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1.
Cytometry A ; 103(7): 575-583, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36823754

RESUMO

Peripheral blood mononuclear cells (PBMCs) are a useful model for biochemical assays, particularly for etiological studies. We describe here a method for measuring DNA repair capacity (DRC) in archival cryogenically preserved PBMCs. To model DRC, we measured γ-H2AX repair kinetics in thawed PBMCs after irradiation with 3 Gy gamma rays. Time-dependent fluorescently labeled γ-H2AX levels were measured at five time points from 1 to 20 h, yielding an estimate of global DRC repair kinetics as well as a measure of unrepaired double strand breaks at 20 h. While γ-H2AX levels are traditionally measured by either microscopy or flow-cytometry, we developed a protocol for imaging flow cytometry (IFC) that combines the detailed information of microscopy with the statistical power of flow methods. The visual imaging component of the IFC allows for monitoring aspects such as cellular health and apoptosis as well as fluorescence localization of the γ-H2AX signal, which ensures the power and significance of this technique. Application of a machine-learning based image classification improved flow cytometry fluorescent measurements by identifying apoptotic cells unable to undergo DNA repair. We present here DRC repair parameters from 18 frozen archival PBMCs and 28 fresh blood samples collected from a demographically diverse cohort of women measured in a high-throughput IFC format. This thaw method and assay can be used alone or in conjunction with other assays to measure etiological phenotypes in cryogenic biobanks of PBMCs.


Assuntos
Histonas , Leucócitos Mononucleares , Feminino , Animais , Leucócitos Mononucleares/metabolismo , Histonas/genética , Histonas/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Criopreservação
2.
Biochemistry ; 58(48): 4827-4841, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31682418

RESUMO

Biofilms form when bacteria aggregate in a self-secreted exopolysaccharide matrix; they are resistant to antibiotics and implicated in disease. Nitric oxide (NO) is known to mediate biofilm formation in many bacteria via ligation to H-NOX (heme-NO/oxygen binding) domains. Most NO-responsive bacteria, however, lack H-NOX domain-containing proteins. We have identified another NO-sensing protein (NosP), which is predicted to be involved in two-component signaling and biofilm regulation in many species. Here, we demonstrate that NosP participates in the previously described H-NOX/NO-responsive multicomponent c-di-GMP signaling network in Shewanella oneidensis. Strains lacking either nosP or its co-cistronic kinase nahK (previously hnoS) produce immature biofilms, while hnoX and hnoK (kinase responsive to NO/H-NOX) mutants result in wild-type biofilm architecture. We demonstrate that NosP regulates the autophosphorylation activity of NahK as well as HnoK. HnoK and NahK have been shown to regulate three response regulators (HnoB, HnoC, and HnoD) that together comprise a NO-responsive multicomponent c-di-GMP signaling network. Here, we propose that NosP/NahK adds regulation on top of H-NOX/HnoK to modulate this c-di-GMP signaling network, and ultimately biofilm formation, by governing the flux of phosphate through both HnoK and NahK. In addition, it appears that NosP and H-NOX act to counter each other in a push-pull mechanism; NosP/NahK promotes biofilm formation through inhibition of H-NOX/HnoK signaling, which itself reduces the extent of biofilm formation. Addition of NO results in a reduction of c-di-GMP and biofilm formation, primarily through disinhibition of HnoK activity.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/análogos & derivados , Óxido Nítrico/metabolismo , Shewanella/fisiologia , Proteínas de Bactérias/genética , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Shewanella/genética , Transdução de Sinais
3.
Biochemistry ; 57(43): 6187-6200, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30272959

