Mitochondrial Permeability Transition, Cell Death and Neurodegeneration.

Neurodegenerative diseases are chronic conditions occurring when neurons die in specific brain regions that lead to loss of movement or cognitive functions. Despite the progress in understanding the mechanisms of this pathology, currently no cure exists to treat these types of diseases: for some of them the only help is alleviating the associated symptoms. Mitochondrial dysfunction has been shown to be involved in the pathogenesis of most the neurodegenerative disorders. The fast and transient permeability of mitochondria (the mitochondrial permeability transition, mPT) has been shown to be an initial step in the mechanism of apoptotic and necrotic cell death, which acts as a regulator of tissue regeneration for postmitotic neurons as it leads to the irreparable loss of cells and cell function. In this study, we review the role of the mitochondrial permeability transition in neuronal death in major neurodegenerative diseases, covering the inductors of mPTP opening in neurons, including the major ones-free radicals and calcium-and we discuss perspectives and difficulties in the development of a neuroprotective strategy based on the inhibition of mPTP in neurodegenerative disorders.

Detection of NADH and NADPH levels in vivo identifies shift of glucose metabolism in cancer to energy production.

Profound changes in the metabolism of cancer cells have been known for almost 100 years, and many aspects of these changes have continued to be actively studied and discussed. Differences in the results of various studies can be explained by the diversity of tumours, which have differing processes of energy metabolism, and by limitations in the methods used. Here, using fluorescence lifetime needle optical biopsy in a hepatocellular carcinoma (HCC) mouse model and patients with HCC, we measured reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in control liver, and in HCC tumours and their adjacent regions. We found that NADH level (mostly responsible for energy metabolism) is increased in tumours but also in adjacent regions of the same liver. NADPH level is significantly decreased in the tumours of patients but increased in the HCC mouse model. However, in the ex vivo tumour slices of mouse HCC, reactive oxygen species production and glutathione level (both dependent on NADPH) were significantly suppressed. Thus, glucose-dependent NADH and NADPH production in tumours changed but with a more pronounced shift to energy production (NADH), rather than NADPH synthesis for redox balance.

Deconvolution of the fluorescence spectra measured through a needle probe to assess the functional state of the liver.

OBJECTIVES: Currently, one of the most pressing issues for surgeons in the treatment of obstructive jaundice is the ability to assess the functional state of the liver and to detect and determine the degree of liver failure in a timely manner with simple and objective techniques. In this regard, the use of fluorescence spectroscopy method can be considered as one of the ways to improve the informativity of existing diagnostic algorithms in clinical practice and to introduce new diagnostic tools. Thus, the aim of the work was to study in vivo the functional state of liver parenchyma by the method of fluorescence spectroscopy implemented through a needle probe and assess the contribution of the main tissue fluorophores to reveal new diagnostic criteria. MATERIALS AND METHODS: We compared data from 20 patients diagnosed with obstructive jaundice and 11 patients without this syndrome. Measurements were performed using a fluorescence spectroscopy method at excitation wavelengths of 365 and 450 nm. Data were collected using a 1 mm fiber optic needle probe. The analysis was based on the comparison of the results of deconvolution with the combinations of Gaussian curves reflecting the contribution of the pure fluorophores in the liver tissues. RESULTS: The results showed a statistically significant increase in the contribution of curves reflecting NAD(P)H fluorescence, bilirubin, and flavins in the group of patients with obstructive jaundice. This and the calculated redox ratio values indicated that the energy metabolism of the hepatocytes may have shifted to glycolysis due to hypoxia. An increase in vitamin A fluorescence was also observed. It may also serve as a marker of liver damage, indicating impaired vitamin A mobilization from the liver due to cholestasis. CONCLUSIONS: The results obtained reflect changes associated with shifts in the content of the main fluorophores characterizing hepatocyte dysfunction caused by accumulation of bilirubin and bile acids and after disturbance of oxygen utilization. The contributions of NAD(P)H, flavins, and bilirubin as well as vitamin A can be used for further studies as promising diagnostic and prognostic markers for the course of liver failure. Further work will include collecting fluorescence spectroscopy data in patients with different clinical effects of obstructive jaundice on postoperative clinical outcome after biliary decompression.

