Diabetes Mellitus/epidemiologia , Endoftalmite/terapia , Infecções Oculares Bacterianas/terapia , Feminino , Humanos , Hipertensão/epidemiologia , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , República da Coreia/epidemiologia , Estudos Retrospectivos , Estações do Ano , Distribuição por Sexo , Acuidade Visual , Vitrectomia/métodos
12.
Opt Express ; 22(23): 28954-65, 2014 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-25402134

RESUMO

We report that the Fano resonance of self-collimated beams can be achieved in a two-dimensional photonic crystal by introducing a Fano resonator that is composed of zigzag line defects. An asymmetric Fano line shape in a transmission spectrum is generated by the interference between radiated light beams from the resonator and self-collimated beams that directly pass through the resonator without resonance. It is shown that the Fano profile increases in sharpness as the number of zigzag line defects increases because the phase values of the radiated light beams change more rapidly when the number of defects increases. The Fano resonance of self-collimated beams could provide an efficient approach to manipulate light propagation and increase the possibility of application of self-collimated beams.


Assuntos
Fenômenos Ópticos , Fótons , Simulação por Computador , Cristalização , Eletricidade , Campos Magnéticos , Análise Espectral
13.
Infection ; 42(1): 113-8, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24072645

RESUMO

PURPOSE: To investigate the microorganisms in culture-proven endophthalmitis and their susceptibilities to antimicrobial agents commonly used in South Korea. METHODS: Medical records of consecutive patients with culture-proven endophthalmitis at eight institutions between 1 January 2004 and 31 July 31 2010 were reviewed. Four categories of endophthalmitis were studied: postoperative, posttraumatic, endogenous, and unspecified. Outcome measures were culture-proven infectious organisms, antimicrobial susceptibilities, and final visual acuity in the patients. RESULTS: A total of 93 microorganisms were identified from 103 patients during the study period. The positive culture rate was 59.2 % (103/174). The most common organisms identified were Enterococcus faecalis (in 20.8 % of patients, 20/96), Staphylococcus epidermidis (18.8 %, 18/96), other coagulase-negative staphylococci (10.4 %, 10/96), Pseudomonas aeruginosa (6.3 %, 6/96), and Klebsiella pneumoniae (6.3 %, 6/96). Two cases of Enterococcus faecium (2.1 %) were recognized. Overall, 70 of 96 (73.0 %) isolates were Gram-positive bacteria, 22 (23.0 %) were Gram-negative bacteria, and 4 (4.2 %) were fungi. The most common organisms resulting in reduced light perception were E. faecalis and K. pneumoniae. CONCLUSIONS: The emergence of E. faecalis in endophthalmitis is mainly caused by the high incidence of E. faecalis in postoperative endophthalmitis. This increase also impacts the final visual acuity of the patients.


Assuntos
Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , Endoftalmite/epidemiologia , Endoftalmite/microbiologia , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Antibacterianos/farmacologia , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , República da Coreia/epidemiologia , Estudos Retrospectivos
14.
Biosci Biotechnol Biochem ; 78(7): 1103-11, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25229843

RESUMO

Effective HPLC-DAD and HPLC-ESI-MS/MS methods have been developed for the analysis of eight benzo[c]phenanthridine alkaloids (sanguinarine, chelirubine, macarpine, chelerythrine, dihydrosanguinarine, dihydrochelirubine, dihydromacarpine and dihydrochelerythrine), which are important metabolites in Eschscholtzia californica cell culture. By adopting a ternary gradient pump system, the dihydro-form alkaloids hardly separable from each other could be successfully separated, and all the target alkaloids could be simultaneously quantified with the LOD values of 0.01-0.79 µg/mL and the LOQ values of 0.03-3.59 µg/mL. This HPLC-DAD method was further confirmed by HPLC-ESI-MS/MS system in multiple reaction monitoring mode. Each separated HPLC peak was identified as the target alkaloid, showing its relevant ionized molecule and selected fragment ion. By applying the established method, alkaloid production during the E. californica cell culture could be successfully monitored and some valuable information on its metabolism could be deduced.