RESUMO

A novel family of bacterial hemoproteins named NosP has been discovered recently; its members are proposed to function as nitric oxide (NO) responsive proteins involved in bacterial group behaviors such as quorum sensing and biofilm growth and dispersal. Currently, little is known about molecular activation mechanisms in NosP. Here, functional studies were performed utilizing the distinct spectroscopic characteristics associated with the NosP heme cofactor. NosPs from Pseudomonas aeruginosa ( Pa), Vibrio cholerae ( Vc), and Legionella pneumophila ( Lpg) were studied in their ferrous unligated forms as well as their ferrous CO, ferrous NO, and ferric CN adducts. The resonance Raman (rR) data collected on the ferric forms strongly support the existence of a distorted heme cofactor, which is a common feature in NO sensors. The ferrous spectra exhibit a 213 cm-1 feature, which is assigned to the Fe-Nhis stretching mode. The Fe-C and C-O frequencies in the spectra of ferrous CO NosP complexes are inversely correlated with relatively similar frequencies, consistent with a proximal histidine ligand and a relatively hydrophobic environment. The rR spectra obtained for isotopically labeled ferrous NO adducts provide evidence of formation of a 5-coordinate NO complex, resulting from proximal Fe-Nhis cleavage, which is believed to play a role in biological heme-NO signal transduction. Additionally, we found that of the three NosPs studied, Lpg NosP contains the most electropositive ligand binding pocket, while Pa NosP has the most electronegative ligand binding pocket. This pattern is also observed in the measured heme reduction potentials for these three proteins, which may indicate distinct functions for each.


Assuntos
Hemeproteínas/química , Hemeproteínas/metabolismo , Ferro/metabolismo , Legionella pneumophila/enzimologia , Óxido Nítrico/metabolismo , Pseudomonas aeruginosa/enzimologia , Vibrio cholerae/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Percepção de Quorum
4.
Radiat Res ; 202(3): 541-551, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39034036

RESUMO

After a large-scale radiological or nuclear event, hundreds of thousands of people may be exposed to ionizing radiation and require subsequent medical management. Acute exposure to moderate doses (2-6 Gy) of radiation can lead to the hematopoietic acute radiation syndrome, in which the bone marrow (BM) is severely compromised, and severe hemorrhage and infection are common. Previously, we have developed a panel of intracellular protein markers (FDXR, ACTN1, DDB2, BAX, p53 and TSPYL2), designed to reconstruct absorbed radiation dose from human peripheral blood (PB) leukocyte samples in humanized mice up to 3 days after exposure. The objective of this work was to continue to use the humanized mouse model to evaluate biomarker dose-/time- kinetics in human PB leukocytes in vivo, at an earlier (day 2) and later (day 7) time point, after exposure to total-body irradiation (TBI) doses of 0 to 2 Gy of X rays. In addition, to assess hematological sensitivity and radiation-induced injury, PB leukocyte cell counts, human BM hematopoietic stem cell (HSC) and progenitor cell [multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte myeloid progenitor (GMP), megakaryocyte/erythrocyte progenitor (MEP) and multi-lymphoid progenitor (MLP)] levels were measured, and their correlation was also examined as the BM damages are difficult to assess by routine tests. Peripheral blood B-cells were significantly lower after TBI doses of 0.5 Gy on day 2 and 2 Gy on days 2 and 7; T-cells were significantly reduced only on day 2 after 2 Gy TBI. Bone marrow HSCs and MPP cells showed a dose-dependent depletion after irradiation with 0.5 Gy and 2 Gy on day 2, and after 1 Gy and 2 Gy on day 7. Circulating B cells correlated with HSCs, MPP and MLP cells on day 2, whereas T cells correlated with MPP, and myeloid cells correlated with MLP cells. On day 7, B cells correlated with MPP, CMP, GMP and MEP, while myeloid cells correlated with CMP, GMP and MEP. The intracellular leukocyte biomarkers were able to discriminate unirradiated and irradiated samples at different time points calculated by receiver operating characteristic (ROC) curve. Using machine learning algorithm methods, combining ACTN1, p53, TSPYL2 and PB-T cell and PB-B cell counts served as a strong predictor (area under the ROC >0.8) to distinguish unirradiated and irradiated samples independent of the days after TBI. The results further validated our biomarker-based triage assay and additionally evaluated the radiation sensitivity of the hematopoietic system after TBI exposures.