Laser-induced singlet oxygen selectively triggers oscillatory mitochondrial permeability transition and apoptosis in melanoma cell lines.

Singlet oxygen (1O2) is an electronically excited state of triplet oxygen which is less stable than molecular oxygen in the electronic ground state and produced by photochemical, thermal, chemical, or enzymatic activation of O2. Although the role of singlet oxygen in biology and medicine was intensively studied with photosensitisers, using of these compounds is limited due to toxicity and lack of selectivity. We generated singlet oxygen in the skin fibroblasts and melanoma cell lines by 1267 nm laser irradiation. It did not induce production of superoxide anion, hydrogen peroxide or activation of lipid peroxidation in these cells confirming high selectivity of 1267 nm laser to singlet oxygen. 1O2 did not change mitochondrial membrane potential (ΔΨm) in skin fibroblasts but induced fluctuation in ΔΨm and complete mitochondrial depolarisation due to opening permeability transition pore in B16 melanoma cells. 1267 nm irradiation did not change the percentage of fibroblasts with necrosis but significantly increased the number of B16 melanoma cells with apoptosis. Thus, singlet oxygen can induce apoptosis in cancer B16 melanoma cells by opening of mitochondrial permeability transition pore (PTP) but not in control fibroblasts.

Evaluation of Wheat Resistance to Snow Mold Caused by Microdochium nivale (Fr) Samuels and I.C. Hallett under Abiotic Stress Influence in the Central Non-Black Earth Region of Russia.

Microdochium nivale is one of the most harmful fungal diseases, causing colossal yield losses and deteriorating grain quality. Wheat genotypes from the world collection of the N.I. Vavilov Institute (VIR) were evaluated for fifty years to investigate their resistance to biotic stress factors (M. nivale). Between 350 to 1085 of winter wheat genotypes were investigated annually. Ten out of fifty years were identified as rot epiphytotics (1978, 1986, 1989, 1990, 1993, 1998, 2001, 2003, 2005 and 2021). The wheat collection was investigated by following the VIR methodological requirements and CMEA unified classification of Triticum aestivum L. The field investigations were carried out in the early spring during fixed-route observations and data collection was included on the spread and development degree of the disease, followed by microbiological and microscopic pathogen identifications. The observations revealed that the primary reason for pink snow mold to infect the wheat crops was abiotic stress factors, such as thawed soil covered in snow that increased the soil temperature by 1.0-4.6 °C above normal. Under these conditions, the plants kept growing, quickly exhausting their carbohydrate and protein resources, thus weakening their immune systems, which made them an easy target for different infections, mainly cryophilic fungi, predominantly Microdochium nivale in the Moscow region. In some years, the joint effect of abiotic and biotic stresses caused crop failure, warranting the replanting of the spring wheat. The investigated wheat genotypes exhibited variable resistance to pink snow mold. The genotypes Mironovskaya 808 (k-43920) from Ukraine;l Nemchinovskaya 846 (k-56861), from Russia; Novobanatka (k-51761) from Yugoslavia; Liwilla (k-57580) from Poland; Zdar (UH 7050) from the Czech Republic; Maris Plowman (k-57944) from the United Kingdom; Pokal (k-56827) from Austria; Hvede Sarah (k-56289) from Denmark; Moldova 83 (k-59750) from Romania; Compal (k-57585) from Germany; Linna (k-45889) from Finland and Kehra (k-34228) from Estonia determined the sources, stability and tolerance to be used in advanced breeding programs.

Fluorescence lifetime needle optical biopsy discriminates hepatocellular carcinoma.

