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1.
Artículo en Inglés | MEDLINE | ID: mdl-38421741

RESUMEN

OBJECTIVES: The research examined the motivation to integrate and perceived discrimination as antecedents of cultural identity styles, the cognitive and behavioral strategies that bicultural individuals use for decision making in managing and maintaining their ethnic and national identities. Two major cultural identity styles have been distinguished: the alternating identity style (AIS, changing cultural identities depending on the circumstances) and the hybrid identity style (HIS, blending selected aspects of these identities in a unique way). Based on earlier cross-sectional research, we tested the hypotheses that the motivation to integrate would predict greater use of both styles and that perceived discrimination would predict greater use of the AIS, but not the HIS, over time. METHOD: A community sample of 493 Chinese Americans (56% female, 51.5% first generation, Mage = 53.27 years) completed an online survey at two points in time with approximately a 1-month interval. Path modeling controlling for demographic factors (age, generation) and cultural identity style (AIS and HIS at T1) was used to test our hypotheses. RESULTS: Analyses revealed that younger Chinese Americans made greater use of the AIS and that both the motivation to integrate and perceived discrimination were significant predictors of the AIS at T2. In contrast, only the motivation to integrate predicted the HIS at T2, confirming our hypotheses. CONCLUSIONS: The results demonstrate that both personal and situational factors affect the management of cultural identities. The findings are discussed in relation to research on acculturation and integration and theories of social and situated identity identities. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

2.
Artículo en Inglés | MEDLINE | ID: mdl-37917482

RESUMEN

OBJECTIVE: The present study identified unique profiles of cultural stressors (i.e., bicultural stress, discrimination, and negative context of reception) and acculturative strategies (i.e., heritage practices, heritage identification, U.S. practices, and U.S. identification), in Hispanic/Latinx (HL) emerging adults. Additionally, we examined associations between positive and negative psychosocial functioning, with profiles of acculturative strategies and cultural stressors. METHOD: The present study utilized a baseline sample of 779 HL college students (75.8% female, Mage = 20.80 years, SD = 2.66) drawn from a daily diary study on acculturation. RESULTS: Latent profile analysis identified four distinct profiles. The Bicultural and Low Cultural Stressors (B-LowCS; 53.55%) was marked by strong heritage and U.S. cultural orientation and low levels across all cultural stressors. The Marginalization and High Acculturative Stressors (M-HighAS; 20.13%) was marked by weak heritage and U.S. cultural orientation, high acculturative stressors, and low discrimination. The third profile, the Heritage Rejection and Low Cultural Stressors (HR-LowCS; 16.05%) was marked by rejection of heritage culture and low cultural stressors. Finally, the Separation and High Cultural Stressors (S-HighCS; 10.26%) was marked by weak U.S. cultural orientation and high cultural stressors. Consistent with past research, the B-LowCS profile was marked by the highest level of positive psychosocial functioning and the lowest levels of internalizing and externalizing symptoms. CONCLUSIONS: The results of the present study highlight the usefulness of person-centered approaches for understanding the interplay between acculturative strategies and cultural stressors, and the implications of these distinct profiles on psychosocial functioning in HL emerging adults. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

3.
Int J Mol Sci ; 24(4)2023 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-36835524

RESUMEN

Migraine and neuropathic pain (NP) are both painful, disabling, chronic conditions which exhibit some symptom similarities and are thus considered to share a common etiology. The calcitonin gene-related peptide (CGRP) has gained credit as a target for migraine management; nevertheless, the efficacy and the applicability of CGRP modifiers warrant the search for more effective therapeutic targets for pain management. This scoping review focuses on human studies of common pathogenic factors in migraine and NP, with reference to available preclinical evidence to explore potential novel therapeutic targets. CGRP inhibitors and monoclonal antibodies alleviate inflammation in the meninges; targeting transient receptor potential (TRP) ion channels may help prevent the release of nociceptive substances, and modifying the endocannabinoid system may open a path toward discovery of novel analgesics. There may exist a potential target in the tryptophan-kynurenine (KYN) metabolic system, which is closely linked to glutamate-induced hyperexcitability; alleviating neuroinflammation may complement a pain-relieving armamentarium, and modifying microglial excitation, which is observed in both conditions, may be a possible approach. Those are several potential analgesic targets which deserve to be explored in search of novel analgesics; however, much evidence remains missing. This review highlights the need for more studies on CGRP modifiers for subtypes, the discovery of TRP and endocannabinoid modulators, knowledge of the status of KYN metabolites, the consensus on cytokines and sampling, and biomarkers for microglial function, in search of innovative pain management methods for migraine and NP.