Assuntos
Biomarcadores , Animais , Camundongos , Humanos , Biomarcadores/sangue , Radiometria/métodos , Irradiação Corporal Total/efeitos adversos , Células-Tronco Hematopoéticas/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Relação Dose-Resposta à Radiação , Modelos Animais de Doenças
5.
PLoS One ; 18(8): e0289634, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37561730

RESUMO

In the event of a widespread radiological incident, thousands of people may be exposed to a wide range of ionizing radiation. In this unfortunate scenario, there will be a need to quickly screen a large number of people to assess the amount of radiation exposure and triage for medical treatment. In our earlier work, we previously identified and validated a panel of radiosensitive protein biomarkers in blood leukocytes, using the humanized-mouse and non-human primate (NHP) models. The objective of this work was to develop a high-throughput imaging flow-cytometry (IFC) based assay for the rapid measurement of protein biomarker expression in human peripheral blood samples irradiated ex vivo. In this assay design, peripheral human blood samples from healthy adult donors were exposed to 0-5 Gy X-irradiation ex vivo and cultured for up to 2 days. Samples were stained with a cocktail of surface antigens (CD66b, CD20, and CD3), fixed and permeabilized, and intracellularly stained for BAX (Bcl-2-associated X) protein, used here as a representative biomarker. Samples were interrogated by IFC, and a uniform analysis template was created to measure biomarker expression in heterogeneous and specific leukocyte subtype populations at each time point. In this human blood ex vivo model, we show that within gated populations of leukocyte subtypes, B-cells are highly radiosensitive with the smallest surviving fraction, followed by T-cells and granulocytes. Dose-dependent biomarker responses were measured in the lymphocytes, B-, and T-cell populations, but not in the granulocytes, with dose-response curves showing increasing fold changes in BAX protein expression up to Day 2 in lymphocyte populations. We present here the successful use of this ex vivo model for the development of radiation dose-response curves of a candidate protein biomarker towards future applications of dose reconstruction and biodosimetry.


Assuntos
Linfócitos , Primatas , Adulto , Humanos , Animais , Camundongos , Relação Dose-Resposta à Radiação , Doses de Radiação , Linfócitos/metabolismo , Biomarcadores/metabolismo , Radiometria/métodos
6.
Sci Rep ; 13(1): 949, 2023 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-36653416

RESUMO

During a large-scale radiological event such as an improvised nuclear device detonation, many survivors will be shielded from radiation by environmental objects, and experience only partial-body irradiation (PBI), which has different consequences, compared with total-body irradiation (TBI). In this study, we tested the hypothesis that applying machine learning to a combination of radiation-responsive biomarkers (ACTN1, DDB2, FDXR) and B and T cell counts will quantify and distinguish between PBI and TBI exposures. Adult C57BL/6 mice of both sexes were exposed to 0, 2.0-2.5 or 5.0 Gy of half-body PBI or TBI. The random forest (RF) algorithm trained on ½ of the data reconstructed the radiation dose on the remaining testing portion of the data with mean absolute error of 0.749 Gy and reconstructed the product of dose and exposure status (defined as 1.0 × Dose for TBI and 0.5 × Dose for PBI) with MAE of 0.472 Gy. Among irradiated samples, PBI could be distinguished from TBI: ROC curve AUC = 0.944 (95% CI: 0.844-1.0). Mouse sex did not significantly affect dose reconstruction. These results support the hypothesis that combinations of protein biomarkers and blood cell counts can complement existing methods for biodosimetry of PBI and TBI exposures.


Assuntos
Exposição à Radiação , Irradiação Corporal Total , Masculino , Feminino , Camundongos , Animais , Camundongos Endogâmicos C57BL , Biomarcadores , Irradiação Corporal Total/efeitos adversos , Contagem de Células Sanguíneas , Exposição à Radiação/efeitos adversos , Relação Dose-Resposta à Radiação , Doses de Radiação
7.
Sci Rep ; 10(1): 12716, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728041

