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1.
Forensic Sci Int ; 307: 110116, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31881371

RESUMO

The estimation of the time elapsed since a biological stain was deposited at a crime scene can provide crucial information to a forensic investigation, indicating either when a crime was committed, or whether the biological evidence was deposited at the time of a known crime event. This would enable the investigators to limit the number of suspects and to assess alibis. The relative expression ratios (RERs) of body fluid-specific RNA markers are promising molecular tools for indicating the age of biological stains. However, the nature of some forensic samples found at crime scenes could be challenging, as they frequently occur in a mixture of different body fluid types. The research presented here has utilised reverse transcription quantitative PCR (RT-qPCR) to explore the impact of bloodstains being present in mixtures with other body fluids (saliva or semen) on the resulting RERs of blood-specific markers. The expression level of three blood-specific markers (HBA, HBB and miR16) along with two reference genes (18S and U6) were analysed across multiple ageing time points in pure and mixed bloodstains. For some markers, no significant differences were found when comparing RERs in pure and mixed bloodstains, however some RERs were altered in mixed stains. This indicates that the presence of body fluid mixtures may have a significant effect on the RERs of some blood-specific markers. This should therefore be considered when selecting markers for estimating the age of stains, particularly when multiple body fluids are thought to be present.


Assuntos
Manchas de Sangue , Subunidades de Hemoglobina/análise , MicroRNAs/análise , Saliva/química , Sêmen/química , Feminino , Medicina Legal , Marcadores Genéticos , Humanos , Masculino , RNA Ribossômico 18S/análise , RNA Nuclear Pequeno/análise , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
2.
J Assist Reprod Genet ; 36(12): 2515-2523, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31758512

RESUMO

PURPOSE: To investigate the validity, accuracy, and clinical outcomes of Karyomapping in preimplantation genetic testing (PGT) for ß-thalassemia combined with human leukocyte antigen (HLA) matching. METHODS: A total of 128 cycles from January 2014 to December 2017 were identified, and 1205 embryos were biopsied. The case group included 88 cycles using Karyomapping for PGT-HLA, compared with 40 cycles using polymerase chain reaction-short tandem repeat (PCR-STR) as the control group. RESULTS: There were significant differences in the HLA matching rate (21.34 vs. 14.37%), the matched transferable embryo rate (9.79 vs. 14.07%), the clinical pregnancy rate (65.08 vs. 41.86%), and the spontaneous miscarriage rate (2.44 vs. 22.22%) between the case and control groups. In the case group, nearly 1/3 (33.37%) of the embryos showed aneuploidy. According to the results of single nucleotide polymorphism (SNP) haplotype analysis, the recombination rates of HBB (hemoglobin subunit beta) and HLA were 11.46% and 5.61% respectively. HLA gene recombination was mostly distributed between HLA-A and HLA-B and the downstream region of HLA-DQB1. In addition, STR analysis could be considered in the case of copy-neutral loss of heterozygosity (LOH) in the region where the HLA gene is located. CONCLUSION: Karyomapping contributes to accurate selection of matched embryos, along with aneuploidy screening. However, STRs assist identification in cases of LOH in the target region.


Assuntos
Antígenos HLA-A/genética , Antígenos HLA-B/genética , Cariotipagem/métodos , Diagnóstico Pré-Implantação , Talassemia beta/diagnóstico , Adulto , Biópsia , Transferência Embrionária , Feminino , Cadeias beta de HLA-DQ/genética , Subunidades de Hemoglobina/genética , Humanos , Perda de Heterozigosidade/genética , Gravidez , Taxa de Gravidez , Talassemia beta/genética , Talassemia beta/patologia
3.
NPJ Syst Biol Appl ; 5: 26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396396

