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1.
Subcell Biochem ; 94: 499-520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189313

RESUMO

C-reactive protein (CRP) is an evolutionary highly conserved member of the pentraxin superfamily of proteins. CRP is widely used as a marker of inflammation, infection and for risk stratification of cardiovascular events. However, there is now a large body of evidence, that continues to evolve, detailing that CRP directly mediates inflammatory reactions and the innate immune response in the context of localised tissue injury. These data support the concept that the pentameric conformation of CRP dissociates into pro-inflammatory CRP isoforms termed pCRP* and monomeric CRP. These pro-inflammatory CRP isoforms undergo conformational changes that facilitate complement binding and immune cell activation and therefore demonstrate the ability to trigger complement activation, activate platelets, monocytes and endothelial cells. The dissociation of pCRP occurs on the surface of necrotic, apoptotic, and ischaemic cells, regular ß-sheet structures such as ß-amyloid, the membranes of activated cells (e.g., platelets, monocytes, and endothelial cells), and/or the surface of microparticles, the latter by binding to phosphocholine. Therefore, the deposition and localisation of these pro-inflammatory isoforms of CRP have been demonstrated to amplify inflammation and tissue damage in a broad range of clinical conditions including ischaemia/reperfusion injury, Alzheimer's disease, age-related macular degeneration and immune thrombocytopaenia. Given the potentially broad relevance of CRP to disease pathology, the development of inhibitors of CRP remains an area of active investigation, which may pave the way for novel therapeutics for a diverse range of inflammatory diseases.


Assuntos
Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Sequência Conservada , Evolução Molecular , Inflamação/metabolismo , Inflamação/patologia , Biomarcadores/química , Biomarcadores/metabolismo , Humanos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo
2.
Science ; 367(6483): 1240-1246, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32165585

RESUMO

In neurons, the loading of neurotransmitters into synaptic vesicles uses energy from proton-pumping vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases). These membrane protein complexes possess numerous subunit isoforms, which complicates their analysis. We isolated homogeneous rat brain V-ATPase through its interaction with SidK, a Legionella pneumophila effector protein. Cryo-electron microscopy allowed the construction of an atomic model, defining the enzyme's ATP:proton ratio as 3:10 and revealing a homolog of yeast subunit f in the membrane region, which we tentatively identify as RNAseK. The c ring encloses the transmembrane anchors for cleaved ATP6AP1/Ac45 and ATP6AP2/PRR, the latter of which is the (pro)renin receptor that, in other contexts, is involved in both Wnt signaling and the renin-angiotensin system that regulates blood pressure. This structure shows how ATP6AP1/Ac45 and ATP6AP2/PRR enable assembly of the enzyme's catalytic and membrane regions.


Assuntos
Biomarcadores/química , Encéfalo/enzimologia , Receptores de Superfície Celular/química , ATPases Vacuolares Próton-Translocadoras/química , Animais , Proteínas de Bactérias/química , Biocatálise , Membrana Celular/enzimologia , Microscopia Crioeletrônica , Modelos Químicos , Domínios Proteicos , Ratos , Sistema Renina-Angiotensina , Via de Sinalização Wnt
3.
J Agric Food Chem ; 68(7): 2263-2275, 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-31986019

RESUMO

The growth conditions and age of Panax ginseng are vital for determining the quality of the ginseng plant. However, the considerable difference in price according to the cultivation method and period of P. ginseng leads to its adulteration in the trade market. We herein focused on ginseng peptides and the possibility of these peptides to be used as biomarker(s) for discrimination of P. ginseng. We applied an ultraperformance liquid chromatography-high resolution mass spectrometry-based peptidomics approach to characterize ginseng peptides and discover novel peptide biomarkers for authentication of mountain-cultivated ginseng (MCG). We identified 52 high-confidence peptides and screened 20 characteristic peptides differentially expressed between MCG and cultivated ginseng (CG). Intriguingly, 6 differential peptides were expressed significantly in MCG and originated from dehydrins that accumulated during cold or drought conditions. In addition, 14 other differential peptides that were significantly expressed in CG derived from ginseng major protein, an essential protein for nitrogen storage. These biological associations confirmed the reliability and credibility of the differential peptides. Additionally, we determined several robust peptide biomarkers for discrimination of MCG through a precise selection process. These findings demonstrate the potential of peptide biomarkers for identification and quality control of P. ginseng in addition to ginsenoside analysis.


