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1.
Nat Commun ; 10(1): 4185, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519914

RESUMO

Cell migration during the invasion-metastasis cascade requires cancer cells to navigate a spatially complex microenvironment that presents directional choices to migrating cells. Here, we investigate cellular energetics during migration decision-making in confined spaces. Theoretical and experimental data show that energetic costs for migration through confined spaces are mediated by a balance between cell and matrix compliance as well as the degree of spatial confinement to direct decision-making. Energetic costs, driven by the cellular work needed to generate force for matrix displacement, increase with increasing cell stiffness, matrix stiffness, and degree of spatial confinement, limiting migration. By assessing energetic costs between possible migration paths, we can predict the probability of migration choice. Our findings indicate that motility in confined spaces imposes high energetic demands on migrating cells, and cells migrate in the direction of least confinement to minimize energetic costs. Therefore, therapeutically targeting metabolism may limit cancer cell migration and metastasis.


Assuntos
Movimento Celular/fisiologia , Tomada de Decisões , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Engenharia Biomédica , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Glucose/metabolismo , Humanos , Microscopia de Força Atômica , Microscopia Confocal , Microscopia de Contraste de Fase , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
2.
Invest Ophthalmol Vis Sci ; 60(12): 4008-4020, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31560364

RESUMO

Purpose: The injection of cultured human corneal endothelial cells (cHCECs) into the anterior chamber (AC) is a newly developed modality for the successful treatment of corneal endothelium dysfunction. Here, we investigated whether or not cHCECs could be labeled using quantum dots (QDs) composed of semiconductor nanoparticle octa-arginine (R8) to trace injected cHCECs and examined the utility of in vivo fluorescence imaging to analyze the dynamics and accumulation of QD-labeled injected cHCECs in a corneal endothelial dysfunction mouse model. Methods: The cHCECs, either of high quality or with cell-state transition, were labeled by adding a mixture of QDs655 and R8. The labeling efficiency and the unchanging of the cell phenotypes by the labeling was confirmed by flow cytometry. The labeled cHCECs were injected into the AC of either healthy mice or mice with corneal endothelium damaged by cryogenic treatment. The kinetics of the injected cHCECs was traced quantitatively via multiphoton confocal laser microscopy. Results: QD labeling induced no morphologic change in the cHCECs or in the expression of the functional markers of cHCECs (i.e., Na+/K+-ATPase and zonula occludens-1). The injected cHCECs-QDs were quantitatively detected, and the retention of cHCECs-QDs was evident, from 3 to 48 hours post cell injection on the posterior surface in the cryogenically injured corneal endothelium mouse model eyes, yet not in the noninjured healthy control eyes. Conclusions: The findings of this study show that in the field of regenerative medicine, QD labeling of cells presents a convenient and sensitive method of finely monitoring the fate of injected cells in vivo.


Assuntos
Câmara Anterior/metabolismo , Epitélio Posterior/metabolismo , Pontos Quânticos , Adulto , Animais , Biomarcadores/metabolismo , Contagem de Células , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Injeções Intraoculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Medicina Regenerativa/métodos , ATPase Trocadora de Sódio-Potássio/metabolismo , Doadores de Tecidos , Adulto Jovem , Proteína da Zônula de Oclusão-1/metabolismo
4.
Microsc Res Tech ; 82(10): 1802-1809, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31313452

RESUMO

Mature spermatozoa traits and morphology of endangered Albanian water frog, Pelophylax shqipericus, have been characterized for the first time through phase contrast microscopy, as part of successful implementation of in vitro fertilization technique for this species. The basic morphology of P. shqipericus spermatozoa consists of an elongated, thick, smooth-edged, and solid-staining head, continuing with a thin and long tail which usually extends 2.48 times the head length. The acrosome was not clearly discernible so the measurements were done on the head as a whole, while the middle section was better visible. Average length of head, including the acrosome and midsection was estimated to be 11.78 µm ± 0.32, while the tail length resulted 29.24 ± 1.75 µm. The average thickness of the head was shown to be 3.45 µm. The total sperm length resulted to be 41.02 ± 1.83 µm. The average sperm concentration was estimated of 25.5 × 106 /ml. Sperm amount, survival rate and motility were also measured. The sperm survival rate was maximal immediately after preparation of the suspension and tended to decrease over time of storage, reaching 50% after 72 hr. Decreased sperm motility seemed to follow the same trend as sperm viability. Sperm traits resulted to be very similar both in size and in shape with those of "Lessonae" frog group, one of the lineages of Western Palearctic species complex, suggesting a strong phylogenetic relationship among these species.