Asunto(s)
Trastornos Migrañosos , Neuralgia , Canales de Potencial de Receptor Transitorio , Humanos , Péptido Relacionado con Gen de Calcitonina/metabolismo , Endocannabinoides , Trastornos Migrañosos/metabolismo , Neuralgia/tratamiento farmacológico , Analgésicos/uso terapéutico , Canales de Potencial de Receptor Transitorio/metabolismo
4.
Int J Mol Sci ; 24(21)2023 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-37958722

RESUMEN

Revealing the underlying pathomechanisms of neurological and psychiatric disorders, searching for new biomarkers, and developing novel therapeutics all require translational research [...].


Asunto(s)
Trastornos Mentales , Investigación Biomédica Traslacional , Humanos , Trastornos Mentales/terapia , Trastornos Mentales/psicología , Biomarcadores , Ciencia Traslacional Biomédica
5.
J Neural Transm (Vienna) ; 129(5-6): 627-642, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35624406

RESUMEN

Following introduction of the monoamine oxidase type B inhibitor selegiline for the treatment of Parkinson's disease (PD), discovery of the action mechanism of Alzheimer's disease-modifying agent memantine, the role of iron in PD, and the loss of electron transport chain complex I in PD, and development of the concept of clinical neuroprotection, Peter Riederer launched one of the most challenging research project neurodevelopmental aspects of neuropsychiatric disorders. The neurodevelopmental theory holds that a disruption of normal brain development in utero or during early life underlies the subsequent emergence of neuropsychiatric symptoms during later life. Indeed, the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition and the International Classification of Diseases, 11th Revision categorize autism spectrum disorder and attention deficit hyperactivity disorder in neurodevelopmental disorders (NDDs). More and more evidence, especially from preclinical studies, is revealing that neurodevelopmental pathology is not limited to the diagnostic class above, but also contributes to the development of other psychiatric disorders such as schizophrenia, bipolar disorder, and obsessive-compulsive disorder as well as neurodegenerative diseases such as PD and Huntington's disease. Preclinical animal research is taking a lead in understanding the pathomechanisms of NDDs, searching for novel targets, and developing new neuroprotective agents against NDDs. This narrative review discusses emerging evidence of the neurodevelopmental etiology of neuropsychiatric disorders, recent advances in modelling neurodevelopmental pathogenesis, potential strategies of clinical neuroprotection using novel kynurenine metabolites and analogues, and future research direction for NDDs.


Asunto(s)
Trastorno del Espectro Autista , Trastornos del Neurodesarrollo , Fármacos Neuroprotectores , Animales , Trastorno del Espectro Autista/tratamiento farmacológico , Humanos , Quinurenina , Trastornos del Neurodesarrollo/etiología , Trastornos del Neurodesarrollo/prevención & control , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Selegilina
6.
Qual Life Res ; 30(8): 2161-2170, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-33843014

RESUMEN

PURPOSE: Maintaining or improving quality of life (QoL) in later life has become a major policy objective. Yet we currently know little about how QoL develops at older ages. The few studies that have modelled QoL change across time for older adults have used 'averaged' trajectories. However, this ignores the variations in the way QoL develops between groups of older adults. METHODS: We took a theoretically informed 'capabilities approach' to measuring QoL. We used four waves of data, covering 6 years, from the New Zealand Health, Work and Retirement Study (NZHWR) (N = 3223) to explore whether distinct QoL trajectories existed. NZHWR is a nationally representative longitudinal study of community-dwelling adults aged 50 + in New Zealand. Growth mixture modelling was applied to identify trajectories over time and multinomial regressions were calculated to test baseline differences in demographic variables (including age, gender, ethnicity, education and economic living standards). RESULTS: We found five QoL trajectories: (1) high and stable (51.94%); (2) average and declining (22.74%); (3) low and increasing (9.62%); (4) low and declining (10.61%); (5) low and stable (5.09%). Several differences across profiles in baseline demographic factors were identified, with economic living standards differentiating between all profiles. CONCLUSIONS: The trajectory profiles demonstrate that both maintaining and even improving QoL in later life is possible. This has implications for our capacity to develop nuanced policies for diverse groups of older adults.