Assuntos
Panax/química , Peptídeos/química , Sequência de Aminoácidos , Biomarcadores/química , Cromatografia Líquida de Alta Pressão , Análise Discriminante , Contaminação de Alimentos/análise , Espectrometria de Massas , Panax/crescimento & desenvolvimento , Mapeamento de Peptídeos , Raízes de Plantas/química , Raízes de Plantas/crescimento & desenvolvimento , Controle de Qualidade
4.
Nature ; 577(7788): 52-59, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31894146

RESUMO

The proper functioning of living systems and physiological phenotypes depends on molecular composition. Yet simultaneous quantitative detection of a wide variety of molecules remains a challenge1-8. Here we show how broadband optical coherence opens up opportunities for fingerprinting complex molecular ensembles in their natural environment. Vibrationally excited molecules emit a coherent electric field following few-cycle infrared laser excitation9-12, and this field is specific to the sample's molecular composition. Employing electro-optic sampling10,12-15, we directly measure this global molecular fingerprint down to field strengths 107 times weaker than that of the excitation. This enables transillumination of intact living systems with thicknesses of the order of 0.1 millimetres, permitting broadband infrared spectroscopic probing of human cells and plant leaves. In a proof-of-concept analysis of human blood serum, temporal isolation of the infrared electric-field fingerprint from its excitation along with its sampling with attosecond timing precision results in detection sensitivity of submicrograms per millilitre of blood serum and a detectable dynamic range of molecular concentration exceeding 105. This technique promises improved molecular sensitivity and molecular coverage for probing complex, real-world biological and medical settings.


Assuntos
Biomarcadores/sangue , Análise Química do Sangue/métodos , Soro/química , Espectrofotometria Infravermelho , Biomarcadores/química , Análise Química do Sangue/instrumentação , Humanos , Sensibilidade e Especificidade , Água/química
5.
PLoS One ; 15(1): e0227980, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978133

RESUMO

INTRODUCTION: Particles in exhaled air (PEx) provide samples of respiratory tract lining fluid from small airways containing, for example, Surfactant protein A (SP-A) and albumin, potential biomarkers of small airway disease. We hypothesized that there are differences between morning, noon, and afternoon measurements and that the variability of repeated measurements is larger between days than within days. METHODS: PEx was obtained in sixteen healthy non-smoking adults on 11 occasions, within one day and between days. SP-A and albumin were quantified by ELISA. The coefficient of repeatability (CR), intraclass correlation coefficient (ICC), and coefficient of variation (CV) were used to assess the variation of repeated measurements. RESULTS: SP-A and albumin increased significantly from morning towards the noon and afternoon by 13% and 25% on average, respectively, whereas PEx number concentration and particle mean mass did not differ significantly between the morning, noon and afternoon. Between-day CRs were not larger than within-day CRs. CONCLUSIONS: Time of the day influences the contents of SP-A and albumin in exhaled particles. The variation of repeated measurements was rather high but was not influenced by the time intervals between measurements.