Assuntos
Microscopia de Contraste de Fase , Ranidae , Espermatozoides/citologia , Espermatozoides/fisiologia , Animais , Biometria , Sobrevivência Celular , Locomoção , Masculino
5.
Appl Opt ; 58(12): 3104-3114, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-31044784

RESUMO

Modern microscopes are designed with functionalities that are tailored to enhance image contrast. Dark-field imaging, phase contrast, differential interference contrast, and other optical techniques enable biological cells and other phase-only objects to be visualized. Quantitative phase imaging refers to an emerging set of techniques that allow for the complex transmission function of the sample to be measured. With this quantitative phase image available, any optical technique can then be simulated; it is trivial to generate a phase contrast image or a differential interference contrast image. Rheinberg illumination, proposed almost a century ago, is an optical technique that applies color contrast to images of phase-only objects by introducing a type of optical staining via an amplitude filter placed in the illumination path that consists of two or more colors. In this paper, the complete theory of Rheinberg illumination is derived, from which an algorithm is proposed that can digitally simulate the technique. Results are shown for a number of quantitative phase images of diatom cells obtained via digital holographic microscopy. The results clearly demonstrate the potential of the technique for label-free color staining of subcellular features.


Assuntos
Diatomáceas/citologia , Holografia/métodos , Iluminação , Microscopia de Contraste de Fase/métodos , Coloração e Rotulagem/métodos , Algoritmos
6.
Cells ; 8(5)2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31108912

RESUMO

Cytokinesis D is known as the midwife mechanism in which neighboring cells facilitate cell division by crossing the cleavage furrow of dividing cells. Cytokinesis D is thought to be mediated by chemotaxis, where midwife cells migrate toward dividing cells by sensing an unknown chemoattractant secreted from the cleavage furrow. In this study, to validate this chemotaxis model, we aspirated the fluid from the vicinity of the cleavage furrow of a dividing Dictyostelium cell and discharged it onto a neighboring cell using a microcapillary. However, the neighboring cells did not show any chemotaxis toward the fluid. In addition, the cells did not manifest an increase in the levels of intracellular Ca2+, cAMP, or cGMP, which are expected to rise in chemotaxing cells. From several lines of our experiments, including these findings, we concluded that chemotaxis does not contribute to cytokinesis D. As an alternative, we propose a cortical-flow model, where a migrating cell attaches to a dividing cell by chance and is guided toward the furrow by the cortical flow on the dividing cell, and then physically assists the separation of the daughter cells.


Assuntos
Quimiotaxia/fisiologia , Citocinese/fisiologia , Dictyostelium/citologia , Dictyostelium/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Cálcio/metabolismo , Movimento Celular , Rastreamento de Células/métodos , Células Cultivadas , Fatores Quimiotáticos/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Interfase/fisiologia , Microscopia de Contraste de Fase , Mitose/fisiologia , Modelos Biológicos , Domínios de Homologia à Plecstrina/fisiologia
7.
Eur Radiol ; 29(10): 5378-5385, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30937586