This work presents results of in vivo and in situ measurements of hepatocellular carcinoma by a developed optical biopsy system. Here, we describe the technical details of the implementation of fluorescence lifetime and diffuse reflectance measurements by the system, equipped with an original needle optical probe, compatible with the 17.5G biopsy needle standard. The fluorescence lifetime measurements observed by the setup were verified in fresh solutions of NADH and FAD++, and then applied in a murine model for the characterisation of inoculated hepatocellular carcinoma (HCC) and adjacent liver tissue. The technique, applied in vivo and in situ and supplemented by measurements of blood oxygen saturation, made it possible to reveal statistically significant transformation in the set of measured parameters linked with the cellular pools of NADH and NADPH. In the animal model, we demonstrate that the characteristic changes in registered fluorescent parameters can be used to reliably distinguish the HCC tissue, liver tissue in the control, and the metabolically changed liver tissues of animals with the developed HCC tumour. For further transition to clinical applications, the optical biopsy system was tested during the routing procedure of the PNB in humans with suspected HCC. The comparison of the data from murine and human HCC tissues suggests that the tested animal model is generally representative in the sense of the registered fluorescence lifetime parameters, while statistically significant differences between their absolute values can still be observed.

Interaction of Mitochondrial Calcium and ROS in Neurodegeneration.

Neurodegenerative disorders are currently incurable devastating diseases which are characterized by the slow and progressive loss of neurons in specific brain regions. Progress in the investigation of the mechanisms of these disorders helped to identify a number of genes associated with familial forms of these diseases and a number of toxins and risk factors which trigger sporadic and toxic forms of these diseases. Recently, some similarities in the mechanisms of neurodegenerative diseases were identified, including the involvement of mitochondria, oxidative stress, and the abnormality of Ca2+ signaling in neurons and astrocytes. Thus, mitochondria produce reactive oxygen species during metabolism which play a further role in redox signaling, but this may also act as an additional trigger for abnormal mitochondrial calcium handling, resulting in mitochondrial calcium overload. Combinations of these factors can be the trigger of neuronal cell death in some pathologies. Here, we review the latest literature on the crosstalk of reactive oxygen species and Ca2+ in brain mitochondria in physiology and beyond, considering how changes in mitochondrial metabolism or redox signaling can convert this interaction into a pathological event.

Spatial heterogeneity of cutaneous blood flow respiratory-related oscillations quantified via laser speckle contrast imaging.

LSCI technique provides experimental data which can be considered in the context of spatial blood flow coherency. Analysis of vascular tone oscillations gives additional information to ensure a better understanding of the mechanisms affecting microvascular physiology. The oscillations with different frequencies are due to different physiological mechanisms. The reasons for the generation of peripheral blood flow oscillations in the 0.14-0.6 Hz frequency band are as follows: cardio-respiratory interactions, pressure variations in the venous part of the circulatory system, and the effect of the sympathetic nervous system on the vascular tone. Earlier, we described the spatial heterogeneity of around 0.3 Hz oscillations and this motivated us to continue the research to find the conditions for the occurrence of spatial phase synchronization. For this purpose, a number of physiological tests (controlled respiration, breath holder, and venous occlusion tests) which influence the blood flow oscillations of 0.14-0.6 Hz were considered, an appropriate measurement system and the required data processing algorithms were developed. At spontaneous respiration, the oscillations with frequencies around 0.3 Hz were stochastic, whereas all the performed tests induced an increase in spatial coherence. The protocols and methods proposed here can help to clarify whether the heterogeneity of respiratory-related blood flow oscillations exists on the skin surface.

Machine Learning Aided Photonic Diagnostic System for Minimally Invasive Optically Guided Surgery in the Hepatoduodenal Area.