Assuntos
Albuminas/isolamento & purificação , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Proteína A Associada a Surfactante Pulmonar/isolamento & purificação , Sistema Respiratório/química , Adulto , Idoso , Ar/análise , Albuminas/metabolismo , Biomarcadores/química , Testes Respiratórios , Expiração/fisiologia , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Sistema Respiratório/metabolismo , Espirometria/métodos
6.
Biomed Chromatogr ; 34(1): e4640, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31256423

RESUMO

Bioanalysis of unstable compounds such as acyl glucuronide metabolites represents a great analytical challenge owing to poor analyte stability in biological matrices. The primary goal for bioanalytical assay development is to minimize the breakdown of acyl glucuronide metabolite into its parent aglycone during sample collection, transportation, storage and analysis. Samples need to be stabilized ex vivo immediately after sample collection to minimize potential breakdown and thus to ensure accurate concentration measurement of both acyl glucuronide metabolite and its parent aglycone. In this review paper, formation of acyl glucuronide metabolites, the importance of establishing acyl glucuronide exposure measurement and safety coverage, optimization of sample pretreatment to stabilize the acyl glucuronide metabolites, current analytical strategy of assaying them as well as considerations for regulatory filings are discussed. It is important to identify acyl glucuronide metabolites that are capable of undergoing hydrolysis and pH-dependent intra-molecular migration as well as covalently binding to plasma and tissue proteins which can cause toxicity in vivo in the early stages of drug development. Carefully planning analytical experiments, identifying structures of acyl glucuronides and monitoring their concentrations in early drug development can help assess the risks associated with their exposures and potentially predict their concentrations in human circulation.


Assuntos
Cromatografia Líquida/métodos , Glucuronídeos , Espectrometria de Massas em Tandem/métodos , Biomarcadores/análise , Biomarcadores/química , Glucuronídeos/análise , Glucuronídeos/química , Humanos
7.
Int J Vitam Nutr Res ; 90(1-2): 124-130, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30747606

RESUMO

This study aimed to assess the relation between zinc status and inflammation biomarkers in adolescent judokas. This cross-sectional study included 52 male adolescents, aged between 14 and 19 years, who were subdivided into two groups: judoka group (n = 25) and control group (n = 27). Zinc intake was monitored using 3-day food records and the NutWin software version 1.5. The plasma and erythrocyte zinc concentrations were determined by flame atomic absorption spectrophotometry. Analysis of cytokines (IL-1ß, IL-6, and TNF-α) was performed. The mean values of zinc concentration in the diet were found to be higher than those recommended (11.0±3.9 mg/day and 20.3±11.9 mg/day for control group and judokas, respectively) although there was no significant difference between the groups. The mean plasma concentrations of zinc were below the reference range (71.4±16.0 µg/dL and 71.9±13.8 µg/dL for control group and judokas, respectively), without a significant difference between the groups. The mean concentrations of zinc erythrocyte were within the reference range (41.2±8.6 µg/gHb and 42.6±11.3 µg/gHb for control group and judokas, respectively), without a significant difference between the groups. There was no significant difference in the inflammatory biomarkers between the judokas and controls. There was not a significant correlation between biochemical parameters of zinc and inflammation biomarkers in adolescent judokas. Regarding the data found in the study, it can be concluded that the athletes evaluated have low plasma zinc concentrations, normal erythrocyte values, and high dietary intake of zinc. Moreover, the study don't show a relationship between zinc parameters and inflammatory markers evaluated.


Assuntos
Biomarcadores/análise , Estado Nutricional , Zinco , Adolescente , Biomarcadores/química , Estudos Transversais , Dieta , Humanos , Masculino , Adulto Jovem , Zinco/química
8.
Equine Vet J ; 52(1): 144-151, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31120574