RESUMO

OBJECTIVE: To investigate the incidence, CT appearance, and implication for prognosis of the hollow adrenal gland sign (HAGS). METHODS: A total of 194 patients with septic shock and 24 patients with hemorrhagic shock (as control group) were retrospectively included in this study and the patients with septic shock were further divided into four subgroups (digestive tract diseases, DTD, n = 49; biliary and pancreatic diseases, BPD, n = 41; postsurgical infection, PI, n = 64; and other diseases, OD, n = 40). All patients underwent a dual-phase contrast-enhanced CT within 1 week after diagnosis. CT findings and clinical records were reviewed. If in the arterial phase the central zone of adrenal gland showed temporally much lower attenuation than the peripheral zone, it was defined as HAGS positive. The incidence of the HAGS in patients with septic shock and hemorrhagic shock, the demographic features, and mortality between HAGS-positive and HAGS-negative patients in each group were respectively compared. RESULTS: The incidence of the HAGS in the septic shock group was nearly 30%, while it was 0 in the hemorrhagic shock group. There was no significant difference in age or gender between HAGS-positive and HAGS-negative patients in all groups, while the mortality of HAGS-positive patients was significantly higher than that of HAGS-negative patients in each group (p < 0.05). The concordance correlation coefficient value showed excellent reproducibility of the two observers (κ = 0.977). CONCLUSION: The HAGS is specific and common on dual-phase contrast-enhanced CT in patients with septic shock and predicts a poor prognosis. KEY POINTS: • The hollow adrenal gland sign (HAGS) newly described in this study is a special enhancing pattern of adrenal gland on dual-phase contrast-enhanced CT in patients with septic shock. • The HAGS is characterized by the much lower-attenuated central zone of the adrenal gland in arterial phase and it showed excellent reproducibility between different observers. • The HAGS is specific and common on dual-phase contrast-enhanced CT in patients with septic shock and predicts a poor prognosis.


Assuntos
Glândulas Suprarrenais/diagnóstico por imagem , Choque Séptico/diagnóstico por imagem , Adulto , Idoso , Meios de Contraste , Feminino , Humanos , Masculino , Microscopia de Contraste de Fase/métodos , Pessoa de Meia-Idade , Variações Dependentes do Observador , Prognóstico , Reprodutibilidade dos Testes , Estudos Retrospectivos , Choque Séptico/mortalidade , Tomografia Computadorizada por Raios X/métodos
8.
Nat Commun ; 10(1): 1877, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015402

RESUMO

Many microorganisms have evolved chemotactic strategies to exploit the microscale heterogeneity that frequently characterizes microbial habitats. Chemotaxis has been primarily studied as an average characteristic of a population, with little regard for variability among individuals. Here, we adopt a classic tool from animal ecology - the T-maze - and implement it at the microscale by using microfluidics to expose bacteria to a sequence of decisions, each consisting of migration up or down a chemical gradient. Single-cell observations of clonal Escherichia coli in the maze, coupled with a mathematical model, reveal that strong heterogeneity in the chemotactic sensitivity coefficient exists even within clonal populations of bacteria. A comparison of different potential sources of heterogeneity reveals that heterogeneity in the T-maze originates primarily from the chemotactic sensitivity coefficient, arising from a distribution of pathway gains. This heterogeneity may have a functional role, for example in the context of migratory bet-hedging strategies.


Assuntos
Quimiotaxia/genética , Escherichia coli/fisiologia , Modelos Biológicos , Fenótipo , Dimetilpolisiloxanos/química , Microscopia Intravital/métodos , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Contraste de Fase/métodos , Análise de Célula Única/métodos
9.
Methods Mol Biol ; 1981: 291-312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016662

RESUMO

Since HepaRG cells can differentiate into well-polarized mature hepatocyte-like cells that synthesize, conjugate, and secrete bile acids, they represent an appropriate surrogate to primary human hepatocytes for investigations on drug-induced cholestasis mechanisms. In this chapter, culture conditions for obtaining HepaRG hepatocytes and the main methods used to detect cholestatic potential of drugs are described. Assays for evaluation of bile canaliculi dynamics and morphology are mainly based on time-lapse and phase-contrast microscopy analysis. Bile acid uptake, trafficking, and efflux are investigated using fluorescent probes. Individual bile acids are quantified in both culture media and cell layers by high-pressure liquid chromatography/tandem mass spectrometry. Preferential cellular accumulation of toxic hydrophobic bile acids is easily evidenced when exogenous primary and secondary bile acids are added to the culture medium.