Assuntos
Alcaloides/análise , Alcaloides/química , Cromatografia Líquida de Alta Pressão/instrumentação , Eschscholzia/citologia , Fenantridinas/química , Espectrometria de Massas por Ionização por Electrospray , Proliferação de Células , Células Cultivadas
15.
Metabolites ; 14(1)2024 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-38248837

RESUMO

Multi-omics integrates diverse types of biological information from genomic, proteomic, and metabolomics experiments to achieve a comprehensive understanding of complex cellular mechanisms. However, this approach is also challenging due to technical issues such as limited sample quantities, the complexity of data pre-processing, and reproducibility concerns. Furthermore, existing studies have primarily focused on technical performance assessment and the presentation of modified protocols through quantitative comparisons of the identified protein counts. Nevertheless, the specific differences in these comparisons have been minimally investigated. Here, findings obtained from various omics approaches were profiled using various extraction methods (methanol extraction, the Folch method, and Matyash methods for metabolites and lipids) and two digestion methods (filter-aided sample preparation (FASP) and suspension traps (S-Trap)) for resuspended proteins. FASP was found to be more effective for the identification of membrane-related proteins, whereas S-Trap excelled in isolating nuclear-related and RNA-processing proteins. Thus, FASP may be suitable for investigating the immune response and bacterial infection pathways, whereas S-Trap may be more effective for studies focused on the mechanisms of neurodegenerative diseases. Moreover, regarding the choice of extraction method, the single-phase method identified organic compounds and compounds related to fatty acids, whereas the two-phase extraction method identified more hydrophilic compounds such as nucleotides. Lipids with strong hydrophobicity, such as ChE and TG, were identified in the two-phase extraction results. These findings highlight that significant differences among small molecules are primarily identified due to the varying polarities of extraction solvents. These results, obtained by considering variables such as human error and batch effects in the sample preparation step, offer comprehensive and detailed results not previously provided by existing studies, thereby aiding in the selection of the most suitable pre-processing approach.

16.
Nutrients ; 16(3)2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38337695

RESUMO

In this study, Liquid Chromatography-Mass Spectrometry (LC-MS)-based metabolomics profiling was conducted to elucidate the urinary profiles of premature infants during early and late postnatal stages. As a result, we discovered significant excretion of maternal drugs in early-stage infants and identified crucial metabolites like hormones and amino acids. These findings shed light on the maternal impact on neonatal metabolism and underscore the beneficial effects of breastfeeding on the metabolism of essential amino acids in infants. This research not only enhances our understanding of maternal-infant nutritional interactions and their long-term implications for preterm infants but also offers critical insights into the biochemical characteristics and physiological mechanisms of preterm infants, laying a groundwork for future clinical studies focused on neonatal development and health.


Assuntos
Recém-Nascido Prematuro , Espectrometria de Massa com Cromatografia Líquida , Lactente , Feminino , Humanos , Recém-Nascido , Cromatografia Líquida , Espectrometria de Massas em Tandem , Metaboloma , Metabolômica/métodos
17.
Mol Vis ; 19: 935-43, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23687430

RESUMO

PURPOSE: To report a case series of patients with novel forkhead box CI (FOXC1) mutations in a Korean family with Axenfeld-Rieger syndrome (ARS). METHODS: Four members of the same family underwent complete ophthalmologic and systemic examinations and genetic analysis. Genomic DNA was isolated from peripheral blood leukocytes, and all coding exons with flanking intronic regions of the FOXC1 and pituitary homeobox 2 genes were amplified using PCR, and sequenced. RESULTS: The patients were 40, 12, 11, and 10 years old (father, son, and two sisters, respectively). All four had uncontrolled intraocular pressure, glaucomatous visual field defect, retinal nerve fiber layer defect, iridocorneal adhesion on gonioscopy, hypoplasia and marked atrophy of the iris, flattening of the midface, and broad flat noses. A diagnosis of ARS was made based on characteristic ocular and systemic traits. A novel FOXC mutation, c.317delA, was identified in all affected members of the family with ARS. CONCLUSIONS: We found a novel c.317delA mutation in FOXC1 in a Korean family with ARS. We suggest that this FOXC1 mutation causes typical ARS, and that our results may be useful for better understanding of the spectrum of FOXC1 mutations and the role of FOXC1 in the development and progression of ARS.


Assuntos
Povo Asiático/genética , Anormalidades do Olho/genética , Fatores de Transcrição Forkhead/genética , Mutação/genética , Adulto , Segmento Anterior do Olho/anormalidades , Sequência de Bases , Criança , Análise Mutacional de DNA , Anormalidades do Olho/patologia , Oftalmopatias Hereditárias , Família , Feminino , Proteínas de Homeodomínio/genética , Humanos , Recém-Nascido , Masculino , Dados de Sequência Molecular , Linhagem , República da Coreia , Fatores de Transcrição/genética , Proteína Homeobox PITX2
18.
Rapid Commun Mass Spectrom ; 27(24): 2767-76, 2013 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-24214862