RESUMO

BACKGROUND: Allogeneic bone marrow-derived mesenchymal stem cells (BMDMSCs) could provide multiple advantages over autologous BMDMSCs, including creating an 'off-the-shelf' treatment together with the ability to control for donor variation. OBJECTIVES: The objective of the study was to compare the clinical and synovial fluid response of the normal equine joint to autologous and pooled-allogeneic BMDMSCs while controlling for individual variation and joint variations in response to intra-articular injections. We hypothesised that, by controlling for individual animal and joint variation, we could identify differences between allogeneic vs. autologous BMDMSCs in noninflamed joints. STUDY DESIGN: Randomised-controlled experiment. METHODS: Bone marrow was harvested from eight horses. Autologous BMDMSCs were culture expanded, cryopreserved and thawed immediately prior to administration. For allogeneic BMDMSC treatments, four horses' BMDMSCs were culture expanded, pooled, cryopreserved and thawed immediately prior to use. Ten million (autologous or pooled-allogeneic) BMDMSCs were administered into contralateral forelimb metacarpophalangeal joints so that every autologous and allogeneic injection could be compared within the same animal. Clinical parameters included subjective lameness, objective lameness (Lameness Locator™), response to flexion, joint circumference and joint effusion. Arthrocentesis was performed for assessment of the nucleated cell count, differential cell count, total protein, and synovial concentrations of prostaglandin E2 (PGE2) and c-reactive protein (CRP). All parameters were measured at baseline, 6, 12, 24, 72, 168 and 336 h post-injection. RESULTS: No difference was detected in any parameters between forelimb metacarpophalangeal joints administered autologous or pooled-allogeneic BMDMSCs. MAIN LIMITATIONS: This study did not attempt to measure efficacy of BMDMSCs for musculoskeletal disease and should be followed by properly controlled efficacy trials. CONCLUSIONS: The study did not identify any clinical or cytological differences in the normal joint response to allogeneic or autologous BMDMSCs. A larger study to prove equivalence is warranted as allogeneic BMDMSCs may be a feasible alternative to autologous BMDMSCs.


Assuntos
Células da Medula Óssea , Cavalos , Transplante de Células-Tronco Mesenquimais/veterinária , Células-Tronco Mesenquimais/fisiologia , Animais , Biomarcadores/química , Injeções Intra-Articulares , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Líquido Sinovial , Transplante Autólogo , Transplante Homólogo
9.
Biosens Bioelectron ; 147: 111766, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31654821

RESUMO

Citrus greening, or Huanglongbing (HLB), is currently the most devasting disease of citrus, creating unprecedented crisis for the multibillion-dollar global citrus industry. To-date, there is no effective cure and disease management relies on early detection and removal of infected trees. Thus, it is imperative that accurate, timely, and robust disease detection and diagnosis technologies are available to minimize the spread of disease. This study reports a sensitive and selective label-free biosensor that combines the physical and chemical advantages of carbon nanomaterials like single-walled carbon nanotubes (SWNTs) in a field-effect transistor (FET)/chemiresistor architecture with selective antibodies against Sec-delivered effector 1 (SDE1), a secreted protein biomarker, for the detection of HLB. The biosensor detected SDE1 biomarkers for citrus greening in plant tissue extracts with the dynamic range over three orders of magnitude in the low nanomolar to micromolar concentration range and limit of detection of 5 nM. The study also demonstrated the use of the standard additions assay method with the biosensor to attain a 90-percent signal recovery in concentrated plant tissue extract, allowing for quantitative detection without an external calibration. Adopting the novel detection strategy targeting the secreted protein biomarker, SDE1, addresses some of the challenges faced by current methods of nucleic acid-based assays and symptom-based diagnosis, which have been found prone to false negatives and misdiagnoses, respectively.


Assuntos
Biomarcadores/química , Técnicas Biossensoriais , Citrus/genética , Nanotubos de Carbono/química , Animais , Citrus/parasitologia , Hemípteros/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Folhas de Planta/química , Folhas de Planta/parasitologia
10.
J Pharm Biomed Anal ; 177: 112889, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31568966