Assuntos
Colestase/metabolismo , Colestase/patologia , Hepatócitos/citologia , Linhagem Celular , Células Cultivadas , Humanos , Microscopia de Contraste de Fase
10.
Analyst ; 144(9): 3136-3143, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30941383

RESUMO

Nosema bombycis (Nb) is the pathogen that causes pebrine in silkworms. Aldehydes are effective disinfectants commonly used in sericulture. However, the precise mechanism of their action on Nb spores remains unclear. Here, we used laser tweezers Raman spectroscopy to investigate the effects of glutaraldehyde and formaldehyde on individual Nb spores, as well as phase contrast microscopy imaging to monitor the germination dynamics of individual treated spores, to acquire a deeper understanding of the mechanism of action of aldehydes and to provide a theoretical reference for establishing an effective strategy for disease control in sericulture. The positions of the Raman peaks remained constant during treatment. The Raman intensity was enhanced and the germination rate of the spores significantly decreased with treatment time. Tlag, the time when individual spores begin to germinate, and Tgerm, the time for complete germination, increased with enhanced treatment. The germination time (ΔTgerm) showed no significant difference from that for untreated spores. Heterogeneity was shown, which is relevant to the resistance of Nb spores to aldehydes. The results indicate that glutaraldehyde and formaldehyde do not destroy the spore wall and plasma membrane, do not cause the leakage of intracellular components, and might not damage the extrusion apparatus. The effects of aldehydes on Nb spores are mainly on the spore coat. They may block the external factors that stimulate spore germination. Single-cell analysis based on novel optical techniques reveals the action of chemical sporicides on microsporidia spores in real time and explains the heterogeneity of cell stress resistance. These applications of new techniques offer new insight into traditional disinfectants.


Assuntos
Desinfetantes/farmacologia , Formaldeído/farmacologia , Glutaral/farmacologia , Nosema/efeitos dos fármacos , Esporos Fúngicos/efeitos dos fármacos , Microscopia de Contraste de Fase/métodos , Pinças Ópticas , Análise de Célula Única/métodos , Análise Espectral Raman/métodos
11.
J Low Genit Tract Dis ; 23(2): 176-181, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30907778

RESUMO

OBJECTIVES: Wet mount microscopy (WMM) is an ancillary test in the diagnosis of vulvovaginitis. However, there are little data about the impact of the sampling site. Our main objective was to determine the agreement between samples collected from different vaginal sites. MATERIALS AND METHODS: Five vaginal samples (one from each of the fornices and one from the lower third) were collected from 50 consecutive women and evaluated using phase-contrast WMM, including the following: lactobacillary grade, presence of other bacteria, clue cells, inflammation, and atrophy. Global percentage of agreement, κ index [poor (<0), slight (0.01-0.20), fair (0.21-0.40), moderate (0.41-0.60), substantial (0.61-0.80), and almost perfect (0.81-1)], sensitivity and negative predictive value were calculated. RESULTS: Global percentage of agreement was high for all individual parameters (>75%), but low for the final diagnosis (57%). Agreement rate was substantial or almost perfect for the evaluation of normal versus abnormal flora, substantial for the presence of absence of bacteria other than lactobacilli and for the presence of clue cells, and moderate to almost perfect for the presence of dysbiosis and cytolysis. Agreement rates were worse for inflammation, atrophy, and the presence of Candida. The lower third of the vagina had the highest sensitivity for Candida (0.6, 95% CI = 0.41-0.86) and bacterial vaginosis (0.92, 95% CI = 0.73-1.00), whereas the anterior fornix performed better for cytolytic vaginosis (0.75, 95% CI = 0.43-0.93). CONCLUSIONS: There is some variation in the results according to the sampling site, which may be overcome by collecting 2 samples (anterior fornix and lower third of the vagina).


Assuntos
Microscopia de Contraste de Fase/métodos , Manejo de Espécimes/métodos , Vagina/patologia , Vulvovaginite/diagnóstico , Vulvovaginite/patologia , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Adulto Jovem
12.
Biotechniques ; 66(3): 128-133, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30869548

RESUMO

The need for technologies to monitor cell health is increasing with advancements in the field of cell therapy and regenerative medicine. In this study, we demonstrated unlabeled optical metabolic imaging of cultured living cells. This imaging technique is based on motion vector analysis with a block-matching algorithm to compare sequential time-lapse images. Motion vector analysis evaluates the movement of intracellular granules observed with a phase-contrast microscope. We demonstrated that the motion speed of intracellular movement reflects adenosine triphosphate (ATP)-dependent intracellular trafficking in cells. We also confirmed that intracellular motion speed is correlated with the ATP concentrations of the cells. This assay can measure cellular viability at a single-cell level without requiring any reagents.