RESUMO

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated inflammatory response to pathogens. Bioinformatics and transcriptomics studies contribute to get a better understanding of the pathogenesis of sepsis. These studies revealed differentially expressed genes (DEGs) in sepsis involved in several pathways. Here we investigated the gene expression profiles of blood leukocytes using three microarray datasets of sepsis secondary to pneumonia, focusing on the heme/hemoglobin metabolism pathway. We demonstrate that the heme/hemoglobin metabolism pathway was found to be enriched in these three cohorts with four common genes (ALAS2, AHSP, HBD, and CA1). Several studies show that these four genes are involved in the cytoprotection of non-erythrocyte cells in response to different stress conditions. The upregulation of heme/hemoglobin metabolism in sepsis might be a protective response of white cells to the hostile environment present in septic patients (follow-up samples).


Assuntos
Heme/metabolismo , Hemoglobinas/metabolismo , Sepse/genética , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Biologia Computacional/métodos , Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Heme/genética , Subunidades de Hemoglobina/genética , Subunidades de Hemoglobina/metabolismo , Hemoglobinas/genética , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Pneumonia/complicações , Pneumonia/genética , Sepse/sangue , Sepse/metabolismo , Transcriptoma/genética
4.
Hemoglobin ; 43(3): 182-187, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31298599

RESUMO

This is the first report of quadrupole time-of-flight (Q-TOF) mass spectrometric identification of the hemoglobin (Hb) subunits, α, ß, δ and γ peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the cation exchange chromatography of Hb. The objectives were to identify the unknown high performance liquid chromatography (HPLC) peaks in healthy subjects and in patients with ß-thalassemia (ß-thal). The results demonstrate the existence of pools of free globin chains in red blood cells (RBCs). The α-, ß-, δ- and γ-globin peptides were identified in the unknown HPLC peaks. The quantification and role of the free globin pool in patients with ß-thal requires further investigation. Identification of all types of Hb subunits in the retention time (RT) before 1 min. suggests that altered Hbs is the nature of these fast-eluting peaks. Relevancy of thalassemias to the protein-aggregation disorders will require review of the role of free globin in the pathology of the disease.


Assuntos
Cromatografia Líquida de Alta Pressão , Subunidades de Hemoglobina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Talassemia beta/sangue , Talassemia beta/diagnóstico , Adolescente , Adulto , Sequência de Aminoácidos , Criança , Pré-Escolar , Feminino , Subunidades de Hemoglobina/química , Hemoglobinas Anormais/análise , Hemoglobinas Anormais/química , Humanos , Masculino , Adulto Jovem , alfa-Globinas/análise , alfa-Globinas/química , Globinas beta/análise , Globinas beta/química , Globinas delta/análise , Globinas delta/química , gama-Globinas/análise , gama-Globinas/química
5.
Forensic Sci Int ; 298: 161-168, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30909103

RESUMO

We report the preliminary observations of the peptide content of decomposition fluid produced under controlled laboratory conditions and in the absence of a soil matrix. Four domestic pig (Sus scrofa domesticus) cadavers were used to model human decomposition over a four-week trial period; physical characteristics were recorded and the peptide components of decomposition fluid was analysed using high performance liquid chromatography-time of flight mass spectrometry. Preliminary data analysis indicated that a range of peptides were consistently detected across the course of the trial period and 27 of these were common to all four cadavers; 22 originating from haemoglobin. The peptides associated with haemoglobin subunit alpha and beta displayed a breakdown pattern that remained consistent for all cadavers for the duration of the trial. Though identification of peptides during decomposition has potential for estimating the time since death, quantification of selected peptides is likely to be essential to identify time-dependent trends.