RESUMO

This work presents the development of a methodology for the accurate and precise quantification of the renal biomarker Cystatin C in human urine by Isotope Dilution Mass Spectrometry (IDMS). The procedure is based on the addition of a known quantity of the proteotypic peptide ALDFAVG*EYNK labelled with 13C2-glycine to the urine sample followed by protein hydrolysis using trypsin. Then, preconcentration and purification of the isotope diluted peptide was carried out by a selective monoclonal antibody bound to magnetic beads and final measurement was done after injection of the sample in a HPLC-MS/MS triple quadrupole instrument. The isotopic distribution of the isotope diluted proteotypic peptide was measured by low resolution selected reaction monitoring. Using this aquisition mode, the bandpass of the first quadrupole was widened (FWHM =13 u) so the whole isotopic clusters for both the natural abundance and the labelled peptides entered the collision cell. The proposed acquisition mode provided similar accuracy and precision than the regular SRM mode (FWHM =0.7 u) but a higher sensitivity was observed. The purification of the sample by antibody based enrichment of the target peptide was shown to remove interfering compounds more efficiently in comparison with a sample purification based on semipreparative liquid chromatography. Using 5 ng of the labelled peptide it was possible to quantify Cystatin C in human urine in patients with normal and impaired renal function. Recoveries from 100 to 104% were obtained in samples containing from 90 to 700 µg L-1 of Cystatin C with relative standard deviations from 0.5 to 6%. The stability of Cystatin C in urine samples was evaluated under different storage conditions showing that only when the urine samples were stored at room temperature during more than 10 days, a significant degradation of Cystatin C was observed.


Assuntos
Cistatina C/urina , Nefropatias/diagnóstico , Manejo de Espécimes/efeitos adversos , Espectrometria de Massas em Tandem/métodos , Biomarcadores/química , Biomarcadores/urina , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão , Cistatina C/química , Taxa de Filtração Glomerular/fisiologia , Humanos , Técnicas de Diluição do Indicador , Rim/fisiopatologia , Nefropatias/fisiopatologia , Nefropatias/urina , Estabilidade Proteica , Manejo de Espécimes/métodos , Temperatura , Fatores de Tempo
11.
Talanta ; 207: 120346, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594588

RESUMO

Acute myocardial infarction (AMI) causes significant mortality and morbidity. The determination of multiple AMI biomarkers is very important for the timely diagnosis of AMI. In this work, simultaneous determination of three AMI biomarkers were achieved by virtue of a three-dimensional (3D) microfluidic paper-analytical device (µPAD) with temporally resolved chemiluminescence (CL) emissions for the first time. A dual-signal amplification strategy was introduced including by employing primary antibody functionalized gold nanoparticles (Ab1-GNPs) immobilized on the detection zone as amplified capture probes, and Co(II) catalyst, secondary antibody, luminol multifunctionalized gold nanoparticles (Co(II)-Ab2-luminol-GNPs) with excellent CL activity as amplified signal probes. CL immunoreactions were performed at three detection zone of the fabricated 3D µPAD by assembling Ab1-GNPs, antigen, and Co(II)-Ab2-luminol-GNPs to form sandwich-type immunocomplexes. Auto separated CL signals with temporal resolution were obtained by time delayed transport of H2O2 to different detection zones for multiplexed analysis. The CL signal obtained by using Co(II)-Ab2-luminol-GNPs as signal probe (10576 a.u.) were about 20-fold higher than that by using conventional horseradish peroxidase labeled antibody modified luminol-GNPs as signal probe (531 a.u.). Finally, three AMI biomarkers including heart-type fatty acid-binding protein (H-FABP), cardiac troponin I (cTnI) and copeptin were quantitatively analyzed in one CL detection run by reading the CL intensity of the obtained three CL emission peaks. The detection range were ultra-wide ranged from 0.1 pg/mL to 1 µg/mL, 0.5 pg/mL to 1 µg/mL and 1 pg/mL to 1 mg/mL with the detection limits down to 0.06 pg/mL, 0.3 pg/mL and 0.4 pg/mL for H-FABP, cTnI and copeptin detection, respectively. The developed µPAD based immunoassay performing multiplexed analysis ability, high sensitivity, ultra-wide dynamic range, favorable selectivity, accessible accuracy and reproducibility, have great application potential for the early diagnosis of AMI.