Abdominal cancer is a widely prevalent group of tumours with a high level of mortality if diagnosed at a late stage. Although the cancer death rates have in general declined over the past few decades, the mortality from tumours in the hepatoduodenal area has significantly increased in recent years. The broader use of minimal access surgery (MAS) for diagnostics and treatment can significantly improve the survival rate and quality of life of patients after surgery. This work aims to develop and characterise an appropriate technical implementation for tissue endogenous fluorescence (TEF) and assess the efficiency of machine learning methods for the real-time diagnosis of tumours in the hepatoduodenal area. In this paper, we present the results of the machine learning approach applied to the optically guided MAS. We have elaborated tissue fluorescence approach with a fibre-optic probe to record the TEF and blood perfusion parameters during MAS in patients with cancers in the hepatoduodenal area. The measurements from the laser Doppler flowmetry (LDF) channel were used as a sensor of the tissue vitality to reduce variability in TEF data. Also, we evaluated how the blood perfusion oscillations are changed in the tumour tissue. The evaluated amplitudes of the cardiac (0.6-1.6 Hz) and respiratory (0.2-0.6 Hz) oscillations was significantly higher in intact tissues (p < 0.001) compared to the cancerous ones, while the myogenic (0.2-0.06 Hz) oscillation did not demonstrate any statistically significant difference. Our results demonstrate that a fibre-optic TEF probe accompanied with ML algorithms such as k-Nearest Neighbours or AdaBoost is highly promising for the real-time in situ differentiation between cancerous and healthy tissues by detecting the information about the tissue type that is encoded in the fluorescence spectrum. Also, we show that the detection can be supplemented and enhanced by parallel collection and classification of blood perfusion oscillations.

Optical percutaneous needle biopsy of the liver: a pilot animal and clinical study.

This paper presents the results of the experiments which were performed using the optical biopsy system specially developed for in vivo tissue classification during the percutaneous needle biopsy (PNB) of the liver. The proposed system includes an optical probe of small diameter acceptable for use in the PNB of the liver. The results of the feasibility studies and actual tests on laboratory mice with inoculated hepatocellular carcinoma and in clinical conditions on patients with liver tumors are presented and discussed. Monte Carlo simulations were carried out to assess the diagnostic volume and to trace the sensing depth. Fluorescence and diffuse reflectance spectroscopy measurements were used to monitor metabolic and morphological changes in tissues. The tissue oxygen saturation was evaluated using a recently developed approach to neural network fitting of diffuse reflectance spectra. The Support Vector Machine Classification was applied to identify intact liver and tumor tissues. Analysis of the obtained results shows the high sensitivity and specificity of the proposed multimodal method. This approach allows to obtain information before the tissue sample is taken, which makes it possible to significantly reduce the number of false-negative biopsies.

Interaction of Oxidative Stress and Misfolded Proteins in the Mechanism of Neurodegeneration.

Aggregation of the misfolded proteins ß-amyloid, tau, huntingtin, and α-synuclein is one of the most important steps in the pathology underlying a wide spectrum of neurodegenerative disorders, including the two most common ones-Alzheimer's and Parkinson's disease. Activity and toxicity of these proteins depends on the stage and form of aggregates. Excessive production of free radicals, including reactive oxygen species which lead to oxidative stress, is proven to be involved in the mechanism of pathology in most of neurodegenerative disorders. Both reactive oxygen species and misfolded proteins play a physiological role in the brain, and only deregulation in redox state and aggregation of the proteins leads to pathology. Here, we review the role of misfolded proteins in the activation of ROS production from various sources in neurons and glia. We discuss if free radicals can influence structural changes of the key toxic intermediates and describe the putative mechanisms by which oxidative stress and oligomers may cause neuronal death.

Wavelet Analysis of the Temporal Dynamics of the Laser Speckle Contrast in Human Skin.

OBJECTIVE: Spectral analysis of laser Doppler flowmetry (LDF) signals has been widely used in studies of physiological vascular function regulation. An alternative to LDF is the laser speckle contrast imaging method (LSCI), which is based on the same physical principle. In contrast to LDF, LSCI provides non-scanning full-field imaging of a relatively wide skin area and offers high spatial and temporal resolutions, which allows visualization of microvascular structure. This circumstance, together with a large number of works which had shown the effectiveness of temporal LSCI analysis, gave impetus to experimental studies of the relation between LDF and LSCI used to monitor the temporal dynamics of blood flow. METHODS: Continuous wavelet transform was applied to construct a time-frequency representation of a signal. RESULTS: Analysis of 10 minute LDF and LSCI output signals recorded simultaneously revealed rather high correlation between oscillating components. It was demonstrated for the first time that the spectral energy of oscillations in the 0.01-2 Hz frequency range of temporal LSCI recordings carries the same information as the conventional LDF recordings and hence it reflects the same physiological vascular tone regulation mechanisms. CONCLUSION: The approach proposed can be used to investigate speckle pattern dynamics by LSCI in both normal and pathological conditions. SIGNIFICANCE: The results of research on the influence of spatial binning and averaging on the spectral characteristics of perfusion monitored by LSCI are of considerable interest for the development of LSCI systems optimized to evaluate temporal dynamics.