Assuntos
Peptídeos/análise , Mudanças Depois da Morte , Animais , Biomarcadores/análise , Creatina Quinase/análise , Patologia Legal , Subunidades de Hemoglobina , Humanos , Modelos Animais , Fosfopiruvato Hidratase/análise , Proteólise , Piruvato Quinase/análise , Suínos
6.
Front Biosci (Landmark Ed) ; 24: 1085-1096, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844732

RESUMO

Red blood cells (RBCs) are exposed to exogenous reactive oxygen species in the circulatory system. To this end, the interactions between the different hemoglobin (Hb) subunits and peroxiredoxin 2, which is a ubiquitous member of the antioxidant enzymes that also controls the cytokine-induced peroxide levels, were assessed. We predicted by the increment of diversity with quadratic discriminant analysis (IDQD) that peroxiredoxin2 (Prx2) could interact with the hemoglobin alpha, beta and gamma subunits but not with the delta subunit. Coimmunoprecipitation (co-IP), electrospray ionization quadrupole time of flight (ESI-Q-TOF) mass spectrometry, Western blotting and X-ray absorption fine structure (XAFS) spectroscopy were performed to verify these predictions. The results showed that Prx2 was a member of the beta-globin immunoprecipitating complex that existed in hemoglobin A, hemolysate-hemoglobin A, hemoglobin A-hemoglobin A2, hemolysate-hemoglobin A-hemoglobin A2 and hemoglobin A2 but not in hemolysate-hemoglobin A2. Adding Prx2 to hemoglobin A altered the second shell of iron embedded in hemoglobin A. Therefore, Prx2 interacts with hemoglobin A (Alpha2Beta2) and hemoglobin F (Alpha2Gamma2) but not with hemoglobin A2 (Alpha2Delta2).


Assuntos
Subunidades de Hemoglobina/química , Proteínas de Homeodomínio/química , Peroxirredoxinas/química , Algoritmos , Cromatografia Líquida , Eritrócitos/química , Hemoglobinas/química , Hemólise , Humanos , Imunoprecipitação , Espectrometria de Massas , Estresse Oxidativo , Fragmentos de Peptídeos/química , Ligação Proteica , Domínios Proteicos , Espectrometria de Massas por Ionização por Electrospray
7.
Anal Bioanal Chem ; 410(18): 4371-4378, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29696299

RESUMO

Identifying body fluids from forensic samples can provide valuable evidence for criminal investigations. Messenger RNA (mRNA)-based body fluid identification was recently developed, and highly sensitive parallel identification using reverse transcription polymerase chain reaction (RT-PCR) has been described. In this study, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a simple, rapid assay for identifying three common forensic body fluids, namely blood, semen, and saliva, and evaluated its specificity and sensitivity. Hemoglobin beta (HBB), transglutaminase 4 (TGM4), and statherin (STATH) were selected as marker genes for blood, semen, and saliva, respectively. RT-LAMP could be performed in a single step including both reverse transcription and DNA amplification under an isothermal condition within 60 min, and detection could be conveniently performed via visual fluorescence. Marker-specific amplification was performed in each assay, and no cross-reaction was observed among five representative forensically relevant body fluids. The detection limits of the assays were 0.3 nL, 30 nL, and 0.3 µL for blood, semen, and saliva, respectively, and their sensitivities were comparable with those of RT-PCR. Furthermore, RT-LAMP assays were applicable to forensic casework samples. It is considered that RT-LAMP is useful for body fluid identification.


Assuntos
Líquidos Corporais/química , Genética Forense/métodos , Marcadores Genéticos , Técnicas de Amplificação de Ácido Nucleico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sangue , DNA/genética , Subunidades de Hemoglobina/genética , Humanos , Cinética , Limite de Detecção , Masculino , Saliva/química , Proteínas e Peptídeos Salivares/genética , Espectrometria de Fluorescência , Espermatozoides/química , Transglutaminases/genética
8.
Gene ; 641: 55-62, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29031777

RESUMO

The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management.


