Alteration in Fluidity of Cell Plasma Membrane in Huntington Disease Revealed by Spectral Phasor Analysis.

Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN. Using phasor approach, we characterized the fluorescence of LAURDAN that is sensitive to the polarity of the immediate environment. LAURDAN is affected by the physical order of phospholipids (lipid order) and reports the membrane fluidity. We also validated our results using a different fluorescent membrane probe, Nile Red (NR). The plasma membrane in the cells expressing expanded polyQ shows a shift toward increased membrane fluidity revealed by both LAURDAN and NR spectral phasors. This finding brings a new perspective in the understanding of the early stages of HD that can be used as a target for drug screening

The phasor-FLIM fingerprints reveal shifts from OXPHOS to enhanced glycolysis in Huntington Disease.

Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher free NADH suggesting a possible synergism between metabolic dysfunction and transcriptional regulation. Our results indicate that metabolic dysfunction in HD shifts to increased glycolysis leading to oxidative stress and cell death. This powerful label free method can be used to screen native HD tissue samples and for potential drug screening

Reversibility of Age-related Oxidized Free NADH Redox States in Alzheimer's Disease Neurons by Imposed External Cys/CySS Redox Shifts.

Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/NAD+) are critical for maintaining redox homeostasis. Aging as a major risk factor for Alzheimer's disease (AD) is associated with oxidative shifts, decreases in anti-oxidant protection and dysfunction of mitochondria. Here, we examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. We used phasor fluorescence lifetime imaging microscopy (FLIM) to distinguish free and bound NADH in mitochondria, nuclei and cytoplasm. Under an external oxidative stress, a lower capacity for maintaining mitochondrial free NADH levels was found in old compared to young neurons and a further decline with genetic load. Remarkably, an imposed Cys/CySS reductive state rejuvenated the mitochondrial free NADH levels of old NTg neurons by 71% and old 3xTg-AD neurons by 89% to levels corresponding to the young neurons. Using FLIM as a non-invasive approach, we were able to measure the reversibility of aging subcellular free NADH levels in live neurons. Our results suggest a potential reductive treatment to reverse the loss of free NADH in old and Alzheimer's neurons

Method measuring oxygen tension and transport within subcutaneous devices

Cellular therapies hold promise to replace the implantation of whole organs in the treatment of disease. For most cell types, in vivo viability depends on oxygen delivery to avoid the toxic effects of hypoxia. A promising approach is the in situ vascularization of implantable devices which can mediate hypoxia and improve both the lifetime and utility of implanted cells and tissues. Although mathematical models and bulk measurements of oxygenation in surrounding tissue have been used to estimate oxygenation within devices, such estimates are insufficient in determining if supplied oxygen is sufficient for the entire thickness of the implanted cells and tissues. We have developed a technique in which oxygen-sensitive microparticles (OSMs) are incorporated into the volume of subcutaneously implantable devices. Oxygen partial pressure within these devices can be measured directly in vivo by an optical probe placed on the skin surface. As validation, OSMs have been incorporated into alginate beads, commonly used as immunoisolation devices to encapsulate pancreatic islet cells. Alginate beads were implanted into the subcutaneous space of Sprague–Dawley rats. Oxygen transport through beads was characterized from dynamic OSM signals in response to changes in inhaled oxygen. Changes in oxygen dynamics over days demonstrate the utility of our technology

Pulmonary Manifestations of SARS Co V 2 Infection in Mild/Severe Patients

The coronavirus disease 2019 (COVID-19) caused viral pneumonia in Wuhan City in China in December of 2019. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily targets the lungs with severe hypoxia, which usually results in death. COVID-19 is highly heterogeneous regarding severity, clinical phenotype, and more importantly, global dispersal. The respiratory system in all aspects such as respiratory airways, endothelium of pulmonary vessels, conducting airways, the alveoli, neuromuscular breathing structure, and pulmonary circulation are affected by this virus. A comprehensive concept of the source and dynamic action of the SARS-CoV-2 and the possible causes of heterogeneity in COVID-19 is required for predicting and managing the illness in acute and chronic stages of the pulmonary sign

Multiphoton imaging and phasor approach to identify new biomarkers in Huntington Disease

