Non-destructive, label free identification of cell cycle phase in cancer cells by multispectral microscopy of autofluorescence

Background: Cell cycle analysis is important for cancer research. However, available methodologies have drawbacks including limited categorisation and reliance on fixation, staining or transformation. Multispectral analysis of endogenous cell autofluorescence has been shown to be sensitive to changes in cell status and could be applied to the discrimination of cell cycle without these steps. Methods: Cells from the MIA-PaCa-2, PANC-1, and HeLa cell lines were plated on gridded dishes and imaged using a multispectral fluorescence microscope. They were then stained for proliferating cell nuclear antigen (PCNA) and DNA intensity as a reference standard for their cell cycle position (G1, S, G2, M). The multispectral data was split into training and testing datasets and models were generated to discriminate between G1, S, and G2 + M phase cells. A standard decision tree classification approach was taken, and a two-step system was generated for each line. Results: Across cancer cell lines accuracy ranged from 68.3% (MIA-PaCa-2) to 73.3% (HeLa) for distinguishing G1 from S and G2 + M, and 69.0% (MIA-PaCa-2) to 78.0% (PANC1) for distinguishing S from G2 + M. Unmixing the multispectral data showed that the autofluorophores NADH, FAD, and PPIX had significant differences between phases. Similarly, the redox ratio and the ratio of protein bound to free NADH were significantly affected. Conclusions: These results demonstrate that multispectral microscopy could be used for the non-destructive, label free discrimination of cell cycle phase in cancer cells. They provide novel information on the mechanisms of cell-cycle progression and control, and have practical implications for oncology research.Jared M. Campbell, Abbas Habibalahi, Saabah Mahbub, Martin Gosnell, Ayad G. Anwer, Sharon Paton, Stan Gronthos and Ewa Goldy

Non-invasive real-time imaging of reactive oxygen species (ROS) using auto-fluorescence multispectral imaging technique: a novel tool for redox biology

Detecting reactive oxygen species (ROS) that play a critical role as redox modulators and signalling molecules in biological systems currently requires invasive methods such as ROS -specific indicators for imaging and quantification. We developed a non-invasive, real-time, label-free imaging technique for assessing the level of ROS in live cells and thawed cryopreserved tissues that is compatible with in-vivo imaging. The technique is based on autofluorescence multispectral imaging (AFMI) carried out in an adapted fluorescence microscope with an expanded number of spectral channels spanning specific excitation (365 nm-495 nm) and emission (420 nm-700 nm) wavelength ranges. We established a strong quantitative correlation between the spectral information obtained from AFMI and the level of ROS obtained from CellROX staining. The results were obtained in several cell types (HeLa, PANC1 and mesenchymal stem cells) and in live kidney tissue. Additioanly,two spectral regimes were considered: with and without UV excitation (wavelengths > 400 nm); the latter being suitable for UV-sensitive systems such as the eye. Data were analyzed by linear regression combined with an optimization method of swarm intelligence. This allowed the calibration of AFMI signals to the level of ROS with excellent correlation (R = 0.84, p = 0.00) in the entire spectral range and very good correlation (R = 0.78, p = 0.00) in the limited, UV-free spectral range. We also developed a strong classifier which allowed us to distinguish moderate and high levels of ROS in these two regimes (AUC = 0.91 in the entire spectral range and AUC = 0.78 for UV-free imaging). These results indicate that ROS in cells and tissues can be imaged non-invasively, which opens the way to future clinical applications in conditions where reactive oxygen species are known to contribute to progressive disease such as in ophthalmology, diabetes, kidney disease, cancer and neurodegenerative diseases.Abbas Habibalahi, Mahdieh Dashtbani Moghari, Jared M. Campbell, Ayad G. Anwer, Saabah B. Mahbub, Martin Gosnell, Sonia Saad, Carol Pollock, Ewa M. Goldy

Impact of dietary incorporation of Spirulina (Arthrospira platensis) and exogenous enzymes on broiler performance, carcass traits and meat quality

