RESUMEN
Holo-tomographic microscopy (HTM) is a label-free microscopy method reporting the fine changes of a cell's refractive indices (RIs) in three dimensions at high spatial and temporal resolution. By combining HTM with epifluorescence, we demonstrate that mammalian cellular organelles such as lipid droplets (LDs) and mitochondria show specific RI 3D patterns. To go further, we developed a computer-vision strategy using FIJI, CellProfiler3 (CP3), and custom code that allows us to use the fine images obtained by HTM in quantitative approaches. We could observe the shape and dry mass dynamics of LDs, endocytic structures, and entire cells' division that have so far, to the best of our knowledge, been out of reach. We finally took advantage of the capacity of HTM to capture the motion of many organelles at the same time to report a multiorganelle spinning phenomenon and study its dynamic properties using pattern matching and homography analysis. This work demonstrates that HTM gives access to an uncharted field of biological dynamics and describes a unique set of simple computer-vision strategies that can be broadly used to quantify HTM images.
Asunto(s)
Microscopía Fluorescente/métodos , Orgánulos/fisiología , Refractometría/métodos , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Metabolismo de los Lípidos , Mitocondrias/metabolismoRESUMEN
The life history, distribution, and acoustic ecology of the sei whale (Balaenoptera borealis) in the western North Atlantic Ocean remains poorly understood. In this study an array of bottom-mounted recorders captured previously undocumented low frequency 50 to 30-Hz triplet and singlet down sweep vocalizations in close association with signature 82 to 34-Hz sei whale down sweep vocalizations. Spatiotemporal correlations of acoustically tracked sei whales confirm the original vocalizations are produced by sei whales. The 50 to 34-Hz down sweep call types were characterized with a suite of five spectral and temporal measurements. The pattern and repetition of the full acoustic suite is suggestive of song structure and warrants further investigation. The discovery of vocalizations attributed specifically to sei whales enables historic acoustic records to be re-evaluated for the presence of this species throughout its range.
Asunto(s)
Acústica , Balaenoptera/fisiología , Vocalización Animal/fisiología , Animales , Océano Atlántico , Espectrografía del Sonido/métodosRESUMEN
In melanoma, vascular endothelial growth factor-C (VEGF-C) expression and consequent lymphangiogenesis correlate with metastasis and poor prognosis. VEGF-C also promotes tumor immunosuppression, suggesting that lymphangiogenesis inhibitors may be clinically useful in combination with immunotherapy. We addressed this concept in mouse melanoma models with VEGF receptor-3 (VEGFR-3)-blocking antibodies and unexpectedly found that VEGF-C signaling enhanced rather than suppressed the response to immunotherapy. We further found that this effect was mediated by VEGF-C-induced CCL21 and tumor infiltration of naïve T cells before immunotherapy because CCR7 blockade reversed the potentiating effects of VEGF-C. In human metastatic melanoma, gene expression of VEGF-C strongly correlated with CCL21 and T cell inflammation, and serum VEGF-C concentrations associated with both T cell activation and expansion after peptide vaccination and clinical response to checkpoint blockade. We propose that VEGF-C potentiates immunotherapy by attracting naïve T cells, which are locally activated upon immunotherapy-induced tumor cell killing, and that serum VEGF-C may serve as a predictive biomarker for immunotherapy response.
Asunto(s)
Inmunoterapia , Linfangiogénesis , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Linfocitos T/patología , Animales , Proliferación Celular , Quimiocina CCL21/metabolismo , Supervivencia sin Enfermedad , Epítopos/inmunología , Humanos , Melanoma Experimental/patología , Ratones , Metástasis de la Neoplasia , Receptores CCR7/metabolismo , Transducción de Señal , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
RATIONALE: Spirometry plays a major role in the diagnosis and assessment of severity of lung disease. Determining which lung function values are normal and which are below the lower limit of normal depends on reference equations derived from an appropriate population. OBJECTIVES: The purpose of this study was to derive spirometric reference equations for the Canadian population. METHODS: The Canadian Health Measures Survey consisted of a respiratory questionnaire, urinary cotinine measurements, and spirometry performed in the sitting position with rigorous quality control standards. Of the 16,606 respondents between 6 and 79 years of age, 11,145 were eliminated for positive responses to the respiratory questionnaire, tobacco exposure, or inability to provide high-quality spirograms. Of the remaining 5,461, roughly half were less than 18 years of age. Quantile regression was used to derive predictive (median) and lower limit of normal equations for males and females for FEV1, FVC, and FEV1/FVC ratio for those with ages greater and less than 18 years. MEASUREMENTS AND MAIN RESULTS: The resulting equations were compared with those from the Global Lung Initiative (GLI) and National Health and Nutrition Examination Survey (NHANES) III by using an ideal subject on the 50th percentile for height and between the ages of 6 and 79 years; the comparison showed minor and inconsistent discrepancies among the predictive equations. A plot of residuals (predicted minus measured value for each subject) suggested a marginally better fit compared with the GLI and NHANES III equations, although differences among the equations were small and unlikely to have any clinical significance. CONCLUSIONS: This study provides spirometric reference equations for the Canadian population that were measured under the recommended clinical conditions and with rigorous quality control.
Asunto(s)
Pulmón/fisiología , Espirometría/métodos , Adolescente , Adulto , Distribución por Edad , Anciano , Canadá , Niño , Femenino , Volumen Espiratorio Forzado/fisiología , Encuestas Epidemiológicas , Humanos , Masculino , Persona de Mediana Edad , Valores de Referencia , Análisis de Regresión , Distribución por Sexo , Encuestas y Cuestionarios , Capacidad Vital/fisiología , Adulto JovenRESUMEN
Low frequency (<100 Hz) downsweep vocalizations were repeatedly recorded from ocean gliders east of Cape Cod, MA in May 2005. To identify the species responsible for this call, arrays of acoustic recorders were deployed in this same area during 2006 and 2007. 70 h of collocated visual observations at the center of each array were used to compare the localized occurrence of this call to the occurrence of three baleen whale species: right, humpback, and sei whales. The low frequency call was significantly associated only with the occurrence of sei whales. On average, the call swept from 82 to 34 Hz over 1.4 s and was most often produced as a single call, although pairs and (more rarely) triplets were occasionally detected. Individual calls comprising the pairs were localized to within tens of meters of one another and were more similar to one another than to contemporaneous calls by other whales, suggesting that paired calls may be produced by the same animal. A synthetic kernel was developed to facilitate automatic detection of this call using spectrogram-correlation methods. The optimal kernel missed 14% of calls, and of all the calls that were automatically detected, 15% were false positives.