Assuntos
Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Contraste de Fase/métodos , Análise de Célula Única/métodos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/isolamento & purificação , Algoritmos , Linhagem Celular , Movimento Celular/genética , Humanos
13.
J Biol Regul Homeost Agents ; 33(2): 511-516, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30915828

RESUMO

To explore the possible cytological mechanism underlying the role of Astragaloside IV in promoting the repair of bone defects, osteoblasts were cultured in vitro and identified using inverted phase contrast microscopy, alkaline phosphatase (ALP) staining and alizarin red staining.


Assuntos
Osteoblastos/efeitos dos fármacos , Saponinas/farmacologia , Triterpenos/farmacologia , Fosfatase Alcalina , Antraquinonas , Osso e Ossos/patologia , Diferenciação Celular , Células Cultivadas , Humanos , Microscopia de Contraste de Fase
14.
Asian Pac J Cancer Prev ; 20(3): 757-765, 2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30909682

RESUMO

Aim: Newcastle disease virus (NDV) is a member of genus Avulavirus within the family Paramyxoviridae. Interest of using NDV as an anticancer agent has arisen from its ability to kill tumor cells with limited toxicity to normal cells. Methods: In this investigation, the proliferation of brain tumor cell line, glioblastoma multiform (DBTRG.05MG) induced by NDV strain AF2240 was evaluated in-vitro, by using MTT proliferation assay. Furthermore, Cytological observations were studied using fluorescence microscopy and transmission electron microscopy, DNA laddering in agarose gel electrophoresis assay used to detect the mode of cell death and analysis of the cellular DNA content by flowcytometery. Results: MTT proliferation assay, Cytological observations using fluorescence microscopy and transmission electron microscopy show the anti-proliferation effect and apoptogenic features of NDV on DBTRG.05MG. Furthermore, analysis of the cellular DNA content showed that there was a loss of treated cells in all cell cycle phases (G1, S and G2/M) accompanied with increasing in sub-G1 region (apoptosis peak). Conclusion: It could be concluded that NDV strain AF2240 is a potent antitumor agent that induce apoptosis and its cytotoxicity increasing while increasing of time and virus titer.


Assuntos
Apoptose , Proliferação de Células , Glioblastoma/patologia , Microscopia de Contraste de Fase/métodos , Vírus da Doença de Newcastle/fisiologia , Terapia Viral Oncolítica/métodos , Ciclo Celular , Glioblastoma/terapia , Humanos , Células Tumorais Cultivadas
15.
Appl Opt ; 58(5): A26-A31, 2019 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-30873988

RESUMO

While simultaneous phase-contrast and two-photon fluorescence imaging in microscopy can bring abundant biomedical information, it is difficult to retrieve phase information from conventional two-photon microscopes. To realize low-cost, in situ phase-contrast and two-photon fluorescence imaging, we propose Schlieren two-photon microscopy, a method that implements phase-contrast imaging on two-photon microscopes. This method involves spatially modulated fluorescence plates, which are made of two-photon fluorescence dyes or upconversion nanoparticles. We demonstrate that the fluorescence intensity fluctuation reflects the phase gradients of the specimen via theoretical analysis, simulations, and experiments. The proposed method is fully compatible with commercial two-photon microscopes, thus enabling widespread applications in live tissue imaging.


Assuntos
Células HeLa/citologia , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Contraste de Fase/métodos , Corantes Fluorescentes , Humanos
16.
Biofactors ; 45(3): 401-415, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30854715