This study assessed the effect of Spirulina (Arthrospira platensis), individually and in combination with exogenous enzymes, on growth performance, carcass traits, and meat quality of broiler chickens. One hundred and twenty Ross 308 male chickens were allocated into 40 battery brooders, with 3 birds per cage, and fed ad libitum a corn-based diet during the first 21 D of the trial. The experimental period lasted from day 21 to 35, during which birds were fed 4 different diets: a corn-soybean basal diet, taken as the control group, a basal diet containing 15% Spirulina (MA), a basal diet containing 15% Spirulina plus 0.005% Rovabio Excel AP (MAR), and a basal diet containing 15% Spirulina plus 0.01% lysozyme (MAL). Body weight gain (P , 0.001) and feed conversion rate (P , 0.001) were improved in control chickens, when compared with those fed with Spirulina. In addition, Spirulina increased the length of duodenum plus jejunum in relation to the other treatment (P , 0.01). Chickens on the MAL diet showed a considerable increase in digesta viscosity (P , 0.05) compared with the control group. Breast and thigh meats from chickens fed with Spirulina, with or without the addition of exogenous enzymes, had higher values of yellowness (b*) (P , 0.001), total carotenoids (P , 0.001), and saturated fatty acids (P , 0.001), whereas n-3 polyunsaturated fatty acid (P , 0.01) and a-tocopherol (P , 0.001) decreased, when compared with the control. In conclusion, the incorporation of 15% Spirulina in broiler diets, individually or combined with exogenous enzymes, reduced birds’ performance through a higher digesta viscosity, which is likely associated with the gelation of microalga indigestible proteins. In addition, cell wall of Spirulina was successfully broken by the addition of lysozyme, but not by Rovabio Excel AP. Therefore, we anticipate that the combination of lysozyme with an exogenous specific peptidase could improve the digestibility of proteins from this microalga and avoid their detrimental gelationinfo:eu-repo/semantics/publishedVersio

Non-invasive monitoring of functional state of articular cartilage tissue with label-free unsupervised hyperspectral imaging

Damage and degradation of articular cartilage leads to severe pain and loss of mobility. The development of new therapies for cartilage regeneration for monitoring their effect requires further study of cartilage, ideally at a molecular level and in a minimally invasive way. Hyperspectral microscopy is a novel technology which utilises endogenous fluorophores to non-invasively assess the molecular composition of cells and tissue. In this study, we applied hyperspectral microscopy to healthy bovine articular cartilage and osteoarthritic human articular cartilage to investigate its capacity to generate informative molecular data and characterise disease state and treatment effects. We successfully demonstrated label-free fluorescence identification of collagen type I and II - isolated in cartilage here for the first time and the co-enzymes free NADH and FAD which together give the optical redox ratio that is an important measure of metabolic activity. The intracellular composition of chondrocytes was also examined. Differences were observed in the molecular ratios within the superficial and transitional zones of the articular cartilage which appeared to be influenced by disease state and treatment. These findings show that hyperspectral microscopy could be useful for investigating the molecular underpinnings of articular cartilage degradation and repair. As it is non-invasive and non-destructive, samples can be repeatedly assessed over time, enabling true time-course experiments with in-depth molecular data. Additionally, there is potential for the hyperspectral approach to be adapted for patient examination to allow the investigation of cartilage state. This could be of advantage for assessment and diagnosis as well as treatment monitoring.Saabah B. Mahbub, Anna Guller, Jared M. Campbell, Ayad G. Anwer, Martin E. Gosnell, Graham Vesey, Ewa M. Goldy

Crescimento da parte aérea de cana crua e queimada Shoot growth of green and burned canes