Assuntos
Ouro/química , Imunoensaio/instrumentação , Dispositivos Lab-On-A-Chip , Medições Luminescentes/instrumentação , Nanopartículas Metálicas/química , Infarto do Miocárdio/metabolismo , Papel , Doença Aguda , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/química , Técnicas Biossensoriais/instrumentação , Humanos
12.
Dis Markers ; 2019: 6741518, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31885741

RESUMO

Human saliva is increasingly being used and validated as a biofluid for diagnosing, monitoring systemic disease status, and predicting disease progression. The discovery of biomarkers in saliva biofluid offers unique opportunities to bypass the invasive procedure of blood sampling by using oral fluids to evaluate the health condition of a patient. Saliva biofluid is clinically relevant since its components can be found in plasma. As salivary lipids are among the most essential cellular components of human saliva, there is great potential for their use as biomarkers. Lipid composition in cells and tissues change in response to physiological changes and normal tissues have a different lipid composition than tissues affected by diseases. Lipid imbalance is closely associated with a number of human lifestyle-related diseases, such as atherosclerosis, diabetes, metabolic syndromes, systemic cancers, neurodegenerative diseases, and infectious diseases. Thus, identification of lipidomic biomarkers or key lipids in different diseases can be used to diagnose diseases and disease state and evaluate response to treatments. However, further research is needed to determine if saliva can be used as a surrogate to serum lipid profiles, given that highly sensitive methods with low limits of detection are needed to discover salivary biomarkers in order to develop reliable diagnostic and disease monitoring salivary tests. Lipidomic methods have greatly advanced in recent years with a constant advance in mass spectrometry (MS) and development of MS detectors with high accuracy and high resolution that are able to determine the elemental composition of many lipids.


Assuntos
Biomarcadores/química , Lipidômica/métodos , Saliva/química , Humanos , Estilo de Vida , Limite de Detecção , Espectrometria de Massas , Estresse Fisiológico
13.
Int J Mol Sci ; 21(1)2019 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-31877774

RESUMO

In order to effectively develop personalized medicine for kidney diseases we urgently need to develop highly accurate biomarkers for use in the clinic, since current biomarkers of kidney damage (changes in serum creatinine and/or urine albumin excretion) apply to a later stage of disease, lack accuracy, and are not connected with molecular pathophysiology. Analysis of urine peptide content (urinary peptidomics) has emerged as one of the most attractive areas in disease biomarker discovery. Urinary peptidome analysis allows the detection of short and long-term physiological or pathological changes occurring within the kidney. Urinary peptidomics has been applied extensively for several years now in renal patients, and may greatly improve kidney disease management by supporting earlier and more accurate detection, prognostic assessment, and prediction of response to treatment. It also promises better understanding of kidney disease pathophysiology, and has been proposed as a "liquid biopsy" to discriminate various types of renal disorders. Furthermore, proteins being the major drug targets, peptidome analysis may allow one to evaluate the effects of therapies at the protein signaling pathway level. We here review the most recent findings on urinary peptidomics in the setting of the most common kidney diseases.


Assuntos
Nefropatias/urina , Peptídeos/urina , Proteômica/métodos , Biomarcadores/química , Biomarcadores/urina , Humanos , Nefropatias/patologia , Espectrometria de Massas/métodos , Peptídeos/química , Medicina de Precisão/métodos , Urinálise/métodos
14.
Chem Commun (Camb) ; 55(95): 14339-14342, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31720594

RESUMO

Exosomes are emerging as a promising source of disease biomarkers. However, glycans from exosomes have been less studied. Here, for the first time, the N-glycome of human serum exosomes is reported and the potential of N-glycans from exosomes as a source for biomarker discovery is revealed.