RESUMO

Tumor hypoxia is positively correlated with tumor aggressiveness and hence is a negative prognostic factor in cancer. As normal cells usually do not experience such low oxygen levels, hypoxic cell signaling has attracted significant attention for the development of tumor-selective treatment strategies. In response to hypoxia, the master transcriptional regulator, HIF-1α plays central role in cellular adaptation by transactivating several crucial downstream target genes, which are involved in angiogenesis, metastasis, and EMT. In this study, we investigated the effect of cinnamaldehyde (CA), the main active ingredient of Cinnamon cassia bark extract, on hypoxia-induced angiogenesis and metastasis. The study in vitro comprised two cell lines, viz, sarcoma 180 and B16F10 melanoma, which were further confirmed in their respective transplantable in vivo models. Results show that CA administration inhibited tumor angiogenesis, EMT, and metastasis. At the molecular level, this was accompanied by a reduction in VEGF secretion, VEGF receptor (FLK) phosphorylation, matrix metalloproteinase (MMP) expression, and activity as well as a reduction in the EMT-related factors TWIST and ZEB1. Next, we focused our study particularly on the modulation of HIF-1 α by CA, which revealed that CA decreased HIF-1 α protein level by inhibiting its synthesis without affecting its proteasomal degradation. Furthermore, the PI3/Akt/mTOR pathway, which plays an important role in HIF-1α transcription and translation, was also inhibited by CA both in vitro and in vivo. Thus, it can be concluded that CA decreased angiogenesis and metastasis in tumor cells by inhibiting HIF-1α protein accumulation probably by targeting the PI3/Akt/mTOR pathway. © 2019 BioFactors, 45(3):401-415, 2019.


Assuntos
Acroleína/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Neovascularização Patológica/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Acroleína/uso terapêutico , Animais , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaio de Imunoadsorção Enzimática , Hipóxia/complicações , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Microscopia de Contraste de Fase , Neovascularização Patológica/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos
17.
Clin Chim Acta ; 491: 126-131, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30703345

RESUMO

BACKGROUND: The use of phase contrast in urinalysis has been highly recommended. A new system, sediMAX conTRUST PRO, is now available providing simultaneous automated phase contrast and bright field microscopy. This study aimed to evaluate both analytical and diagnostic performance of this new analyzer. METHODS: Results from 504 samples evaluated with the sediMAX conTRUST PRO were compared to those obtained from the same samples by manual microscopy (MM). Analytical and diagnostic performance were assessed according to established guidelines. RESULTS: The concentration of red blood cells (RBCs)and white blood cells (WBCs) at which the LoQ satisfied a CV< 25% was 12 particles per µL (p/µL) and 8 p/µL, respectively. Within one grade of agreement concordance was quite high, 97.8% for RBCs and 98.0% for WBCs, and above 90% for all other particles. Overall, diagnostic sensitivity and specificity were good (>80%) for the particles considered, although lower sensitivities, 70.6% and 61.8%, were respectively found for hyaline and pathological casts. CONCLUSIONS: The sediMAX conTRUST PRO provides very good performance in terms of RBC and WBC recognition and enumeration, and quite good performance for all other particles. Hyaline cast and pathological cast identification is fine and comparable to other automated systems, but could use further improvement.


Assuntos
Microscopia de Contraste de Fase , Urinálise/instrumentação , Automação , Humanos , Limite de Detecção , Modelos Lineares
18.
Exp Parasitol ; 197: 57-64, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30677395

RESUMO

Leishmaniasis is a vector borne parasitic disease affecting millions of people worldwide and is spreading into further areas because of global warming. The development of new active substances against these single-cell eukaryotic parasites is of great importance. Leishmania tarentolae promastigotes (LtP) are non-pathogenic for mammals and serve as model organisms for pathogenic Leishmania in basic research. However, it is important to refine methods to study the process of the infection of mammalian macrophages by LtP and pathogenic Leishmania. Important stages of the infection are phagocytosis by macrophages and multiplication of Leishmania amastigotes in the phagolysosome of macrophages. In this study, advanced methods using electron spin resonance (ESR) spectroscopy and genetically manipulated LtP were used to monitor the infection of adherent J774 macrophages with LtP. An ESR method was established to detect the formation of superoxide radicals directly in adherent J774 cells and to investigate the effect of LtP on this activity. J774 cells responded with a burst of superoxide radicals in the presence of phorbol myristate acetate as positive control. In contrast, challenging J774 cells with LtP resulted in a much lower burst of superoxide radicals. To facilitate LtP detection in the phagolysosome of J774 macrophages, LtP expressing enhanced green fluorescent protein (EGFP-LtP) were constructed. After different infection times with EGFP-LtP, the J774 cells were visualized by phase contrast microscopy and the cell number was determined. The intramacrophage Leishmania tarentolae amastigotes (LtA) expressing EGFP were detected by fluorescence microscopy and then counted with ImageJ. These experiments showed that LtP are taken up by J774 cells and form intraphagolysosomal amastigotes. LtA under our conditions multiplied intracellularly and were able to persist about 48 h in J774 cells. These experiments showed that ESR spectroscopy of attached macrophages and the use of the EGFP-LtP are suitable methods to study the initial phase of Leishmania infection in vitro.