RESUMO

Following a large-scale radiological incident, there is a need for FDA-approved biodosimetry devices and biomarkers with the ability to rapidly determine past radiation exposure with sufficient accuracy for early population triage and medical management. Towards this goal, we have developed FAST-DOSE (Fluorescent Automated Screening Tool for Dosimetry), an immunofluorescent, biomarker-based system designed to reconstruct absorbed radiation dose in peripheral blood samples collected from potentially exposed individuals. The objective of this study was to examine the performance of the FAST-DOSE assay system to quantify intracellular protein changes in blood leukocytes for early biodosimetry triage from humanized NOD-scid-gamma (Hu-NSG) mice and non-human primates (NHPs) exposed to ionizing radiation up to 8 days after radiation exposure. In the Hu-NSG mice studies, the FAST-DOSE biomarker panel was able to generate delivered dose estimates at days 1, 2 and 3 post exposure, whereas in the NHP studies, the biomarker panel was able to successfully classify samples by dose categories below or above 2 Gy up to 8 days after total body exposure. These results suggest that the FAST-DOSE bioassay has large potential as a useful diagnostic tool for rapid and reliable screening of potentially exposed individuals to aid early triage decisions within the first week post-exposure.


Assuntos
Leucócitos Mononucleares/química , Exposição à Radiação/análise , Radiometria/métodos , Irradiação Corporal Total/métodos , Animais , Linhagem Celular , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Camundongos , Camundongos SCID , Modelos Animais , Primatas , Doses de Radiação
8.
Antioxid Redox Signal ; 29(18): 1872-1887, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-28847157

RESUMO

SIGNIFICANCE: The molecule nitric oxide (NO) has been shown to regulate behaviors in bacteria, including biofilm formation. NO detection and signaling in bacteria is typically mediated by hemoproteins such as the bis-(3',5')-cyclic dimeric adenosine monophosphate-specific phosphodiesterase YybT, the transcriptional regulator dissimilative nitrate respiration regulator, or heme-NO/oxygen binding (H-NOX) domains. H-NOX domains are well-characterized primary NO sensors that are capable of detecting nanomolar NO and influencing downstream signal transduction in many bacterial species. However, many bacteria, including the human pathogen Pseudomonas aeruginosa, respond to nanomolar concentrations of NO but do not contain an annotated H-NOX domain, indicating the existence of an additional nanomolar NO-sensing protein (NosP). Recent Advances: A newly discovered bacterial hemoprotein called NosP may also act as a primary NO sensor in bacteria, in addition to, or in place of, H-NOX. NosP was first described as a regulator of a histidine kinase signal transduction pathway that is involved in biofilm formation in P. aeruginosa. CRITICAL ISSUES: The molecular details of NO signaling in bacteria are still poorly understood. There are still many bacteria that are NO responsive but do encode either H-NOX or NosP domains in their genomes. Even among bacteria that encode H-NOX or NosP, many questions remain. FUTURE DIRECTIONS: The molecular mechanisms of NO regulation in many bacteria remain to be established. Future studies are required to gain knowledge about the mechanism of NosP signaling. Advancements on structural and molecular understanding of heme-based sensors in bacteria could lead to strategies to alleviate or control bacterial biofilm formation or persistent biofilm-related infections.


Assuntos
Bactérias/metabolismo , Heme/metabolismo , Óxido Nítrico/metabolismo
9.
Adv Microb Physiol ; 70: 1-36, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28528645

RESUMO

Low concentrations of nitric oxide (NO) modulate varied behaviours in bacteria including biofilm dispersal and quorum sensing-dependent light production. H-NOX (haem-nitric oxide/oxygen binding) is a haem-bound protein domain that has been shown to be involved in mediating these bacterial responses to NO in several organisms. However, many bacteria that respond to nanomolar concentrations of NO do not contain an annotated H-NOX domain. Nitric oxide sensing protein (NosP), a newly discovered bacterial NO-sensing haemoprotein, may fill this role. The focus of this review is to discuss structure, ligand binding, and activation of H-NOX proteins, as well as to discuss the early evidence for NO sensing and regulation by NosP domains. Further, these findings are connected to the regulation of bacterial biofilm phenotypes and symbiotic relationships.


Assuntos
Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Óxido Nítrico/metabolismo , Bactérias/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Heme/química , Hemeproteínas/química , Percepção de Quorum/fisiologia , Transdução de Sinais/fisiologia
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