Este trabalho teve como objetivos: 1. comparar o crescimento de cana colhida crua, mecanizada e de cana após a queima, colhida manualmente; 2. avaliar a influência do clima sobre as duas condições de crescimento e 3. analisar o comportamento do crescimento de cana crua e cana queimada nos 1º e 2º anos de rebrota, através de curvas adaptadas. A pesquisa foi realizada no município de Morro Agudo, SP, de julho de 1995 a julho de 1997. A variedade cultivada foi a SP 70 -1143. Utilizaram-se como indicadores de crescimento os seguintes índices biométricos: número de perfilhos, número de folhas, matéria seca de colmos e de folhas, IAF e avaliou-se a influência das temperaturas e das umidades do ar, do solo e das folhas. Adotou-se regressão polinomial e regressão não-linear para se adaptar os dados às curvas de crescimento. O crescimento no primeiro ciclo foi semelhante para cana crua e cana queimada. No início do segundo ciclo ocorreu maior crescimento em cana crua, enquanto que no final, foi maior em cana queimada. O perfilhamento da cana crua não apresentou diferenças significativas que confirmem a influência negativa da palha na rebrota. Os fatores climatológicos, isoladamente, não provocaram mudanças nos ciclos de crescimento de maneira que se identificasse uma tendência geral. As diferenças expressas na curva de crescimento do 1º para o 2º ano são devidas aos fatores climatológicos, tanto para cana crua como para cana queimada.<br>This work had as objectives: 1. to compare the shoot growth between green cane, mechanically harvested and burned cane, harvested manually; 2. to evaluate the influence of the weather on the two conditions of growth and 3. to analyze the growth of green cane and cane burned in the 1st and 2nd years of second ratoon cane crop, by means of adapted curves. The research was performed in Morro Agudo, SP, Brazil from July 1995 to July 1997. The cultivar was the SP 70 -1143. As growth indicators the following biometric indexes were used: number of tillers, number of leaves, dry matter of stalks, and leaves and leaf area index (LAI). For each treatment, 4 areas were used, each one being considered a replication. Polynomial regression and non-linear regression were used to adjust the data to the growth curves. The growth in the first cycle was similar for the green and the burned canes. At the beginning of the second cycle the green cane growth was greater, while at the end, the growth was greater for the burned cane. Tillering of the green cane did not present significant differences that confirm the negative influence of the straw in the ratoon cane crop. The climatological factors, separately, did not promote changes in the growth cycles, that could identify a general tendency. The differences in the development expressed on the curve of growth from the 1st to the 2nd year are due to the climatological factors, for both, the green and the burned cane

Um simulador dinâmico do crescimento de uma cultura de cana-de-açúcar A dynamic simulator of the sugarcane crop growth

Este trabalho descreve a primeira versão de um simulador matemático-fisiológico do crescimento diário de uma cultura de cana-de-açúcar (SIMCANA) em resposta às condições do ambiente durante a estação de crescimento. SIMCANA resume a maior parte das informações disponíveis concernentes aos processos fisiológicos da cultura de cana-de-açúcar. Esta sua versão não incluí os processos degerminação e florescimento, havendo necessidade de especificar as condições da cultura no primeiro dia de simulação. Em função das condições diárias de radiação solar global, temperatura máxima e mínima, umidade relativa do ar, SIMCANA calcula as taxas de fotossíntese, respiração e crescimento da cultura, as taxas de senescência das folhas e raízes, a massa seca das folhas, colmos e raízes, e o índice de área foliar. Embora várias relações empíricas tenham sido usadas, SIMCANA parece ser capaz de simular o crescimento da cultura de cana-de-açúcar.<br>The first version of a mathematical-physiological simulator of the daily growth of a sugarcane crop (SIMCANA) as a function of the environmental conditions during the growing season is described. SIMCANA summarizes most of the available information regarding to the physiological processes of the sugarcane crop. This version does not include the germination and flowering processes, therefore it is necessary to specify the crop conditions at the first day of simulation. Given the daily conditions of global solar radiation, maximum and minimum temperature, and the relative humidity, SIMCANA computes the rates of crop photosynthesis, respiration, and growth, the senescence rates for leaves and roots, the dry mass of leaves, stems, and roots, and the leaf area index. Although several empirical relations have been used, SIMCANA seems to be able to simulate the sugarcane crop growth