Lipofuscin-mediated photodynamic stress induces adverse changes in nanomechanical properties of retinal pigment epithelium cells

Abstract Retinal pigment epithelium (RPE) is an important part of the blood-retina barrier (BRB) that separates the retina from the choroid. Although melanin granules contribute to the mechanical stability of the BRB complex, it is unknown if the age pigment lipofuscin affects mechanical properties of the tissue. To address this issue the effect of sub-lethal photic stress mediated by phagocytized lipofuscin granules, isolated from RPE of human donors, on morphology and mechanical properties of ARPE-19 cells was investigated. Nanomechanical analysis using atomic force spectroscopy revealed that irradiation of cells containing lipofuscin granules with blue light induced significant softening of the cells, which was accompanied by substantial reorganization of the cell cytoskeleton due to peroxidation of cellular proteins. Our results indicate that lipofuscin-mediated photic stress can cause significant modification of the RPE cells with the potential to disturb biological function of the BRB complex

The effect of antioxidants on photoreactivity and phototoxic potential of RPE melanolipofuscin granules from human donors of different age

One of the most prominent age-related changes of retinal pigment epithelium (RPE) is the accumulation of melanolipofuscin granules, which could contribute to oxidative stress in the retina. The purpose of this study was to determine the ability of melanolipofuscin granules from younger and older donors to photogenerate reactive oxygen species, and to examine if natural antioxidants could modify the phototoxic potential of this age pigment. Electron paramagnetic resonance (EPR) oximetry, EPR-spin trapping, and time-resolved detection of near-infrared phosphorescence were employed for measuring photogeneration of superoxide anion and singlet oxygen by melanolipofuscin isolated from younger and older human donors. Phototoxicity mediated by internalized melanolipofuscin granules with and without supplementation with zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells by determining cell survival, oxidation of cellular proteins, organization of the cell cytoskeleton, and the cell specific phagocytic activity. Supplementation with antioxidants reduced aerobic photoreactivity and phototoxicity of melanolipofuscin granules. The effect was particularly noticeable for melanolipofuscin mediated inhibition of the cell phagocytic activity. Antioxidants decreased the extent of melanolipofuscin-dependent oxidation of cellular proteins and disruption of the cell cytoskeleton. Although melanolipofuscin might be involved in chronic phototoxicity of the aging RPE, natural antioxidants could partially ameliorate these harmful effects

Bronchial fibroblasts from asthmatic patients display impaired responsiveness to direct current electric fields (dcEFs)

Accumulating evidence suggests that an important role is played by electric signals in modifying cell behaviour during developmental, regenerative and pathological processes. However, their role in asthma has not yet been addressed. Bronchial fibroblasts have recently been identified having important roles in asthma development. Therefore, we adapted an experimental approach based on the lineages of human bronchial fibroblasts (HBF) derived from non-asthmatic (NA) donors and asthmatic (AS) patients to elucidate whether their reactivity to direct current electric fields (dcEF) could participate in the asthmatic process. The efficient responsiveness of NA HBF to an electric field in the range of 2-4 V/cm was illustrated based on the perpendicular orientation of long axes of the cells to the field lines and their directional movement towards the anode. These responses were related to the activity of TGF-$\beta$ signalling, as the electrotaxis and re-orientation of NA HBF polarity was impaired by the inhibitors of canonical and non-canonical TGF-$\beta$ -dependent pathways. A similar tendency towards perpendicular cell-dcEF orientation was observed for AS HBF. However, their motility remained insensitive to the electric field applied at 2-4 V/cm. Collectively, these observations demonstrate the sensitivity of NA HBF to dcEF, as well as the inter-relations between this parameter and the canonical and non-canonical TGF-$\beta$ pathways, and the differences between the electrotactic responses of NA and AS HBF point to the possible role of their dcEFs in desensitisation in the asthmatic process. This process may impair the physiologic behaviour of AS HBF functions, including cell motility, ECM deposition, and contractility, thus promoting bronchial wall remodelling, which is a characteristic of bronchial asthma

Fenofibrate reduces the asthma-related fibroblast-to-myofibroblast transition by TGF-Β/Smad2/3 signaling attenuation and connexin 43-dependent phenotype destabilization

The activation of human bronchial fibroblasts by transforming growth factor-β1 (TGF-β1) leads to the formation of highly contractile myofibroblasts in the process of the fibroblast–myofibroblast transition (FMT). This process is crucial for subepithelial fibrosis and bronchial wall remodeling in asthma. However, this process evades current therapeutic asthma treatment strategies. Since our previous studies showed the attenuation of the TGF-β1-induced FMT in response to lipid-lowering agents (e.g., statins), we were interested to see whether a corresponding effect could be obtained upon administration of hypolipidemic agents. In this study, we investigated the effect of fenofibrate on FMT efficiency in populations of bronchial fibroblasts derived from asthmatic patients. Fenofibrate exerted a dose-dependent inhibitory effect on the FMT, even though it did not efficiently affect the expression of α-smooth muscle actin (α-SMA; marker of myofibroblasts); however, it considerably reduced its incorporation into stress fibers through connexin 43 regulation. This effect was accompanied by disturbances in the actin cytoskeleton architecture, impairments in the maturation of focal adhesions, and the fenofibrate-induced deactivation of TGF-β1/Smad2/3 signaling. These data suggest that fenofibrate interferes with myofibroblastic differentiation during asthma-related subepithelial fibrosis. The data indicate the potential application of fenofibrate in the therapy and prevention of bronchial remodeling during the asthmatic process