Assuntos
Sistemas CRISPR-Cas/genética , DNA/análise , Receptores ErbB/genética , Subunidades de Hemoglobina/genética , Reação em Cadeia da Polimerase/métodos , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Carcinoma Pulmonar de Células não Pequenas , DNA/genética , Desoxirribonuclease I/genética , Células HEK293 , Humanos , Neoplasias Pulmonares , Mutação/genética , Taxa de Mutação , Células Tumorais Cultivadas
9.
Biochemistry ; 56(46): 6125-6136, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29064674

RESUMO

Following a previous detailed investigation of the ß subunit of α2ß2 human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α(Fe2+-deoxy/O2)ß(Fe3+) hemoglobin M (Hb M) in which O2 cannot bind to the ß subunit: Hb M Hyde Park (ß92His → Tyr), Hb M Saskatoon (ß63His → Tyr), and Hb M Milwaukee (ß67Val → Glu). In contrast with the ß subunit that exhibited a clear correlation between O2 affinity and Fe2+-His stretching frequencies, the Fe2+-His stretching mode of the α subunit gave two Raman bands only in the T quaternary structure. This means the presence of two tertiary structures in α subunits of the α2ß2 tetramer with T structure, and the two structures seemed to be nondynamical as judged from terahertz absorption spectra in the 5-30 cm-1 region of Hb M Milwaukee, α(Fe2+-deoxy)ß(Fe3+). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α(Fe2+-deoxy)ß(Co2+) and, therefore, seems to be universal among α subunits of Hb A. Unexpectedly, the two Fe-His frequencies were hardly changed with a large alteration of O2 affinity by pH change, suggesting no correlation of frequency with O2 affinity for the α subunit. Instead, a new Fe2+-His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O2 affinity increased. The high-frequency counterpart was also observed for a partially O2-bound form, α(Fe2+-deoxy)α(Fe2+-O2)ß(Fe3+)ß(Fe3+), of the present Hb M, consistent with our previous finding that binding of O2 to one α subunit of T structure α2ß2 tetramer changes the other α subunit to the R structure.


Assuntos
Hemoglobina M/química , Subunidades de Hemoglobina/química , Hemoglobinas Anormais/química , Oxigênio/metabolismo , Hemoglobina M/metabolismo , Subunidades de Hemoglobina/metabolismo , Hemoglobinas Anormais/metabolismo , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Multimerização Proteica , Análise Espectral Raman , Espectroscopia Terahertz
10.
Expert Rev Hematol ; 9(12): 1129-1137, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27801605

RESUMO

INTRODUCTION: Stress erythropoiesis induces fetal hemoglobin (HbF) expression in ß-thalassemias, however the level of expression is highly variable. The last decade has seen dramatic advances in our understanding of the molecular regulators of HbF production and the genetic factors associated with HbF levels, leading to the promise of new methods of the clinical induction of HbF. Areas covered: This article will review the heterogeneity and genetic modifiers of HbF and HbF induction therapy in ß-thalassemia. Expert commentary: One promising curative ß-thalassemia therapy is to induce HbF synthesis in ß-thalassemic erythrocytes to therapeutic levels before clinical symptom occurs. Further understanding of HbF level variation and regulation is needed in order to predict the response from HbF-inducing approaches.


Assuntos
Hemoglobina Fetal/genética , Regulação da Expressão Gênica , Talassemia beta/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Epistasia Genética , Hemoglobina Fetal/química , Hemoglobina Fetal/metabolismo , Edição de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Heterogeneidade Genética , Terapia Genética , Subunidades de Hemoglobina/química , Subunidades de Hemoglobina/genética , Subunidades de Hemoglobina/metabolismo , Humanos , Hidroxiureia/farmacologia , Hidroxiureia/uso terapêutico , Família Multigênica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Multimerização Proteica , Proteínas Repressoras , Talassemia beta/diagnóstico , Talassemia beta/metabolismo , Talassemia beta/terapia
11.
Zoolog Sci ; 33(1): 106-15, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26853876