Asunto(s)
Calidad de Vida/psicología , Jubilación , Anciano , Envejecimiento , Humanos , Estudios Longitudinales , Persona de Mediana Edad , Nueva Zelanda , Jubilación/psicología , Factores Socioeconómicos
7.
J Clin Psychol ; 77(1): 121-144, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32632991

RESUMEN

OBJECTIVE: The present study introduces a daily, micro-level perspective on acculturation using a sample of Hispanic college students in Miami. METHODS: We conducted a 12-day diary study with a sample of first- and second-generation Hispanic college students in Miami. Outcome variables were measured on Days 1 and 12, and acculturation components (practices, identities, and values) were measured on Days 2-11. Daily fluctuations in acculturation components between Days 2 and 11 were examined as predictors of well-being, internalizing symptoms, and externalizing problems on Day 12. RESULTS: Fluctuations in comfort with speaking English negatively predicted three of the four well-being outcomes and positively predicted all of the internalizing and externalizing indicators. Fluctuations in collectivist values predicted two of the well-being outcomes and both anxiety and depressive symptoms, and fluctuations in ethnic identity predicted anxiety and depressive symptoms. CONCLUSION: Daily volatility in comfort with English, collectivist values, and ethnic identity appear to be most distressing.


Asunto(s)
Aculturación , Hispánicos o Latinos , Ansiedad , Etnicidad , Humanos , Estudiantes
8.
Int J Psychol ; 55(3): 465-471, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31148148

RESUMEN

Hybrid and alternating identity styles are dynamic strategies that members of immigrant and ethnic minority groups use to maintain multiple cultural identities. Although research shows that the two strategies predict different outcomes for cultural identity development and psychological well-being, less is known about their antecedents. The present study investigated the temporal relationship between intercultural abilities (i.e. intercultural effectiveness) and the activation of hybrid and alternating identity styles in a community sample of Filipino and Indian New Zealanders. Cross-lagged analysis indicated that intercultural abilities positively predicted the hybrid identity style and negatively predicted the alternating identity style. Cultural identity styles were not predictive of intercultural abilities over time. Multigroup analysis indicated equivalence of regression paths across ethnic groups. Findings suggest that intercultural abilities function as an antecedent of cultural identity styles.


Asunto(s)
Etnicidad/psicología , Adulto , Comparación Transcultural , Femenino , Humanos , Estudios Longitudinales , Masculino
9.
Anal Chem ; 91(9): 6378-6382, 2019 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-30993981

RESUMEN

Although Förster resonance energy transfer (FRET) is one of the most widely used biophysical methods in biology, the effect of high excitation intensity, leading to donor and acceptor saturation, has not been addressed previously. Here, we present a formalism for the experimental determination of the FRET efficiency at high excitation intensity when saturation of both the donor and the acceptor significantly affect conventional FRET calculations. We show that the proposed methodology significantly reduces the dependence of the FRET efficiency on excitation intensity, which otherwise significantly distorts FRET calculations at high excitation intensities commonly used in experiments. The work presented here adds additional rigor to the FRET-based investigation of protein interactions and strengthens the device independence of such results.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Receptor ErbB-2/aislamiento & purificación , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/farmacología , Línea Celular Tumoral , Humanos , Receptor ErbB-2/agonistas , Receptor ErbB-2/química , Trastuzumab/química , Trastuzumab/farmacología
10.
Age Ageing ; 48(2): 267-272, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30379990