RESUMO

RATIONALE: Protein post-translational modifications (PTMs) are directly involved in protein function and cellular activities. Among them, glycosylation and phosphorylation are particularly important modifications on proteins located at extracellular and intracellular domains, respectively. However, the combined detection using phospho- and glycoproteomics is limited mainly due to protocol differences. METHODS: In this study, we developed a novel method for both phospho- and glycoproteome detection from a single sample batch, in which a titanium dioxide cartridge was used to capture the phosphoproteome, and the flow-through solution was processed for capturing N-linked glycopeptides using hydrazide resin. RESULTS: By using 1 mg of protein from kidney tissue lysates from normal and diseased rats, we concurrently identified 437 glycosites/358 phosphosites and 468 glycosites/369 phosphosites in normal and disease kidneys, respectively, by liquid chromatography/tandem mass spectrometric analysis. CONCLUSIONS: Compared with individual PTM analyses, the combined PTM analysis clearly provides more broad implications for PTMs related to the pathological status and discovery of biomarker candidates. Furthermore, the combined protocol thoroughly showed its advantages in enrichment efficiency and biological interpretation compared with current methods.


Assuntos
Glicopeptídeos/análise , Nefrocalcinose/induzido quimicamente , Fosfopeptídeos/análise , Ácido Fítico/toxicidade , Proteoma/efeitos dos fármacos , Proteômica/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Feminino , Rim/química , Rim/efeitos dos fármacos , Dados de Sequência Molecular , Nefrocalcinose/metabolismo , Ácido Fítico/administração & dosagem , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem
19.
Mol Cell Proteomics ; 10(10): M111.011023, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21799047

RESUMO

Prediction of the responses to neoadjuvant chemotherapy (NACT) can improve the treatment of patients with advanced breast cancer. Genes and proteins predictive of chemoresistance have been extensively studied in breast cancer tissues. However, noninvasive serum biomarkers capable of such prediction have been rarely exploited. Here, we performed profiling of N-glycosylated proteins in serum from fifteen advanced breast cancer patients (ten patients sensitive to and five patients resistant to NACT) to discover serum biomarkers of chemoresistance using a label-free liquid chromatography-tandem MS method. By performing a series of statistical analyses of the proteomic data, we selected thirteen biomarker candidates and tested their differential serum levels by Western blotting in 13 independent samples (eight patients sensitive to and five patients resistant to NACT). Among the candidates, we then selected the final set of six potential serum biomarkers (AHSG, APOB, C3, C9, CP, and ORM1) whose differential expression was confirmed in the independent samples. Finally, we demonstrated that a multivariate classification model using the six proteins could predict responses to NACT and further predict relapse-free survival of patients. In summary, global N-glycoproteome profile in serum revealed a protein pattern predictive of the responses to NACT, which can be further validated in large clinical studies.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Farmacológicos/sangue , Proteínas Sanguíneas/análise , Neoplasias da Mama/tratamento farmacológico , Glicoproteínas/análise , Terapia Neoadjuvante , Proteômica , Adulto , Proteínas Sanguíneas/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Glicoproteínas/sangue , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Coloração e Rotulagem
20.
J Environ Manage ; 129: 274-82, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23968913

RESUMO

Lab-scale High Efficiency Digestion (HED) systems containing a Mesophilic Anaerobic Reactor (MAR), Thermophilic Aerobic Reactor (TAR), liquid/solid separation unit, and thermal-alkaline treatment were developed to evaluate the efficiencies of sludge reduction and methane production. The HED process was divided into three phases to examine the influence of sludge pretreatment and pretreated sludge recirculation using TCOD and VSS reduction, COD solubilization, and methane production. The VSS removal with a solid/liquid separation unit, sludge recirculation, and thermal-alkaline treatment drastically increased up to 95% compared to the feed concentration. In addition, the results of COD solubilization and VSS/TSS showed that the solubilization of cells and organic matters by the thermal-alkaline treatment was highly increased, which was also consistent with the SEM images. In particular, the methane production rate increased 24-fold when the feed sludge and recirculated sludge were pretreated together. Collectively, the HED experiments performed with sludge recirculation and thermal-alkaline treatment demonstrated that the HED systems can be successfully employed for highly efficient sewage sludge reduction and methane gas production.


Assuntos
Biocombustíveis/análise , Recuperação e Remediação Ambiental/métodos , Esgotos/análise , Eliminação de Resíduos Líquidos/métodos , Aerobiose , Anaerobiose , Biodegradação Ambiental , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Monitoramento Ambiental , Temperatura Alta , Microscopia Eletrônica de Varredura
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