Conformational landscape of a virus by single-particle X-ray scattering.

Using a manifold-based analysis of experimental diffraction snapshots from an X-ray free electron laser, we determine the three-dimensional structure and conformational landscape of the PR772 virus to a detector-limited resolution of 9 nm. Our results indicate that a single conformational coordinate controls reorganization of the genome, growth of a tubular structure from a portal vertex and release of the genome. These results demonstrate that single-particle X-ray scattering has the potential to shed light on key biological processes.

Retinal oxidative stress at the onset of diabetes determined by synchrotron FTIR widefield imaging: towards diabetes pathogenesis.

Diabetic retinopathy is a microvascular complication of diabetes that can lead to blindness. In the present study, we aimed to determine the nature of diabetes-induced, highly localized biochemical changes in the neuroretina at the onset of diabetes. High-resolution synchrotron Fourier transform infrared (s-FTIR) wide field microscopy coupled with multivariate analysis (PCA-LDA) was employed to identify biomarkers of diabetic retinopathy with spatial resolution at the cellular level. We compared the retinal tissue prepared from 6-week-old Ins2Akita/+ heterozygous (Akita/+, N = 6; a model of diabetes) male mice with the wild-type (control, N = 6) mice. Male Akita/+ mice become diabetic at 4-weeks of age. Significant differences (P < 0.001) in the presence of biomarkers associated with diabetes and segregation of spectra were achieved. Differentiating IR bands attributed to nucleic acids (964, 1051, 1087, 1226 and 1710 cm-1), proteins (1662 and 1608 cm-1) and fatty acids (2854, 2923, 2956 and 3012 cm-1) were observed between the Akita/+ and the WT samples. A comparison between distinctive layers of the retina, namely the photoreceptor retinal layer (PRL), outer plexiform layer (OPL), inner nucleus layer (INL) and inner plexiform layer (IPL) suggested that the photoreceptor layer is the most susceptible layer to oxidative stress in short-term diabetes. Spatially-resolved chemical images indicated heterogeneities and oxidative-stress induced alterations in the diabetic retina tissue morphology compared with the WT retina. In this study, the spectral biomarkers and the spatial biochemical alterations in the diabetic retina and in specific layers were identified for the first time. We believe that the conclusions drawn from these studies will help to bridge the gap in our understanding of the molecular and cellular mechanisms that contribute to the pathobiology of diabetic retinopathy.

Quantitative Assessment of Retinopathy Using Multi-parameter Image Analysis.

A multi-parameter quantification method was implemented to quantify retinal vascular injuries in microscopic images of clinically relevant eye diseases. This method was applied to wholemount retinal trypsin digest images of diabetic Akita/+, and bcl-2 knocked out mice models. Five unique features of retinal vasculature were extracted to monitor early structural changes and retinopathy, as well as quantifying the disease progression. Our approach was validated through simulations of retinal images. Results showed fewer number of cells (P = 5.1205e-05), greater population ratios of endothelial cells to pericytes (PCs) (P = 5.1772e-04; an indicator of PC loss), higher fractal dimension (P = 8.2202e-05), smaller vessel coverage (P = 1.4214e-05), and greater number of acellular capillaries (P = 7.0414e-04) for diabetic retina as compared to normal retina. Quantification using the present method would be helpful in evaluating physiological and pathological retinopathy in a high-throughput and reproducible manner.

Optical Cryoimaging Reveals a Heterogeneous Distribution of Mitochondrial Redox State in ex vivo Guinea Pig Hearts and Its Alteration During Ischemia and Reperfusion.