A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond

According to the World Health Organization (WHO), almost 2 billion people each year are infected worldwide with flu-like pathogens including influenza. This is a contagious disease caused by viruses belonging to the family Orthomyxoviridae. Employee absenteeism caused by flu infection costs hundreds of millions of dollars every year. To successfully treat influenza virus infections, detection of the virus during the initial development phase of the infection is critical, when tens to hundreds of virus-associated molecules are present in the patient’s pharynx. In this study, we describe a novel universal diamond biosensor, which enables the specific detection of the virus at ultralow concentrations, even before any clinical symptoms arise. A diamond electrode is surface-functionalized with polyclonal anti-M1 antibodies, which then serve to identify the universal biomarker for the influenza virus, M1 protein. The absorption of the M1 protein onto anti-M1 sites of the electrode change its electrochemical impedance spectra. We achieved a limit of detection of 1 fg/ml in saliva buffer for the M1 biomarker, which corresponds to 5–10 viruses per sample in 5 minutes. Furthermore, the universality of the assay was confirmed by analyzing different strains of influenza A virus

A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond

According to the World Health Organization (WHO), almost 2 billion people each year are infected worldwide with flu-like pathogens including influenza. This is a contagious disease caused by viruses belonging to the family Orthomyxoviridae. Employee absenteeism caused by flu infection costs hundreds of millions of dollars every year. To successfully treat influenza virus infections, detection of the virus during the initial development phase of the infection is critical, when tens to hundreds of virus-associated molecules are present in the patient’s pharynx. In this study, we describe a novel universal diamond biosensor, which enables the specific detection of the virus at ultralow concentrations, even before any clinical symptoms arise. A diamond electrode is surface-functionalized with polyclonal anti-M1 antibodies, which then serve to identify the universal biomarker for the influenza virus, M1 protein. The absorption of the M1 protein onto anti-M1 sites of the electrode change its electrochemical impedance spectra. We achieved a limit of detection of 1 fg/ml in saliva buffer for the M1 biomarker, which corresponds to 5–10 viruses per sample in 5 minutes. Furthermore, the universality of the assay was confirmed by analyzing different strains of influenza A virus

The basis of cell cryopreservation

Techniki krioprezerwacji (bankowania) znane są naturze od wieków. Wiele gatunków owadów, płazów, ryb, a nawet gadów stosuje naturalne metody krioprezerwacji do przetrwania trudnych warunków zimy lub do życia w ekstremalnie niskich temperaturach. Kriokonserwacja i marzenia o nieśmiertelności od lat intrygowały ludzkość. Pierwsze doniesienia dotyczące obserwacji skutków zamrażania plemników (przechowywanych w śniegu) pochodzą z 1776 r. W 1866 roku Montegazza jako pierwszy zasugerował na ów czas wizję kompletnie niewyobrażalną: „człowiek umierający na polu bitwy może spłodzić następcę prawnego z zamrożonego i przechowywanego w domu nasienia”. Pierwsze, wówczas jeszcze nieskuteczne, doniesienia o laboratoryjnym zamrażaniu ludzkiej spermy pochodzą z lat ’30 ubiegłego stulecia [1]. Wreszcie ludzkość „nauczyła się” krioprezerwacji w połowie XX wieku, kiedy to 15 października 1949 ukazał się drukiem w czasopiśmie Nature artykuł „Odrodzenie plemników po zeszkleniu i odwodnieniu w niskich temperaturach” (oryg. Revival of spermatozoa after vitrification and dehydration at low temperatures) podsumowujący pionierskie badania naukowców z Narodowego Instytutu Badań Medycznych w Mill Hill w Londynie [2]. Dotyczyły one mrożenia nasienia ptactwa domowego w obecności glicerolu, glikolu etylenowego i propylenowego w taki sposób, że po rozmrożeniu było ono zdolne do efektywnego zapłodnienia jaj. Późniejsze użycie dimetylosulfotlenku (DMSO) zrewolucjonizowało współczesną kriobiologię [3-5]. Tak rozpoczęła się era kriokonserwacji, bez której dzisiaj trudno sobie wyobrazić pracę laboratoriów biologii komórki, współczesną hodowlę zwierząt, czy też rozwój współczesnej medycyny.Cryopreservation (banking) techniques have been known to nature for centuries. Many species of insects, amphibians, fish and even reptiles use natural cryopreservation methods to survive the harsh conditions of winter or to live in extremely cold temperatures. Cryopreservation and dreams of immortality have intrigued humanity for years. The first reports of observing the effects of freezing sperm (stored in snow) date back to 1776. In 1866, Montegazza was the first to suggest a vision completely unimaginable for the time: "a man dying on the battlefield can conceive an heir from sperm frozen and stored at home". The first, at that time still unsuccessful, reports of laboratory freezing of human sperm date back to the 1930s [1]. Finally, mankind "learned" cryopreservation in the middle of the twentieth century, when on October 15, 1949, the article "Revival of spermatozoa after vitrification and dehydration at low temperatures" appeared in print in the Nature journal, summarising the pioneering research of scientists from the National Institute for Medical Research, Mill Hill, London [2]. This concerned the freezing of fowl sperm in the presence of glycerol, ethylene glycol and propylene glycol in such a way that after thawing it was able to fertilise eggs effectively. The subsequent use of dimethyl sulfoxide (DMSO) revolutionised modern cryobiology [3-5]. Thus began the era of cryopreservation, without which today it is difficult to imagine the work of cell biology laboratories, modern animal breeding, or the development of modern medicine