RESUMO

Water fleas (Daphnia pulex) normally produce subitaneous eggs that initiate development immediately after oviposition. However, in response to habitat degradation, resting eggs are produced, which are enclosed in a sturdy outer envelope (ephippium) and can survive in harsh environments for an extended time. To understand the molecular mechanism underlying resting egg production in D. pulex, we investigated the genes whose expression patterns played a role in the production and identified the following six candidate genes: Dpfa-1, Dpfa-2, Dpep-1, Dpep-2, Dpep-3, and Dpep-4. These six genes displayed > 40-fold higher expression levels in resting egg-producing animals compared with those in subitaneous egg-producing animals at the period when the ovaries were mature. Dpfa-1 and Dpfa-2 were expressed in the fat cells, and their expression patterns were synchronized with the development of resting egg oocytes in the ovary. In contrast, Dpep-1-4 were expressed in the morphologically altered epidermal cells of the brood chamber with the formation of the ephippium, and their expression patterns were also related to ephippium formation. Our results suggest that the former two genes encode the resting egg-specific components produced by fat cells and that the latter four genes encode the components related to the ephippium formation synthesized by epidermal cells.


Assuntos
Daphnia/fisiologia , Regulação da Expressão Gênica/fisiologia , Óvulo/fisiologia , Sequência de Aminoácidos , Animais , Feminino , Subunidades de Hemoglobina/genética , Subunidades de Hemoglobina/metabolismo , Regulação para Cima
12.
J Am Soc Mass Spectrom ; 27(3): 532-41, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26733405

RESUMO

Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the ß-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R(2) > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate ß-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.


Assuntos
Hemoglobina A Glicada/análise , Subunidades de Hemoglobina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia Líquida de Alta Pressão/métodos , Diabetes Mellitus/diagnóstico , Humanos , Modelos Lineares
13.
Mol Biol Evol ; 32(4): 978-97, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25556236

RESUMO

Major challenges for illuminating the genetic basis of phenotypic evolution are to identify causative mutations, to quantify their functional effects, to trace their origins as new or preexisting variants, and to assess the manner in which segregating variation is transduced into species differences. Here, we report an experimental analysis of genetic variation in hemoglobin (Hb) function within and among species of Peromyscus mice that are native to different elevations. A multilocus survey of sequence variation in the duplicated HBA and HBB genes in Peromyscus maniculatus revealed that function-altering amino acid variants are widely shared among geographically disparate populations from different elevations, and numerous amino acid polymorphisms are also shared with closely related species. Variation in Hb-O2 affinity within and among populations of P. maniculatus is attributable to numerous amino acid mutations that have individually small effects. One especially surprising feature of the Hb polymorphism in P. maniculatus is that an appreciable fraction of functional standing variation in the two transcriptionally active HBA paralogs is attributable to recurrent gene conversion from a tandemly linked HBA pseudogene. Moreover, transpecific polymorphism in the duplicated HBA genes is not solely attributable to incomplete lineage sorting or introgressive hybridization; instead, it is mainly attributable to recurrent interparalog gene conversion that has occurred independently in different species. Partly as a result of concerted evolution between tandemly duplicated globin genes, the same amino acid changes that contribute to variation in Hb function within P. maniculatus also contribute to divergence in Hb function among different species of Peromyscus. In the case of function-altering Hb mutations in Peromyscus, there is no qualitative or quantitative distinction between segregating variants within species and fixed differences between species.


Assuntos
Evolução Molecular , Subunidades de Hemoglobina/genética , Família Multigênica , Mutação , Peromyscus/genética , Polimorfismo Genético , Sequência de Aminoácidos , Animais , Conversão Gênica , Dados de Sequência Molecular
14.
Int J Lab Hematol ; 37(2): 279-86, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25130136

