Martin E. Gosnell

Oxidative stress, mitochondrial perturbations and fetal programming of renal disease induced by maternal smoking.

An adverse in-utero environment is increasingly recognized to predispose to chronic disease in adulthood. Maternal smoking remains the most common modifiable adverse in-utero exposure leading to low birth weight, which is strongly associated with chronic kidney disease (CKD) in later life. In order to investigate underlying mechanisms for such susceptibility, female Balb/c mice were sham or cigarette smoke-exposed (SE) for 6 weeks before mating, throughout gestation and lactation. Offspring kidneys were examined for oxidative stress, expression of mitochondrial proteins, mitochondrial structure as well as renal functional parameters on postnatal day 1, day 20 (weaning) and week 13 (adult age). From birth throughout adulthood, SE offspring had increased renal levels of mitochondrial-derived reactive oxygen species (ROS), which left a footprint on DNA with increased 8-hydroxydeoxyguanosin (8-OHdG) in kidney tubular cells. Mitochondrial structural abnormalities were seen in SE kidneys at day 1 and week 13 along with a reduction in oxidative phosphorylation (OXPHOS) proteins and activity of mitochondrial antioxidant Manganese superoxide dismutase (MnSOD). Smoke exposure also resulted in increased mitochondrial DNA copy number (day 1-week 13) and lysosome density (day 1 and week 13). The appearance of mitochondrial defects preceded the onset of albuminuria at week 13. Thus, mitochondrial damage caused by maternal smoking may play an important role in development of CKD at adult life.

Visible 532 nm laser irradiation of human adipose tissue-derived stem cells: Effect on proliferation rates, mitochondria membrane potential and autofluorescence†

The photobiological effect of laser light on cells and tissues originates from light absorption by endogenous chromophores and hence it depends on the wavelength of light source and cell type. Earlier studies regarding the biostimulation effects of green laser light investigated a wide variety of cells but not adipose tissue‐derived stem cells (ADSCS). In this study we reported the in vitro effect of 532‐nm Nd:YAG laser on proliferation, mitochondrial activity of these mesenchymal stem cells (MSCs) on the autofluorescence emission at wavelengths associated with nicotinamide adenine dinucleotide (NADH) and flavoproteins.

l-Carnitine reverses maternal cigarette smoke exposure-induced renal oxidative stress and mitochondrial dysfunction in mouse offspring

Maternal smoking is associated with metabolic disorders, renal underdevelopment, and a predisposition to chronic kidney disease in offspring, yet the underlying mechanisms are unclear. By exposing female Balb/c mice to cigarette smoke for 6 wk premating and during gestation and lactation, we showed that maternal smoke exposure induced glucose intolerance, renal underdevelopment, inflammation, and albuminuria in male offspring. This was associated with increased renal oxidative stress and mitochondrial dysfunction at birth and in adulthood. Importantly, we demonstrated that dietary supplementation of l-carnitine, an amino acid shown to increase antioxidant defenses and mitochondrial function in numerous diseases, in smoke-exposed mothers during pregnancy and lactation significantly reversed the detrimental maternal impacts on kidney pathology in these male offspring. It increased SOD2 and glutathione peroxidase 1, reduced ROS accumulation, and normalized levels of mitochondrial preprotein translocases of the outer membrane, and oxidative phosphorylation complexes I-V in the kidneys of mouse progeny after intrauterine cigarette smoke exposure. These findings support the hypothesis that oxidative stress and mitochondrial dysfunction are closely linked to the adverse effects of maternal smoking on male offspring renal pathology. The results of our study suggest that l-carnitine administration in cigarette smoke-exposed mothers mitigates these deleterious renal consequences.

Quantitative non-invasive cell characterisation and discrimination based on multispectral autofluorescence features.

Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous autofluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from autofluorescence imaging has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent. Label-free classifications are validated by the analysis of Classification Determinant (CD) antigen expression. The versatility of our method is illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes, and assessing the condition of preimplantation embryos.

Functional hyperspectral imaging captures subtle details of cell metabolism in olfactory neurosphere cells, disease-specific models of neurodegenerative disorders

Hyperspectral imaging uses spectral and spatial image information for target detection and classification. In this work hyperspectral autofluorescence imaging was applied to patient olfactory neurosphere-derived cells, a cell model of a human metabolic disease MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like syndrome). By using an endogenous source of contrast subtle metabolic variations have been detected between living cells in their full morphological context which made it possible to distinguish healthy from diseased cells before and after therapy. Cellular maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant cell subpopulations, in particular a subpopulation with compromised mitochondrial function. Taken together, our results demonstrate that multispectral spectral imaging provides a new non-invasive method to investigate neurodegenerative and other disease models, and it paves the way for novel cellular characterisation in health, disease and during treatment, with proper account of intrinsic cellular heterogeneity.

Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence

Carbonyl cyanide-p-trifluoro methoxyphenylhydrazone (FCCP) is a well-known mitochondrial uncoupling agent. We examined FCCP-induced fluorescence quenching of reduced nicotinamide adenine dinucleotide / nicotinamide adenine dinucleotide phosphate (NAD(P)H) in solution and in cultured HeLa cells in a wide range of FCCP concentrations from 50 to 1000µM. A non-invasive label-free method of hyperspectral imaging of cell autofluorescence combined with unsupervised unmixing was used to separately isolate the emissions of free and bound NAD(P)H from cell autofluorescence. Hyperspectral image analysis of FCCP-treated HeLa cells confirms that this agent selectively quenches fluorescence of free and bound NAD(P)H in a broad range of concentrations. This is confirmed by the measurements of average NAD/NADH and NADP/NADPH content in cells. FCCP quenching of free NAD(P)H in cells and in solution is found to be similar, but quenching of bound NAD(P)H in cells is attenuated compared to solution quenching possibly due to a contribution from the metabolic and/or antioxidant response in cells. Chemical quenching of NAD(P)H fluorescence by FCCP validates the results of unsupervised unmixing of cell autofluorescence.

Scar tissue classification using nonlinear optical microscopy and discriminant analysis

This paper addresses the scar tissue maturation process that occurs stepwise, and calls for reliable classification. The structure of collagen imaged by nonlinear optical microscopy (NLOM) in post-burn hypertrophic and mature scar, as well as in normal skin, appeared to distinguish these maturation steps. However, it was a discrimination analysis, demonstrated here, that automated and quantified the scar tissue maturation process. The achieved scar classification accuracy was as high as 96%. The combination of NLOM and discrimination analysis is believed to be instrumental in gaining insight into the scar formation, for express diagnosis of scar and surgery planning.

Hyperspectral microscopy can detect metabolic heterogeneity within bovine post-compaction embryos incubated under two oxygen concentrations (7% versus 20%)

STUDY QUESTION Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity? SUMMARY ANSWER Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy. WHAT IS KNOWN ALREADY Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment. STUDY DESIGN, SIZE, DURATION The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8; embryo developmental competence) or 5 days (D5; metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40-50 embryos per replicate used for the developmental competency experiment, 10-20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21-22 embryos used for the hyperspectral microscopy study. PARTICIPANTS/MATERIALS, SETTING, METHODS In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy. MAIN RESULTS AND THE ROLE OF CHANCE Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P < 0.05), demonstrating that atmospheric oxygen was a viable model for studying mild metabolic stress. The metabolic profiles of D5 embryos was determined and although metabolic heterogeneity was evident within the cleavage stage (i.e. arrested) embryos exposed to fluorophores, there were no detectable difference in fluorescence intensity and pattern localisation in morula exposed to the two different oxygen concentrations (P > 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P < 0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P < 0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups. LIMITATIONS, REASONS FOR CAUTION Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success. WIDER IMPLICATIONS OF THE FINDINGS Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premiers Science and Research Fund. The authors declare there are no conflict of interest.

Computer-assisted cystoscopy diagnosis of bladder cancer

OBJECTIVES One of the most reliable methods for diagnosing bladder cancer is cystoscopy. Depending on the findings, this may be followed by a referral to a more experienced urologist or a biopsy and histological analysis of suspicious lesion. In this work, we explore whether computer-assisted triage of cystoscopy findings can identify low-risk lesions and reduce the number of referrals or biopsies, associated complications, and costs, although reducing subjectivity of the procedure and indicating when the risk of a lesion being malignant is minimal. MATERIALS AND METHODS Cystoscopy images taken during routine clinical patient evaluation and supported by biopsy were interpreted by an expert clinician. They were further subjected to an automated image analysis developed to best capture cancer characteristics. The images were transformed and divided into segments, using a specialised color segmentation system. After the selection of a set of highly informative features, the segments were separated into 4 classes: healthy, veins, inflammation, and cancerous. The images were then classified as healthy and diseased, using a linear discriminant, the naïve Bayes, and the quadratic linear classifiers. Performance of the classifiers was measured by using receiver operation characteristic curves. RESULTS The classification system developed here, with the quadratic classifier, yielded 50% false-positive rate and zero false-negative rate, which means, that no malignant lesions would be missed by this classifier. CONCLUSIONS Based on criteria used for assessment of cystoscopy images by medical specialists and features that human visual system is less sensitive to, we developed a computer program that carries out automated analysis of cystoscopy images. Our program could be used as a triage to identify patients who do not require referral or further testing.

Statistically strong label-free quantitative identification of native fluorophores in a biological sample

Bioimaging using endogenous cell fluorescence, without any external biomarkers makes it possible to explore cells and tissues in their original native state, also in vivo. In order to be informative, this label-free method requires careful multispectral or hyperspectral recording of autofluorescence images followed by unsupervised extraction (unmixing) of biochemical signatures. The unmixing is difficult due to the scarcity of biochemically pure regions in cells and also because autofluorescence is weak compared with signals from labelled cells, typically leading to low signal to noise ratio. Here, we solve the problem of unsupervised hyperspectral unmixing of cellular autofluorescence by introducing the Robust Dependent Component Analysis (RoDECA). This approach provides sophisticated and statistically robust quantitative biochemical analysis of cellular autofluorescence images. We validate our method on artificial images, where the addition of varying known level of noise has allowed us to quantify the accuracy of our RoDECA analysis in a way that can be applied to real biological datasets. The same unsupervised statistical minimisation is then applied to imaging of mouse retinal photoreceptor cells where we establish the identity of key endogenous fluorophores (free NADH, FAD and lipofuscin) and derive the corresponding molecular abundance maps. The pre-processing methodology of image datasets is also presented, which is essential for the spectral unmixing analysis, but mostly overlooked in the previous studies.