Assuntos
Leishmania/imunologia , Macrófagos/parasitologia , Fagocitose , Animais , Linhagem Celular , Espectroscopia de Ressonância de Spin Eletrônica , Eletroporação , Humanos , Leishmania/genética , Macrófagos/imunologia , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Superóxidos/metabolismo
19.
Environ Sci Pollut Res Int ; 26(7): 6450-6459, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30623323

RESUMO

Asbestos, as with other pollutants in the air, has adverse effects on the health of human beings and animals. Today, the relationship between presence of asbestos fibers in the air breathed by humans and developing serious diseases such as lung cancer (asbestosis) and mesothelioma has been proven. This study was designed and conducted within the time period of August 2017 and June 2018 to determine the concentration of asbestos fiber in the ambient air of Shahryar City and to evaluate their health effects for the general population of the city. For this purpose, samples were taken from four points, and overall 32 air samples were taken along the year. The samples were then analyzed by the phase contrast microscopy (PCM) method. Also, to investigate the type of asbestos and for more accurate counting of fibers, SEM analysis was utilized. Finally, based on the EPA IRIS method, the health effects resulting from asbestos risks were also evaluated. The results of this study indicated that the mean annual concentration of asbestos fiber in the ambient air of Shahryar City was obtained as 0.0019 f/ml PCM and 0.0072 f/ml SEM. Furthermore, the most polluted point was S1 point (0.0119 -0.0026 f/ml, PCM), while the lowest concentration was related to S4 point (0.001 f/ml PCM-0.0021 f/ml SEM). The mean annual risk resulting from airborne asbestos fiber in the ambient air of Shahryar City for all samples was obtained as 1.72 × 10-6 to 2.2 × 10-4, which was higher than the recommended risk range in some points.


Assuntos
Poluentes Atmosféricos/análise , Asbestos/análise , Exposição Ambiental/estatística & dados numéricos , Poluição do Ar/estatística & dados numéricos , Cidades , Monitoramento Ambiental , Humanos , Irã (Geográfico)/epidemiologia , Neoplasias Pulmonares/epidemiologia , Mesotelioma/epidemiologia , Microscopia de Contraste de Fase , Medição de Risco , Estações do Ano
20.
Microsc Res Tech ; 82(7): 953-960, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30636063

RESUMO

A thorough understanding of inner ear anatomy is important for investigators. However, investigation of the mouse inner ear is difficult due to the limitations of imaging techniques. X-ray phase contrast tomography increases contrast 100-1,000 times compared with conventional X-ray imaging. This study aimed to investigate inner ear anatomy in a fresh post-mortem mouse using X-ray phase contrast tomography and to provide a comprehensive atlas of microstructures with less tissue deformation. All experiments were performed in accordance with our institution's guidelines on the care and use of laboratory animals. A fresh mouse cadaver was scanned immediately after sacrifice using an inline phase contrast tomography system. Slice images were reconstructed using a filtered back-projection (FBP) algorithm. Standardized axial and coronal planes were adjusted with a multi-planar reconstruction method. Some three-dimensional (3D) objects were reconstructed by surface rendering. The characteristic features of microstructures, including otoconia masses of the saccular and utricular maculae, superior and inferior macula cribrosae, single canal, modiolus, and osseous spiral lamina, were described in detail. Spatial positions and relationships of the vestibular structures were exhibited in 3D views. This study investigated mouse inner ear anatomy and provided a standardized presentation of microstructures. In particular, otoconia masses were visualized in their natural status without contrast for the first time. The comprehensive anatomy atlas presented in this study provides an excellent reference for morphology studies of the inner ear.


Assuntos
Orelha Interna/anatomia & histologia , Microscopia de Contraste de Fase , Tomografia Computadorizada por Raios X , Animais , Orelha Interna/diagnóstico por imagem , Processamento de Imagem Assistida por Computador , Imagem Tridimensional/instrumentação , Imagem Tridimensional/métodos , Camundongos , Membrana dos Otólitos/anatomia & histologia , Membrana dos Otólitos/diagnóstico por imagem
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