RESUMO

INTRODUCTION: Phenotype studies still occupy a key position in the diagnosis of hemoglobin (Hb) disorders. MATERIAL AND METHODS: In addition to the conventional methods for diagnosis of Hb disorders which are mostly based on differences in charge of the Hb molecules, some progresses have been brought by studying other properties of the globin chains. Among those, difference in hydrophobicity that may be investigated by reversed-phase HPLC (RP-HPLC) discriminates between variants displaying identical charges. RESULTS: In this study, we show how an update of this method allows to recognize an α-chain variant from a γ-chain variant, a problem frequently during neonatal screening. We illustrate that RP-HPLC may also unravel unclear phenotypes which are modified by the presence of an additional variant not detected by the conventional methods, and help to characterize rare mutants. Also we show that it allows a clear distinction between variants with identical electrophoretical charges as exemplified by Hb Lepore Boston-Washington and Lepore Baltimore. CONCLUSIONS: In view of our results, RP-HPLC is a technique that needs to be used as a second step in the general strategy for a correct characterization of Hb variants.


Assuntos
Cromatografia de Fase Reversa , Subunidades de Hemoglobina/química , Hemoglobinopatias/diagnóstico , Fenótipo , Alelos , Substituição de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa/métodos , Subunidades de Hemoglobina/genética , Hemoglobinopatias/genética , Hemoglobinas Anormais/química , Hemoglobinas Anormais/genética , Humanos , Recém-Nascido , Mutação
15.
Inflammation ; 38(1): 394-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25338941

RESUMO

The development of new sepsis-specific biomarkers is mandatory to improve the detection and monitoring of the disease. Hemoglobin is the main oxygen and carbon dioxide carrier in cells of the erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. Hemoglobin subunit beta (HBß) is a component of a larger protein called hemoglobin. The aim of this study was to evaluate blood levels of HBß in septic patients. A prospective study of 82 patients with sepsis was conducted. Furthermore, C57BL/6 mice were subjected to cecal ligation and puncture (CLP) surgery. Alternatively, human umbilical vein endothelial cells (HUVECs) or C57BL/6 mice were exposed to lipopolysaccharide (LPS, 100 ng/ml to HUVECs or 10 mg/kg to mice). The data showed that LPS induced upregulation of the synthesis and secretion of HBß in LPS-treated HUVECs and in LPS-injected and CLP mice. In patients admitted to the intensive care unit with sepsis, circulating levels of HBß were significantly high (sepsis, 64.93-114.76 ng/ml, n = 30; severe sepsis, 157.37-268.69 ng/ml, n = 22; septic shock, 309.98-427.03 ng/ml, n = 30) when compared to the levels of control donors (9.76-12.28 ng/ml, n = 21). Patients with septic shock had higher HBß levels when compared to patients with severe sepsis. Furthermore, the HBß levels in septic patients were higher than those in healthy volunteers. These results suggest that in septic patients, HBß blood level is related to the severity of sepsis and may represent a novel endothelial cell dysfunction marker. Moreover, HBß can be used as a biomarker to determine the severity of sepsis.


Assuntos
Sepse/sangue , Sepse/diagnóstico , Globinas beta/metabolismo , Animais , Biomarcadores/sangue , Células Cultivadas , Diagnóstico Precoce , Subunidades de Hemoglobina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
16.
Eur Ann Allergy Clin Immunol ; 46(5): 164-71, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25224946

RESUMO

BACKGROUND: Rhinitis comprises several diseases with varying causes and different clinical manifestations and pathological features, but treated as a single clinical disorder. As heterogeneous disease, proper differential diagnosis is useful to delineate appropriate therapeutic intervention. Comparative proteomic investigation was aimed to provide information for specific differentially expressed proteins in rhino pathologic state, that could be used for diagnostic purpose and therapeutic monitoring. METHODS: Proteins extracted from nasal mucosa cells of patients with different features of rhinitis and from control subjects, were separated by 2-DE. Proteins differentially expressed were identified by mass spectrometry (MS). RESULTS: Comparative proteomic analyses led to the identification of eighteen proteins differentially expressed in patients with rhinitis, mainly related to cell defense and innate and acquired immunity. From that, at least one protein can be a possible candidate as biomarker of disease.