RESUMEN

OBJECTIVES: the impact of retirement on physical health is an important focus of ageing research; however, research findings vary greatly. To investigate under what conditions retirement might benefit health, we examined physical functioning 8 years pre- and post-retirement. METHODS: using longitudinal data from the New Zealand Health, Work and Retirement Study, multiple linear trajectories of physical functioning were estimated. Growth mixture analysis indicated three distinct trajectory profiles. RESULTS: Profile 1 displayed good but declining physical functioning from 8 years pre-retirement until retirement, which continued to decline more slowly post-retirement. Profile 2 was characterised by poor and declining physical functioning pre-retirement that improved post-retirement. Profile 3 displayed good and stable physical functioning pre-retirement and a slow decline post-retirement. Significant differences were identified across profiles in smoking behaviour, pre-existing chronic conditions, marital status and educational level. Profile 2 also showed increased economic living standards post-retirement. DISCUSSION: findings indicate that retirement can be beneficial for those with poor health and limited resources. For the wealthy and healthy, retirement does not necessarily advantage health. Universal superannuation initiatives may partly address inequalities experienced by older persons in poor health and socio-economic circumstances prior to retirement.


Asunto(s)
Estado de Salud , Jubilación/estadística & datos numéricos , Actividades Cotidianas , Anciano , Anciano de 80 o más Años , Enfermedad Crónica/epidemiología , Escolaridad , Femenino , Humanos , Estudios Longitudinales , Masculino , Estado Civil , Persona de Mediana Edad , Nueva Zelanda/epidemiología , Aptitud Física , Fumar/epidemiología
11.
J Appl Dev Psychol ; 62: 26-37, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-38322153

RESUMEN

We examined two conceptualizations of bicultural identity - the Bicultural Identity Integration (BII) framework (cultural identity blendedness-distance and harmony-conflict) and cultural hybridizing and alternating (mixing one's two cultural identities and/or switching between them). Utilizing data from a 12-day diary study with 873 Hispanic college students, we examined three research questions: (1) cross-sectional and longitudinal inter-correlations among these biculturalism components, (2) links among daily variability in these biculturalism components, and (3) how this daily variability predicts well-being and mental health outcomes over time. Bicultural hybridizing was positively related to, and longitudinally predicted by, both BII blendedness and harmony. Daily fluctuation scores for BII blendedness, BII harmony, and bicultural hybridizing were strongly interrelated. Well-being was negatively predicted by fluctuations in hybridizing, whereas internalizing symptoms were positively predicted by fluctuations in blendedness. These results are discussed in terms of what biculturalism is and how best to promote it.

12.
Biophys J ; 114(3): 688-700, 2018 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-29414714

RESUMEN

Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Carbocianinas/metabolismo , Fluorescencia , Compuestos de Quinolinio/metabolismo , Receptor ErbB-2/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Unión Competitiva , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Carbocianinas/química , Femenino , Polarización de Fluorescencia , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Compuestos de Quinolinio/química , Receptor ErbB-2/inmunología , Espectrometría de Fluorescencia , Células Tumorales Cultivadas
13.
Cytometry A ; 93(11): 1106-1117, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30378727

RESUMEN

The heterodimeric receptor complex of IL-9 consists of the cytokine-specific α-subunit and the common γc -chain shared with other cytokines, including IL-2, a central regulator of T cell function. We have shown previously the bipartite spatial relationship of IL-9 and IL-2 receptors at the surface of human T lymphoma cells: in addition to common clusters, expression of the two receptor kinds could also be observed in segregated membrane areas. Here we analyzed further the mutual cell surface organization of IL-9 and IL-2 receptors. Complementing Pearson correlation data with co-occurrence analysis of confocal microscopic images revealed that a minimum degree of IL-9R/IL-2R co-localization exists at the cell surface regardless of the overall spatial correlation of the two receptor kinds. Moreover, our FRET experiments demonstrated molecular scale assemblies of the elements of the IL-9/IL-2R system. Binding of IL-9 altered the structure and/or composition of these clusters. It is hypothesized, that by sequestering receptor subunits in common membrane areas, the overlapping domains of IL-9R and IL-2R provide a platform enabling both the formation of the appropriate receptor complex as well as subunit sharing between related cytokines. © 2018 International Society for Advancement of Cytometry.


Asunto(s)
Linfoma/inmunología , Receptores de Interleucina-2/inmunología , Receptores de Interleucina-9/inmunología , Linfocitos T/inmunología , Línea Celular , Humanos , Transducción de Señal/inmunología
14.
Orv Hetil ; 158(49): 1953-1959, 2017 Dec.
Artículo en Húngaro | MEDLINE | ID: mdl-29199437

RESUMEN

INTRODUCTION: The currently licensed seasonal influenza vaccines contain split, subunit or whole virions, typically in amounts of 15 µg hemagglutinin per virus strain for adult and up to 60 µg in elderly patients. AIM: The present study reports safety data of the newly licensed, reduced dose vaccine with 6 µg of hemagglutinin per strain produced by Fluart (Hungary) after its first season on the market. The main objective of enhanced safety surveillance was to detect a potential increase in reactogenicity and allergic events that is intrinsic to the product in near real-time in the earliest vaccinated cohorts. METHOD: The study methods were based on the Interim guidance on enhanced safety surveillance for seasonal influenza vaccines in the EU by the European Medicines Agency. STATISTICS: We used the Fisher exact test with 95% confidence intervals. RESULTS: We studied 587 patients and detected a total 24 adverse events, all of which have already been known during the licensing studies of the present vaccine. The frequencies of the adverse events were not different from what had been seen with the previously licensed 15 µg vaccine. CONCLUSIONS: Based on the results, the authors conclude that the new, reduced dose vaccine FluArt is safe and tolerable. Orv Hetil. 2017; 158(49): 1953-1959.


Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Vacunas contra la Influenza/administración & dosificación , Estudios de Cohortes , Glicoproteínas Hemaglutininas del Virus de la Influenza/efectos adversos , Humanos , Hungría , Vacunas contra la Influenza/efectos adversos , Vigilancia de Productos Comercializados/estadística & datos numéricos
16.
Cytometry A ; 89(4): 376-84, 2016 04.
Artículo en Inglés | MEDLINE | ID: mdl-27003481

RESUMEN

Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry.


Asunto(s)
Algoritmos , Transferencia Resonante de Energía de Fluorescencia , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Funciones de Verosimilitud , Fotones , Reproducibilidad de los Resultados
17.
Fogorv Sz ; 109(1): 29-33, 2016 Mar.
Artículo en Húngaro | MEDLINE | ID: mdl-27188159

RESUMEN

Stem cells are present in many tissues, such as dental pulp. Stem cells can be easily isolated from dental pulp because third molars are often removed from patients. Stem cells could be separated from the tissue derived heterogeneous cell population. There are two main methods to separate a cell type from the other ones: the fluorescence activated cell sorting (FACS) and the magnetic activated cell sorting (MACS). The aim of this study was to compare these methods' effect on cell surviving and population growth after sorting on dental pulp cells. The anti-STRO-1 antibody was used as primary antibody to specifically label stem cells. Two secondary antibodies were used: magnetic or fluorescent labelled. We sorted the cells by MACS or by FACS or by combination of both (MACS-FACS). Our results show that the effectivity of MACS and FACS sorting are comparable while of MACS-FACS was significantly higher (MACS 79.53 ± 5.78%, FACS 88.27 ± 3.70%, MACS-FACS 98.43 ± 0.67%). The cell surviving and the post-sorting population growth, on the contrary, are very different. The cell population is growing on first week after MACS but after FACS did not. Moreover, after MACS-FACS, on first week the cell number of population decreased. Taken together, our results suggest to use MACS instead of FACS, at least in case of sorting dental pulp stem cells with anti-STRO-1 antibody.


Asunto(s)
Separación Celular/métodos , Magnetismo , Células Madre , Antígenos de Superficie , Citometría de Flujo , Humanos , Coloración y Etiquetado
19.
Int J Med Microbiol ; 304(3-4): 476-83, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24631212