Optical probe pressure effects on cutaneous blood flow.

The variation of blood flow characteristics caused by the probe pressure during noninvasive studies is of particular interest within the context of fundamental and applied research. It has been shown previously that the weak local pressure induces vasodilation, whereas the increased pressure is able to stop the blood flow in the compressed area, as well as to significantly change optical signals.The blood flow oscillations measured by laser Doppler flowmetry (LDF) characterize the functional state of the microvascular system and can be used for noninvasive diagnostics of its abnormality. This study was intended to identify the patterns of the relationship between the oscillating components of blood flow registered by the LDF method under different levels of pressure applied to an optical fiber probe.For this purpose, we have developed an original optical probe capable of regulating the applied pressure. The developed protocol included six sequential records of the blood perfusion at a pressure within the 0 to 200 mmHg range with unloading at the last stage.Using wavelet analyses, we traced the variation of energy of oscillations for these records in five frequency bands associated with different vascular tone regulation mechanisms. Six young volunteers of the same age (three males and three females) were included in this preliminary study and the protocol was repeated five times in each volunteer. Accordingly, 30 LDF records were available for the analyses. As expected, the LDF signal increases at weak pressure (30 mmHg) and decreases at increased pressure. The statistically stable amplification of endothelial associated blood flow oscillations under the 90 mmHg pressure allowed us to put forward a hypothesis that the endothelial activity increases. The possible causes of this phenomenon are discussed.

Spectral analysis of the blood flow in the foot microvascular bed during thermal testing in patients with diabetes mellitus.

Timely diagnostics of microcirculatory system abnormalities, which are the most severe diabetic complications, is one of the major problems facing modern health care. Functional abnormalities manifest themselves earlier than the structural ones, and therefore their assessment is the issue of primary importance. In this study Laser Doppler flowmetry, a noninvasive technique for the cutaneous blood flow monitoring, was utilized together with local temperature tests and wavelet analysis. The study of the blood flow in the microvascular bed of toes was carried out in the control group of 40 healthy subjects and in two groups of 17 type 1 and 23 type 2 diabetic patients. The local temperature tests demonstrated that the diabetic patients have impaired vasodilation in response to local heating. The tendency for impaired low frequency pulsations of the blood flow associated with endothelial and neurogenic activities in both diabetes groups was observed. Local thermal tests induced variations in perfusion and its spectral characteristics, which were different in the groups under study. In our opinion, the obtained preliminary results can be a basis for further research and provide a deeper understanding of pathological processes that drive microvascular abnormalities caused by diabetes mellitus.

Multimodal optical measurement for study of lower limb tissue viability in patients with diabetes mellitus.

According to the International Diabetes Federation, the challenge of early stage diagnosis and treatment effectiveness monitoring in diabetes is currently one of the highest priorities in modern healthcare. The potential of combined measurements of skin fluorescence and blood perfusion by the laser Doppler flowmetry method in diagnostics of low limb diabetes complications was evaluated. Using Monte Carlo probabilistic modeling, the diagnostic volume and depth of the diagnosis were evaluated. The experimental study involved 76 patients with type 2 diabetes mellitus. These patients were divided into two groups depending on the degree of complications. The control group consisted of 48 healthy volunteers. The local thermal stimulation was selected as a stimulus on the blood microcirculation system. The experimental studies have shown that diabetic patients have elevated values of normalized fluorescence amplitudes, as well as a lower perfusion response to local heating. In the group of people with diabetes with trophic ulcers, these parameters also significantly differ from the control and diabetes only groups. Thus, the intensity of skin fluorescence and level of tissue blood perfusion can act as markers for various degrees of complications from the beginning of diabetes to the formation of trophic ulcers.