Neurodegenerative diseases occur when brain cells (neurons) start to deteriorate. Changes in these cells will lead to dysfunction and eventual cell death. This will lead to mild symptoms like problems with coordination, psychiatric disorders, or memory loss; and as more neurons die the symptoms also progressively worsen. World Health Organization (WHO) indicates up to 1 billion people worldwide are affected by various types of neurodegenerative diseases. In this study, I focused on Huntington disease (HD), a model to study neurodegeneration that is caused by a glitch in a single gene called huntingtin gene (HTT). Huntington disease is an autosomal dominant inherited neurodegenerative disease characterized by movement, cognitive and emotional disorders. We all carry HTT; however, the normal length of DNA trinucleotide, CAG, that codes for glutamine are between 10-35. The expanded repeats of above 40 or more will lead to HD. Using advanced functional imaging technique called Two-Photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM), and spectral and temporal phasor approach, spectro-temporal phasor map in living mammalian cells and animal tissue was obtained. Using this sophisticated imaging technique, I have developed new methods and identified novel biomarkers that can help detect Huntington disease early on. This can also help for evaluating the efficacy of treatment. The novel method established in this work is noninvasive and can be performed at the single cell level. Phasor transformation used here simplifies the FLIM and spectral measurements by providing a graphical global view of the process at each pixel and avoids some of the complexity of the multi-exponential analysis. In this way, using a fit free approach that can be applied to both time and frequency domain measurements, Fluorescence Lifetime and spectral emission can be analyzed. It is hoped that this work shed a light on understanding the mechanism of Huntington disease and for new drug discovery and early diagnosis of the disease. The approach introduced in this work can also be applied as a method for understating similar neurodegenerative diseases. This work is supported in part by NIH grant P41 GM103540, NSF BEST IGERT and UC PDY grant

Multiphoton imaging and phasor approach to identify new biomarkers in Huntington Disease

Neurodegenerative diseases occur when brain cells (neurons) start to deteriorate. Changes in these cells will lead to dysfunction and eventual cell death. This will lead to mild symptoms like problems with coordination, psychiatric disorders, or memory loss; and as more neurons die the symptoms also progressively worsen. World Health Organization (WHO) indicates up to 1 billion people worldwide are affected by various types of neurodegenerative diseases. In this study, I focused on Huntington disease (HD), a model to study neurodegeneration that is caused by a glitch in a single gene called huntingtin gene (HTT). Huntington disease is an autosomal dominant inherited neurodegenerative disease characterized by movement, cognitive and emotional disorders. We all carry HTT; however, the normal length of DNA trinucleotide, CAG, that codes for glutamine are between 10-35. The expanded repeats of above 40 or more will lead to HD. Using advanced functional imaging technique called Two-Photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM), and spectral and temporal phasor approach, spectro-temporal phasor map in living mammalian cells and animal tissue was obtained. Using this sophisticated imaging technique, I have developed new methods and identified novel biomarkers that can help detect Huntington disease early on. This can also help for evaluating the efficacy of treatment. The novel method established in this work is noninvasive and can be performed at the single cell level. Phasor transformation used here simplifies the FLIM and spectral measurements by providing a graphical global view of the process at each pixel and avoids some of the complexity of the multi-exponential analysis. In this way, using a fit free approach that can be applied to both time and frequency domain measurements, Fluorescence Lifetime and spectral emission can be analyzed. It is hoped that this work shed a light on understanding the mechanism of Huntington disease and for new drug discovery and early diagnosis of the disease. The approach introduced in this work can also be applied as a method for understating similar neurodegenerative diseases. This work is supported in part by NIH grant P41 GM103540, NSF BEST IGERT and UC PDY grant

Investigating factors affecting university students\u27 use of subway before and after COVID-19 outbreak: A case study in Tehran

Identifying and examining factors affecting the use of the subway is critical for developing countries as they struggle with high levels of auto use and resulting congestion, noise and air pollution. In this research, we surveyed students of a top-ranked university in the capital of Iran before and after the COVID-19 outbreak to identify the factors affecting their use of the subway. Chi-square tests show that gender, level of education, and being the only child of the family have the highest impact on using a private car. These variables had no significant influence on students\u27 mode choice to university before the COVID-19 pandemic, when students\u27 mode choice was only a function of their residence location. However, the pandemic has affected priorities for mode choice. For instance, hygiene and social distancing, which were previously insignificant to students, are now among their top criteria, and travel time and cost are less important for students than in the past. As a result, subway use has significantly decreased. Based on the results of the research, when making relevant policies, more attention should be paid to the groups of women, undergraduate students and single children that are more likely to use private cars