Assuntos
Mucosa Nasal/imunologia , Mucosa Nasal/patologia , Rinite/genética , Rinite/imunologia , Adulto , Aldeído Desidrogenase/imunologia , Aldeído Desidrogenase 1 , Aldeído-Desidrogenase Mitocondrial/imunologia , Antígenos de Neoplasias/imunologia , Biomarcadores , Eletroforese em Gel Bidimensional , Eosinófilos/patologia , Feminino , Glutationa S-Transferase pi/imunologia , Glutationa Transferase/imunologia , Glicoproteínas/imunologia , Subunidades de Hemoglobina/imunologia , Humanos , Isoenzimas/imunologia , Masculino , Espectrometria de Massas , Mastócitos/patologia , Pessoa de Meia-Idade , Pólipos Nasais/imunologia , Pólipos Nasais/patologia , Neutrófilos/patologia , Peroxirredoxinas/imunologia , Fosfoproteínas/imunologia , Proteômica , Retinal Desidrogenase , Proteínas S100/imunologia , Proteínas de Ligação a Selênio/imunologia , Serpinas/imunologia , Albumina Sérica/imunologia , Tiorredoxinas/imunologia
17.
Stem Cells Transl Med ; 3(7): 792-800, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24873860

RESUMO

Adult hemoglobin composed of α- and ß-globin reflects a change from expression of embryonic ε- and fetal γ-globin to adult ß-globin in human erythroid cells, so-called globin switching. Human pluripotent stem cells (hPSCs) are a potential source for in vitro erythrocyte production, but they show prominent expression of γ-globin with little ß-globin expression, which indicates incomplete globin switching. To examine the mechanism of this impaired globin switching, we optimized multicolor flow cytometry to simultaneously follow expression of different globin subtypes using different immunofluorescent probes. This enabled us to detect upregulation of ß-globin and the corresponding silencing of γ-globin at the single-cell level during cord blood CD34(+) cell-derived erythropoiesis, examined as an endogenous control. Using this approach, we initially characterized the heterogeneous ß-globin expression in erythroblasts from several hPSC clones and confirmed the predominant expression of γ-globin. These hPSC-derived erythroid cells also displayed reduced expression of BCL11A-L. However, doxycycline-induced overexpression of BCL11A-L in selected hPSCs promoted γ-globin silencing. These results strongly suggest that impaired γ-globin silencing is associated with downregulated BCL11A-L in hPSC-derived erythroblasts and that multicolor staining of globin subtypes is an effective approach to studying globin switching in vitro.


Assuntos
Eritropoese , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/metabolismo , Subunidades de Hemoglobina/metabolismo , Células-Tronco Pluripotentes/metabolismo , Globinas beta/metabolismo , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular , Técnicas de Cocultura , Células Alimentadoras , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Subunidades de Hemoglobina/genética , Humanos , Sondas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras , Fatores de Tempo , Transfecção , Globinas beta/genética , gama-Globinas/genética , gama-Globinas/metabolismo
18.
Curr Gastroenterol Rep ; 15(11): 357, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24218070

RESUMO

Colorectal cancer (CRC) is a common, but preventable, disease and is the second most common cause of cancer-related deaths in the U.S. CRC screening has proven effective at reducing both the incidence and mortality of this disease, using any of a number of screening tests available. The test options range from the least invasive and least expensive to more invasive and costly options. Fecal occult blood testing is the oldest, least expensive, and least invasive of these options and has evolved from the poorly sensitive standard guaiac test to the newer and diagnostically superior fecal immunochemical test (FIT) for hemoglobin. This article explores the evolutionary history of fecal occult blood testing, examines test performance characteristics among different FOBTs, and evaluates the role of the FIT in programmatic CRC screening.


Assuntos
Neoplasias Colorretais/diagnóstico , Detecção Precoce de Câncer/métodos , Subunidades de Hemoglobina/análise , Sangue Oculto , Biomarcadores/análise , Colonoscopia , Guaiaco , Humanos , Indicadores e Reagentes
19.
Biochemistry ; 52(47): 8539-55, 2013 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-24224786

RESUMO

The Bohr effect in hemoglobin, which refers to the dependence of the oxygen affinity on the pH, plays an important role in its cooperativity and physiological function. The dominant contribution to the Bohr effect arises from the difference in the pKa values of His residues of the unliganded (deoxy) and liganded (carbonmonoxy) structures. Using recent high resolution structures, the residue pKa values corresponding to the two structures are calculated. The method is based on determining the electrostatic interactions between residues in the protein, relative to those of the residue in solution, by use of the linearized finite difference Poisson-Boltzmann equation and Monte Carlo sampling of protonation states. Given that good agreement is obtained with the available experimental values for the contribution of His residues in HbA to the Bohr effect, the calculated results are used to determine the atomic origin of the pKa shift between deoxy and carbonmonoxy HbA. The contributions to the pKa shift calculated by means of the linear response approximation show that the salt bridge involving His146 plays an important role in the alkaline Bohr effect, as suggested by Perutz but that other interactions are significant as well. A corresponding analysis is made for the contribution of His143 to the acid Bohr effect for which there is no proposed explanation. The method used is summarized and the program by which it is implemented is described in the Appendix .


Assuntos
Hemoglobina A/metabolismo , Histidina/metabolismo , Oxiemoglobinas/metabolismo , Carboxihemoglobina/química , Carboxihemoglobina/metabolismo , Biologia Computacional/métodos , Bases de Dados de Proteínas , Hemoglobina A/química , Subunidades de Hemoglobina/química , Subunidades de Hemoglobina/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Método de Monte Carlo , Oxiemoglobinas/química , Distribuição de Poisson , Conformação Proteica
20.
Fish Shellfish Immunol ; 34(5): 1320-4, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23470816

RESUMO

Hemoglobin (Hb) is the major protein component of erythrocytes in animals with red blood, but it can serve additional functions beyond the transport of oxygen. In this study, we identified polymorphism in the blood clam Tegillarca granosa Hb (Tg-Hb) genes and investigated the association of this polymorphism with resistance/susceptibility to Vibrio parahaemolyticus. Analysis of the 540 sequences revealed 28 SNPs in the coding region of three Tg-Hbs, corresponding to about one SNP per 48 bp. Three SNPS: HbIIA-E2-146, HbIIB-E2-23, HbIIB-E2-121 showed a significant association with resistance/susceptibility to V. parahaemolyticus (P < 0.05). To further demonstrate that three significant SNPs of Tg-Hbs is associated with resistance of clams to V. parahaemolyticus, SNPs were genotyped in V. parahaemolyticus resistant strain clams and the wild base population from which this strain was derived. The results indicated that the nonsynonymous mutation T allele at HbIIA-E2-146 and A allele at HbIIB-E2-23 are associated with V. parahaemolyticus resistance in the blood clam, and its association with disease resistance may be due to its cause changes in amino acid sequences to a functional polymorphism. Together with previous bacterial challenge study, these results provides direct evidence that variation at HbIIA-E2-146 and HbIIB-E2-23 are associated with disease resistance in the blood clam, and these two polymorphic loci could be potential gene markers for the future molecular selection of strains that are resistant to diseases caused by V. parahaemolyticus.


Assuntos
Arcidae/genética , Arcidae/imunologia , Subunidades de Hemoglobina/genética , Subunidades de Hemoglobina/imunologia , Vibrio parahaemolyticus/imunologia , Animais , Arcidae/química , Arcidae/microbiologia , China , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Subunidades de Hemoglobina/química , Imunidade Inata , Dados de Sequência Molecular , Especificidade de Órgãos , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência , Regulação para Cima
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