Oxidation of substrates to generate ATP in mitochondria is mediated by redox reactions of NADH and FADH2. Cardiac ischemia and reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation. We hypothesize that IR alters the metabolic heterogeneity of mitochondrial redox state of the heart that is only evident in the 3-D optical cryoimaging of the perfused heart before, during, and after IR. The study involved four groups of hearts: time control (TC: heart perfusion without IR), global ischemia (Isch), global ischemia followed by reperfusion (IR) and TC with PCP (a mitochondrial uncoupler) perfusion. Mitochondrial NADH and FAD autofluorescence signals were recorded spectrofluorometrically online in guinea pig ex vivo-perfused hearts in the Langendorff mode. At the end of each specified protocol, hearts were rapidly removed and snap frozen in liquid N2 for later 3-D optical cryoimaging of the mitochondrial NADH, FAD, and NADH/FAD redox ratio (RR). The TC hearts revealed a heterogeneous spatial distribution of NADH, FAD, and RR. Ischemia and IR altered the spatial distribution and caused an overall increase and decrease in the RR by 55% and 64%, respectively. Uncoupling with PCP resulted in the lowest level of the RR (73% oxidation) compared with TC. The 3-D optical cryoimaging of the heart provides novel insights into the heterogeneous distribution of mitochondrial NADH, FAD, RR, and metabolism from the base to the apex during ischemia and IR. This 3-D information of the mitochondrial redox state in the normal and ischemic heart was not apparent in the dynamic spectrofluorometric data.

Optical cryoimaging of mitochondrial redox state in bronchopulmonary-dysplasia injury models in mice lungs.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a major cause of morbidity and mortality in premature infants exposed to high levels of oxygen. This is mainly attributed to increased oxidative stress and angiogenesis defects impacting lung alveolarization. METHODS: Here we use optical imaging to investigate the role of Bcl-2 in modulation of oxidative stress and angiogenesis and pathogenesis of BPD. Cryoimaging of the mitochondrial redox state of mouse lungs was applied to determine the metabolic state of the lungs from Bcl-2 +/+ (control), Bcl-2-deleted in the endothelium (Bcl-2 VE-cad) and Bcl-2-deficient (Bcl-2 -/-; global null) using mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide), and FADH2 (Flavin Adenine Dinucleotide) as the primary electron carriers in oxidative phosphorylation. RESULTS: We observed a 47% and 26% decrease in the NADH redox in Bcl-2 deficient lungs, Bcl-2 -/- and Bcl-2 VE-cad, respectively. CONCLUSIONS: Thus, Bcl-2 deficiency is associated with a significant increase in oxidative stress contributing to reduced angiogenesis and enhanced pathogenesis of BPD.

Optical imaging of mitochondrial redox state in rodent model of retinitis pigmentosa.

Oxidative stress (OS) and mitochondrial dysfunction contribute to photoreceptor cell loss in retinal degenerative disorders. The metabolic state of the retina in a rodent model of retinitis pigmentosa (RP) was investigated using a cryo-fluorescence imaging technique. The mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent and can be monitored without exogenous labels using optical techniques. The cryo-fluorescence redox imaging technique provides a quantitative assessment of the metabolism. More specifically, the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), the NADH redox ratio (RR), is a marker of the metabolic state of the tissue. The NADH RR and retinal function were examined in an established rodent model of RP, the P23H rat compared to that of nondystrophic Sprague-Dawley (SD) rats. The NADH RR mean values were 1.11 ± 0.03 in the SD normal and 0.841 ± 0.01 in the P23H retina, indicating increased OS in the P23H retina. Electroretinographic data revealed a significant reduction in photoreceptor function in P23H animals compared to SD nozrmal rats. Thus, cryo-fluorescence redox imaging was used as a quantitative marker of OS in eyes from transgenic rats and demonstrated that alterations in the oxidative state of eyes occur during the early stages of RP.

Surface fluorescence studies of tissue mitochondrial redox state in isolated perfused rat lungs.