Assuntos
Biomarcadores/sangue , Biomarcadores/química , Exossomos/química , Polissacarídeos/sangue , Polissacarídeos/química , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/química , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/química
15.
Forensic Sci Int ; 305: 110027, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31704515

RESUMO

Due the proteins from bone remains are highly resistant to pass of time and environmental conditions, they could tell us about the events that probably happened in the past. In the forensic and physical anthropology context, burnt bone remains are one of the most common pieces of recovered evidence and, generally, they are associated with funerary practices, criminal scenes or massive catastrophic events. In the present study, bone pieces of pigs were calcined at different calcination temperatures, and proteins were searched using biochemical, immunochemical and ultrastructure visualization under these experimentally conditions. For this purpose, it was successfully developed a non-demineralizing protein extraction method from burnt bone remains and the use of specific antibodies permitted the identification of different extracellular matrix and intracellular proteins. While collagen proteins type I and IV were identified and detected under middle and high calcination temperatures (300°C and 600°C); cytoskeletal proteins as actin, tubulin and, the microtubule associated protein Tau, were found under calcination process, even up high calcination temperatures. Under ultrastructural analysis, fibrous materials with a classical disposition of collagens were observed even at high calcination temperatures of the burnt bone remains. The protein identification and characterization in burnt bones as performed in present studies, is clearly demonstrating that using specific strategies for protein characterizations it is possible to found protein biomarkers in burnt bone remains and this strategy could be useful for forensic and anthropological purposes.


Assuntos
Osso e Ossos/química , Proteínas do Citoesqueleto/isolamento & purificação , Proteínas da Matriz Extracelular/isolamento & purificação , Fogo , Animais , Anticorpos/análise , Biomarcadores/química , Western Blotting , Técnica de Desmineralização Óssea , Osso e Ossos/patologia , Colágeno/ultraestrutura , Proteínas do Citoesqueleto/imunologia , Eletroforese , Proteínas da Matriz Extracelular/imunologia , Patologia Legal/métodos , Humanos , Microscopia Eletrônica de Varredura , Suínos , Temperatura
16.
Sensors (Basel) ; 19(19)2019 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-31590208

RESUMO

In this work we present the preparation of graphene material by exfoliation of graphite rods via pulses of current in electrolyte, containing a mixture of boric acid (0.05 M) and sodium chloride (0.05 M). The material was morphologically and structurally characterized by SEM/TEM/HR-TEM, XRD and FTIR techniques. TEM investigation of graphene flakes deposited onto carbon-coated grids allowed the visualization of thin and transparent regions, attributed to few-layer graphene (FLG), as well as thick and dark regions attributed to multi-layer graphene (MLG). The mixed composition of the material was additionally confirmed by XRD, which further indicated that the amount of FLG within the sample was around 83%, while MLG was around 17%. The performance of a screen-printed electrode (SPE) modified with graphene (SPE-Gr) was tested for 8-hydroxy-2'-deoxyguanosine detection. The graphene-modified electrode had a higher sensitivity in comparison with that of SPE, both in standard laboratory solutions (phosphate buffered saline-PBS) and in human saliva.


Assuntos
/isolamento & purificação , Biomarcadores/química , Técnicas Biossensoriais , Técnicas Eletroquímicas , /química , Carbono/química , Grafite/química , Humanos
17.
Anal Chim Acta ; 1089: 115-122, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31627808

RESUMO

Cholesterol is related to many health diseases and is considered as a metabolic disorder biomarker. This compound, present in all food products of animal origin, can also be used as food authentication biomarker. In this work and for the first time, positional 13C isotope contents were determined for such a high molecular weight compound. This was possible by means of NMR using adiabatic refocused INEPT. In order to test the potential of this approach for discrimination, hen eggs from different origins were collected. Quantitative extraction of egg yolk cholesterol was optimized, and partial reduced molar fractions of its different 13C isotopomers were used as predictors in discriminant analysis. Compared with the global 13C isotopic composition determined using isotope ratio monitoring by Mass Spectrometry, the relative content of cholesterol 13C isotopomers added valuable power to sample classifications according to their origins. This study paves the way to isotopomics of other steroids and similar molecular weight compounds.


Assuntos
Colesterol/análise , Criação de Animais Domésticos/classificação , Animais , Biomarcadores/análise , Biomarcadores/química , Isótopos de Carbono/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Galinhas/classificação , Colesterol/química , Colesterol/isolamento & purificação , Gema de Ovo/química , Extração Líquido-Líquido , Reprodutibilidade dos Testes
18.
Anal Chim Acta ; 1089: 56-65, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31627819

RESUMO

A novel all-in-one paper-based sampling concept for mass spectrometric bottom-up protein analysis is here demonstrated in a chip format integrating instant immunocapture, protein reduction, - alkylation and tryptic digestion all in-device. Conventional laboratory grade filter paper was coated with the polymer 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) with a subsequent covalent immobilization of the monoclonal antibody E27 targeting the biomarker human chorionic gonadotropin (hCG). In-device protein reduction and alkylation was optimized with regards to reagent concentration and reaction pH. The sampling concept showed a high degree of performance between 10 and 1000 ng/mL (R2 > 0.99) by a five-point calibration curve sampled with hCG spiked to human serum samples and freshly collected whole blood samples, respectively. LOD (experimentally obtained at 100 pg/mL (2.64 pM/0.9 IU/L)) was demonstrated to be up to ten times lower with more than six times faster sample preparation than what has previously been reported for on-paper analysis of hCG in human serum samples.


Assuntos
Gonadotropina Coriônica/sangue , Teste em Amostras de Sangue Seco/métodos , Papel , Sequência de Aminoácidos , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Biomarcadores/sangue , Biomarcadores/química , Gonadotropina Coriônica/química , Gonadotropina Coriônica/imunologia , Cromatografia Líquida , Teste em Amostras de Sangue Seco/instrumentação , Humanos , Limite de Detecção , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Proteólise , Reprodutibilidade dos Testes , Tripsina/química
19.
Braz Oral Res ; 33: e043, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31508727

RESUMO

Proteomic techniques have become popular in medicine and dentistry because of their widespread use in analyzing bodily fluids such as blood, saliva, urine, and gingival crevicular fluids as well as hard tissues such as enamel, dentine, and cementum. This review is a guide to proteomic techniques in general dentistry, summarizing techniques and their clinical application in understanding and diagnosing diseases and their use in identifying biomarkers of various diseases.


Assuntos
Proteoma , Proteômica/métodos , Saliva/química , Proteínas e Peptídeos Salivares/química , Biomarcadores/química , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Espectrometria de Massas/métodos , Neoplasias Bucais/diagnóstico , Síndrome de Sjogren/diagnóstico
20.
J Chromatogr Sci ; 57(9): 821-827, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31504284

RESUMO

Globally, Tephrosia purpurea (L.) Pers is used as an important component in herbal drug formulations for liver health. The present study is aimed to develop a suitable analytical approach for simultaneous analysis of three flavonoids (rutin, deguelin and rotenone) to establish quality control methods for plant. A novel High-performance liquid chromatography photodiode array detector (HPLC-PDA) method has been developed to quantify these flavonoids in T. purpurea. The method was validated, and data were subjected to chemometric analysis to select most optimal marker compound. The method that was found linear with R2 values ranges from 0.996 to 0.998 with good recoveries. Intra- and inter-day precision values were <2. HPLC analysis revealed high level of chemodiversity. Quantity of all the three chemical markers was found significantly disparate in samples from different locations. Deguelin was detectable only in three out of total eight samples. However, liquid chromatography-mass spectrometry analysis was found sufficiently sensitive to detect all the compounds in all samples. Thus, results suggest to apply combination of approaches to enhance confidence in chromatographic methods for quality control of herbal drugs. Principal component analysis ranked the markers as Rutin>Rotenone>Deguelin. This comprehensive approach employing multichromatography platforms can be successfully utilized in analysis of these bioactive markers and routine standardization of herbal material and formulations containing T. purpurea.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Flavonoides/análise , Espectrometria de Massas/métodos , Extratos Vegetais/química , Tephrosia , Biomarcadores/análise , Biomarcadores/química , Flavonoides/química , Limite de Detecção , Modelos Lineares , Extratos Vegetais/normas , Análise de Componente Principal , Reprodutibilidade dos Testes
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