RESUMEN

Chlamydia muridarum carries a cryptic plasmid (pMoPn) of 7.5kb, which encodes seven genes. Our aims were to describe the transcriptional pattern of the pMoPn genes in C. muridarum-infected mice and to evaluate the host immune responses against pGP3 and pGP4 proteins. BALB/c and C57BL/6N female mice were inoculated intranasally with C. muridarum and sacrificed at different time points, and the total RNA was extracted from the lung suspensions to determine the levels of expression of the different plasmid genes by RT qPCR. The supernatants of the lungs were subjected to the quantitation of recoverable C. muridarum. TCA04 and TCA05, which encode pGP3 and pGP4, respectively, were amplified by PCR and cloned into the pET vector. The proteins were overexpressed in E. coli HB101 and purified. Selected groups of BALB/c and C57BL/6N mice were infected with C. muridarum 1-3 times. The humoral immune responses in the sera of the mice to the proteins encoded by TCA04 and TCA05 were tested by Western blotting, and the cellular immune responses were assessed in lymphocyte proliferation assays. The proteins recognized by the mouse sera were further analysed by a LC/MSMS technique. The kinetics of C. muridarum growth were similar in the mouse strains used, but the pathogen burden was higher in the BALB/c mice in the late phase of infection. All the plasmid genes in the BALB/c mice showed an increased level of expression on day 7, whereas the expression of the same genes did not change on day 7 in the C57BL/6N mice. The levels of expression of the plasmid genes were higher in the C57BL/6N mice at later time points. In Western blot assays, the sera of the singly infected C57BL/6N mice reacted with the monomeric form of pGP3, whereas the sera of the singly infected BALB/c mice reacted with the trimeric form of pGP3. The sera of the multiply infected C57BL/6N mice also recognized pGP4. Similarly to the humoral immune response, cellular immune responses to pGP3 and pGP4 were detected in the C. muridarum-infected C57BL/6N mice, but the spleen cells of BALB/c mice responded with proliferation only to the pGP3 protein. These results suggest that the proteins encoded by pMoPn genes may modulate the host immune response during C. muridarum infection, and that the evolved immune response against plasmid proteins, similarly to that against other chlamydial proteins, depends on the genetic background of the host.


Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Chlamydia muridarum/inmunología , Plásmidos , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/biosíntesis , Proteínas Bacterianas/biosíntesis , Western Blotting , Proliferación Celular , Chlamydia muridarum/genética , Cromatografía Liquida , Clonación Molecular , Escherichia coli/genética , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Leucocitos Mononucleares/inmunología , Pulmón/microbiología , Espectrometría de Masas , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
20.
Cytometry A ; 85(11): 942-52, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25123296

RESUMEN

Ratiometric determination of the efficiency of fluorescence or Förster resonance energy transfer (FRET) is one of the most widespread methods for the characterization of protein clustering and conformation. Low photon numbers, often present in pixel-by-pixel determination of FRET efficiency in digital microscopy, result in large uncertainties in the derived FRET parameter. Here, we propose a method based on maximum likelihood estimation (MLE) of FRET efficiency using photon counting detectors to overcome this limitation. Intensities measured in the donor, FRET, and acceptor channels were all assumed to follow Poisson statistics as a result of detector shot noise. The joint probability of photon numbers detected in the donor, FRET, and acceptor channels was derived using an equation describing the relationship between the three measured intensities. The FRET efficiency generating the measured photon numbers with the largest likelihood was determined iteratively providing a single FRET value for all pixels in the calculation. Since as few as 100 pixels are sufficient to provide a maximum likelihood estimate for FRET, biological variability in FRET values can be revealed by performing the analysis for regions of interests in an image. Since the algorithm provides the probability of a combination of donor, FRET, and acceptor intensities observed in each individual pixel given a certain FRET efficiency, outlier pixels with low probabilities could be excluded from the analysis. Simulations carried out with low photon numbers in the presence and absence of outlier pixels revealed that the proposed approach can reliably and reproducibly estimate FRET efficiency. In addition, systematic evaluation of the simulation results showed that the distribution of pixel-by-pixel FRET efficiencies is skewed, and the mean of these FRET values is a biased and unreliable estimate of the FRET efficiency. In the absence of outlier pixels, FRET calculated from summed donor, FRET, and acceptor intensities proved to be as reliable as MLE. We conclude that MLE of FRET outperforms calculations using summed and pixel-by-pixel intensities in biologically relevant situations involving low photon numbers and outlier pixels. © 2014 International Society for Advancement of Cytometry.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Algoritmos , Línea Celular Tumoral , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Células HeLa , Humanos , Funciones de Verosimilitud , Microscopía Confocal/instrumentación , Microscopía Fluorescente/instrumentación , Distribución de Poisson
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