We designed a fiber-optic-based optoelectronic fluorometer to measure emitted fluorescence from the auto-fluorescent electron carriers NADH and FAD of the mitochondrial electron transport chain (ETC). The ratio of NADH to FAD is called the redox ratio (RR = NADH/FAD) and is an indicator of the oxidoreductive state of tissue. We evaluated the fluorometer by measuring the fluorescence intensities of NADH and FAD at the surface of isolated, perfused rat lungs. Alterations of lung mitochondrial metabolic state were achieved by the addition of rotenone (complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor) and/or pentachlorophenol (PCP, uncoupler) into the perfusate recirculating through the lung. Rotenone- or KCN-containing perfusate increased RR by 21 and 30%, respectively. In contrast, PCP-containing perfusate decreased RR by 27%. These changes are consistent with the established effects of rotenone, KCN, and PCP on the redox status of the ETC. Addition of blood to perfusate quenched NADH and FAD signal, but had no effect on RR. This study demonstrates the capacity of fluorometry to detect a change in mitochondrial redox state in isolated perfused lungs, and suggests the potential of fluorometry for use in in vivo experiments to extract a sensitive measure of lung tissue health in real-time.

OPTICAL IMAGING OF LIPOPOLYSACCHARIDE-INDUCED OXIDATIVE STRESS IN ACUTE LUNG INJURY FROM HYPEROXIA AND SEPSIS.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many acute and chronic pulmonary disorders such as acute lung injury (ALI) in adults and bronchopulmonary dysplasia (BPD) in premature infants. Bacterial infection and oxygen toxicity, which result in pulmonary vascular endothelial injury, contribute to impaired vascular growth and alveolar simplification seen in the lungs of premature infants with BPD. Hyperoxia induces ALI, reduces cell proliferation, causes DNA damage and promotes cell death by causing mitochondrial dysfunction. The objective of this study was to use an optical imaging technique to evaluate the variations in fluorescence intensities of the auto-fluorescent mitochondrial metabolic coenzymes, NADH and FAD in four different groups of rats. The ratio of these fluorescence signals (NADH/FAD), referred to as NADH redox ratio (NADH RR) has been used as an indicator of tissue metabolism in injuries. Here, we investigated whether the changes in metabolic state can be used as a marker of oxidative stress caused by hyperoxia and bacterial lipopolysaccharide (LPS) exposure in neonatal rat lungs. We examined the tissue redox states of lungs from four groups of rat pups: normoxic (21% O2) pups, hyperoxic (90% O2) pups, pups treated with LPS (normoxic + LPS), and pups treated with LPS and hyperoxia (hyperoxic + LPS). Our results show that hyperoxia oxidized the respiratory chain as reflected by a ~31% decrease in lung tissue NADH RR as compared to that for normoxic lungs. LPS treatment alone or with hyperoxia had no significant effect on lung tissue NADH RR as compared to that for normoxic or hyperoxic lungs, respectively. Thus, NADH RR serves as a quantitative marker of oxidative stress level in lung injury caused by two clinically important conditions: hyperoxia and LPS exposure.

Mitochondrial redox studies of oxidative stress in kidneys from diabetic mice.

Chronic hyperglycemia during diabetes leads to increased production of reactive oxygen species (ROS) and increased oxidative stress (OS). Here we investigated whether changes in the metabolic state can be used as a marker of OS progression in kidneys. We examined redox states of kidneys from diabetic mice, Akita(/+) and Akita(/+);TSP1(-/-) mice (Akita mice lacking thrombospondin-1, TSP1) with increasing duration of diabetes. OS as measured by mitochondrial redox ratio (NADH/FAD) was detectable shortly after the onset of diabetes and further increased with the duration of diabetes. Thus, cryo fluorescence redox imaging was used as a quantitative marker of OS progression in kidneys from diabetic mice and demonstrated that alterations in the oxidative state of kidneys occur during the early stages of diabetes.

Optical imaging of tissue mitochondrial redox state in intact rat lungs in two models of pulmonary oxidative stress.

Ventilation with enhanced fractions of O(2) (hyperoxia) is a common and necessary treatment for hypoxemia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low-temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O(2)) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs.