Analysis of Electric Field Stimulation in Blue Light Stressed 661W Cells.

Though electrical stimulation is used as a therapeutic approach to treat retinal and spinal injuries, many protective mechanisms at cellular level have not been elucidated. We performed a detailed analysis of cellular events in blue light (Li) stressed 661W cells, which were subjected to direct current electric field (EF) stimulation. Our findings revealed that EF stimulation induced protective effects in 661W cells from Li-induced stress by multiple defense mechanisms, such as increase in mitochondrial activity, gain in mitochondrial potential, increase in superoxide levels, and the activation of unfolded protein response (UPR) pathways, all leading to an enhanced cell viability and decreased DNA damage. Here, our genetic screen results revealed the UPR pathway to be a promising target to ameliorate Li-induced stress by EF stimulation. Thus, our study is important for a knowledgeable transfer of EF stimulation into clinical application.

[New master's program: multiple sclerosis management]. / Neuer Masterstudiengang: Multiple Sklerose Management.

The new study program "Multiple Sclerosis Management" is aimed at physicians, therapists, nurses, scientists, pharmacists, psychologists and biologists who want to specialize in the field of multiple sclerosis (MS). After successful accreditation in 2019, the first students have been in the master's program offered by Dresden International University (DIU) since 2020. Over a period of four semesters, it can be completed part-time and largely digitally. The master's program is divided into six modules focusing on basics, clinical and diagnostic aspects, MS studies and statistics, disease-modifying and symptomatic therapy, disease monitoring and documentation. The teaching includes theoretical parts and numerous practical units. A further goal is to derive therapeutic intervention plans and problem-solving strategies from scientific publications and clinical studies, to develop them further and to apply them in patient care. The lecturers come from Germany, Austria and Switzerland and are predominantly professors. The German Multiple Sclerosis Society is the patron of the course. This article presents the study program in detail.

VEGF-Trap is a potent modulator of vasoregenerative responses and protects dopaminergic amacrine network integrity in degenerative ischemic neovascular retinopathy.

Retinal hypoxia triggers abnormal vessel growth and microvascular hyper-permeability in ischemic retinopathies. Whereas vascular endothelial growth factor A (VEGF-A) inhibitors significantly hinder disease progression, their benefits to retinal neurons remain poorly understood. Similar to humans, oxygen-induced retinopathy (OIR) mice exhibit severe retinal microvascular malformations and profound neuronal dysfunction. OIR mice are thus a phenocopy of human retinopathy of prematurity, and a proxy for investigating advanced stages of proliferative diabetic retinopathy. Hence, the OIR model offers an excellent platform for assessing morpho-functional responses of the ischemic retina to anti-angiogenic therapies. Using this model, we investigated the retinal responses to VEGF-Trap (Aflibercept), an anti-angiogenic agent recognizing ligands of VEGF receptors 1 and 2 that possesses regulatory approval for the treatment of neovascular age-related macular degeneration, macular edema secondary to retinal vein occlusion and diabetic macular edema. Our results indicate that Aflibercept not only reduces the severity of retinal microvascular aberrations but also significantly improves neuroretinal function. Aflibercept administration significantly enhanced light-responsiveness, as revealed by electroretinographic examinations, and led to increased numbers of dopaminergic amacrine cells. Additionally, retinal transcriptional profiling revealed the concerted regulation of both angiogenic and neuronal targets, including transcripts encoding subunits of transmitter receptors relevant to amacrine cell function. Thus, Aflibercept represents a promising therapeutic alternative for the treatment of further progressive ischemic retinal neurovasculopathies beyond the set of disease conditions for which it has regulatory approval. Cover Image for this issue: doi: 10.1111/jnc.14743.

Photobiomodulation Mediates Neuroprotection against Blue Light Induced Retinal Photoreceptor Degeneration.

Potent neuroprotective effects of photobiomodulation with 670 nm red light (RL) have been demonstrated in several models of retinal disease. RL improves mitochondrial metabolism, reduces retinal inflammation and oxidative cell stress, showing its ability to enhance visual function. However, the current knowledge is limited to the main hypothesis that the respiratory chain complex IV, cytochrome c oxidase, serves as the primary target of RL. Here, we demonstrate a comprehensive cellular, molecular, and functional characterization of neuroprotective effects of 670 nm RL and 810 nm near-infrared light (NIRL) on blue light damaged murine primary photoreceptors. We show that respiratory chain complexes I and II are additional PBM targets, besides complex IV, leading to enhanced mitochondrial energy metabolism. Accordingly, our study identified mitochondria related RL- and NIRL-triggered defense mechanisms promoting photoreceptor neuroprotection. The observed improvement of mitochondrial and extramitochondrial respiration in both inner and outer segments is linked with reduced oxidative stress including its cellular consequences and reduced mitochondria-induced apoptosis. Analysis of regulatory mechanisms using gene expression analysis identified upregulation α-crystallins that indicate enhanced production of proteins with protective functions that point to the rescued mitochondrial function. The results support the hypothesis that energy metabolism is a major target for retinal light therapy.

The human platysma contains numerous muscle spindles.

The mimic muscles are usually described as containing no muscle spindles. In the present publication the human platysma was reinvestigated concerning its content of corpuscular sensors. Serial sections through the platysma of seven donors revealed numerous muscle spindles but no Pacini corpuscules. The muscle spindles were located in the cranial two-thirds of the platysma, and were evenly distributed with a tendency to have more spindles in the lateral part of the muscle. Immunohistochemical staining with S46 antibodies revealed a predominance of nuclear bag fibers. The results point to an extended function of the platysma as an afferent center of the lower face mimic muscles.

Changes in the sympathetic innervation of the gut in rotenone treated mice as possible early biomarker for Parkinson's disease.

INTRODUCTION: Involvement of the peripheral nervous system (PNS) is relatively common in Parkinson's disease (PD) patients. PNS alterations appear early in the course of the disease and are responsible for some of the non-motor symptoms observed in PD patients. In previous studies, we have shown that environmental toxins can trigger the disease by acting on the enteric nervous system. MATERIAL AND METHODS: Here, we analyzed the effect of mitochondrial Complex I inhibition on sympathetic neuritis in vivo and sympathetic neurons in vitro. Combining in vivo imaging and protein expression profiling. RESULTS: we found that rotenone, a widely used mitochondrial Complex I inhibitor decreases the density of sympathetic neurites innervating the gut in vivo, while in vitro, it induces the redistribution of intracellular alpha-synuclein and neurite degeneration. Interestingly, sympathetic neurons are much more resistant to rotenone exposure than mesencephalic dopaminergic neurons. CONCLUSION: Altogether, these results suggest that enteric sympathetic denervation could be an initial pre-motor alteration in PD progression that could be used as an early biomarker of the disease.

Stress responses of human retinal pigment epithelial cells to glyoxal.

PURPOSE: Intracellular formation of advanced glycation end products (AGEs) is a crucial pathological process in retinal diseases such as age-related macular degeneration (AMD) or diabetic retinopathy (DR). Glyoxal is a physiological metabolite produced during formation of AGEs and has also been shown to derive from photodegraded bisretinoid fluorophores in aging retinal pigment epithelial (RPE) cells. METHODS: Flow cytometry was combined with either: 1) immunocytochemical staining to detect glyoxal induced formation of Nε-carboxymethyllysine (CML)-modifications of intracellular proteins (AGEs) and changes in the production of stress response proteins; or 2) vital staining to determine apoptosis rates (annexin V binding), formation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and changes in intracellular pH upon treatment of cells with glyoxal. The percentage of apoptotic cells was further quantified by flow cytometry after staining of fixed cells with propidium iodide to determine cells with a subdiploid (fragmented) DNA content. Apoptosis related activation of caspase 3 was determined by Western blotting. Glyoxal induced changes in VEGF-A165a mRNA expression and protein production were determined by real-time PCR and by flow cytometry after immunocytochemical staining. RESULTS: Increasing glyoxal concentrations resulted in enhanced formation of AGEs, such as CML modifications of proteins. This was associated with elevated levels of intracellular reactive oxygen species, a depolarized MMP, and a decreased intracellular pH, resulting in an increased number of apoptotic cells. Apoptosis related caspase 3 activation increased in a dose dependent manner after glyoxal incubation. In consequence, the cells activated compensatory mechanisms and increased the levels of the anti-oxidative and stress-related proteins heme oxygenase-1, osteopontin, heat shock protein 27, copper/zinc superoxide dismutase, manganese superoxide dismutase, and cathepsin D. Furthermore, VEGF-A165a mRNA expression and VEGF-A protein production were significantly increased after incubation with glyoxal in ARPE-19 cells. CONCLUSIONS: The glyoxal-induced oxidative stress and apoptosis in ARPE-19 cells may provide a suitable in vitro model for studying RPE cellular reactions to AGEs that occur in AMD or in DR.

Phospho-NHE3 forms membrane patches and interacts with beta-actin to sense and maintain constant direction during cell migration.

The Na(+)/H(+) exchanger NHE3 colocalizes with beta-actin at the leading edge of directionally migrating cells. Using human osteosarcoma cells (SaOS-2), rat osteoblasts (calvaria), and human embryonic kidney (HEK) cells, we identified a novel role for NHE3 via beta-actin in anode and cathode directed motility, during electrotaxis. NHE3 knockdown by RNAi revealed that NHE3 expression is required to achieve constant directionality and polarity in migrating cells. Phosphorylated NHE3 (pNHE3) and beta-actin complex formation was impaired by the NHE3 inhibitor S3226 (IC50 0.02µM). Fluorescence cross-correlation spectroscopy (FCCS) revealed that the molecular interactions between NHE3 and beta-actin in membrane protrusions increased 1.7-fold in the presence of a directional cue and decreased 3.3-fold in the presence of cytochalasin D. Data from flow cytometric analysis showed that membrane potential of cells (Vmem) decreases in directionally migrating, NHE3-deficient osteoblasts and osteosarcoma cells whereas only Vmem of wild type osteoblasts is affected during directional migration. These findings suggest that pNHE3 has a mechanical function via beta-actin that is dependent on its physiological activity and Vmem. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels increase while PIP2 remains stable when cells have persistent directionality. Both PI3 kinase (PI3K) and Akt expression levels change proportionally to NHE3 levels. Interestingly, however, the content of pNHE3 level does not change when PI3K/Akt is inhibited. Therefore, we conclude that NHE3 can act as a direction sensor for cells and that NHE3 phosphorylation in persistent directional cell migration does not involve PI3K/Akt during electrotaxis.

NHE3 phosphorylation via PKCη marks the polarity and orientation of directionally migrating cells.

Endogenous electric fields (EF) may provide an overriding cue for directional cell migration during wound closure. Perceiving a constant direction requires active sodium-hydrogen exchanger (pNHE3) at the leading edge of HEK 293 cells but its activation mechanism is not yet fully understood. Because protein kinase C (PKC) is required in electrotaxis, we asked whether NHE3 is activated by PKC during wound healing. Using pharmacological (pseudosubstrate and edelfosine) inhibition, we showed that inhibition of PKCη isoform impairs directional cell migration in HEK 293 cells in the presence of a persistent directional cue (0.25-0.3 V/mm of EF for 2 h). Further, we found that pNHE3 forms complexes with both PKCη and ɣ-tubulin, suggesting that these molecules may regulate the microtubule-organizing center. In addition, cellular pNHE3 content was reduced significantly when PKCη was inhibited during directional cell migration. Taken together, these data suggest that PKCη-dependent phosphorylation of NHE3 and the formation of pNHE3/PKCη/ɣ-tubulin complexes at the leading edge of the cell are required for directional cell migration in an EF.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)-the effect of biomolecular ligands to balance cell adhesion and stimulated detachment.

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

Quality assessment of corneal storage media and their components.

BACKGROUND: To keep the loss of endothelial cell density in donor corneas to a minimum, a storage medium which is adjusted to their nutritional needs is necessary. Different media, used either serum-supplemented or serum-free, are available. The quality of medium- and serum-batches as well as support of endothelial cell viability by the medium are to be tested with a quality assured screening system that allows routine examination. METHODS: A screening system was developed which is based on cell-culture tests with the well-established human corneal endothelial cell line HCEC-12, and therefore can be performed without the need for donor corneas. The cells are plated at a defined density in cell-culture dishes, and are cultured for a defined period of time in the test media. Evaluation is carried out by assaying cell count, activity of cell metabolism (resazurin conversion), and determining the number of apoptotic and necrotic cells (combined vital staining with YO-PRO®-1/propidium iodide and subsequent flow cytometry). RESULTS: Human corneal endothelial cells that are cultured in a medium which is adjusted to their nutritional needs achieve higher cell numbers and show a higher metabolic rate. Simultaneously, the percentage of apoptotic and necrotic cells is lower. The screening system developed in this study allows for easy and reliable detection of slightest differences between different media, different processing steps for same media, and different supplements, as well as different serum batches. CONCLUSIONS: The differentiated results show that the screening system is sensitive enough to show even minor quality differences. Therefore, it is more suitable than the hitherto commonly used growth assay with primary, mostly porcine, corneal endothelial cells.

A Narrative Review Exploring the Similarities between Cilento and the Already Defined "Blue Zones" in Terms of Environment, Nutrition, and Lifestyle: Can Cilento Be Considered an Undefined "Blue Zone"?

Longevity is rightly considered one of the greatest achievements of modern society, not only as a function of lifespan, but, more importantly, as a function of healthspan. There are Longevity Blue Zones (LBZs), regions around the world, such as in Okinawa, Japan; the Nicoya Peninsula, Costa Rica; Loma Linda, California; Icaria, Greece; and Ogliastra, Sardinia, that are characterized by a significant percentage of residents who live exceptionally long lives, often avoiding age-related disability to a significantly higher degree than in the Western way of life. Longevity is not a universal phenomenon, so if there are places in the world with characteristics similar to the LBZs, it is important to identify them in order to better understand what other factors, in addition to the known ones, might contribute to a long and healthy life. This narrative review aims to identify common factors between Cilento and the five LBZs, taking into account environmental, nutritional, and lifestyle factors. Articles from 2004 to the present, limited to studies published in English, German, and Italian, were searched in PubMed/Medline, Scopus, and Google Scholar. The co-authors agreed on 18 final reference texts. In order to evaluate the similarities between Cilento and the LBZs, a descriptive comparative approach was used. Cilento and the LBZs share several common factors, including a hilly altitude ranging from 355 to 600 m; a mild climate throughout the year, with temperatures between 17.4 and 23.5 degrees Celsius; traditional professions, such as agriculture and animal husbandry; and a predominantly Mediterranean or plant-based diet, with typical recipes based on legumes, tubers, vegetables, and extra virgin olive oil. Additionally, maintenance of strong intergenerational family relationships, religious devotion, and social relationships within the community are also prevalent. Given the similarities to Cilento, one might wonder if this is an LBZ waiting to be discovered. The lessons learned from this discovery could be applied to the general population to protect them from non-communicable chronic diseases and help slow the aging process.

An epidemiological qualitative/quantitative SWOT-AHP analysis in order to highlight the positive or critical aspects of dental implants: A pilot study.

OBJECTIVES: In recent years, dental implants are increasing in popularity due to their high success rate, demonstrated functionality, and aesthetic treatment results. Scientific research is very active in proposing improvements in the quality and survival of implants, taking into consideration various aspects. The objective of this study was to provide a holistic epidemiologic view of the state of dental implants, using a systematic approach based on a multimethod SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis and AHP (analytical hierarchical process) qualitative-quantitative analysis to identify the characteristics that can determine their success or failure. MATERIALS AND METHODS: The study used the hybrid method of SWOT-AHP. RESULTS: Analysis of the results showed that among strengths, the skill of the dentist was considered the most important factor, followed by the success of dental implants in the old people; among weaknesses, bruxism and chronic diseases were highlighted; for opportunities, biomechanical behavior, in terms of good mechanical strength and good tribological resistance to chemical and physical agents in the oral cavity, were considered the most important factors; finally, among threats, medical liability and biomechanical problems had equal weight. CONCLUSIONS: This study applied a multimethod SWOT-AHP approach to bring out favorable or critical evidence on the topic of dental implants. In accordance with the result of the strategic vector identified in the Twisting zone Adjustment type section, showed that implant surgery is a widespread technique but always needs improvement to increase the likelihood of success and reduce the complications that can lead to implant failure.

Immunohistochemical localization and characterization of putative mesenchymal stem cell markers in the retinal capillary network of rodents.

Perivascular cells of microvascular niches are the prime candidates for being a reservoire of mesenchymal stem cell (MSC)-like cells in many tissues and organs that could serve as a potential source of cells and a target of novel cell-based therapeutic approaches. In the present study, by utilising typical markers of pericytes (neuronal-glial antigen 2, NG2, a chondroitin sulphate proteoglycan) and those of MSCs (CD146 and CD105) and primitive pluripotent cells (sex-determining region Y-box 2, Sox2), the phenotypic traits and the distribution of murine and rat retinal perivascular cells were investigated in situ. Our findings indicate that retinal microvessels of juvenile rodents are highly covered by NG2-positive branching processes of pericytic (perivascular) cells that are less prominent in mature capillary networks of the adult retina. In the adult rodent retinal vascular bed, NG2 labeling is mainly confined to membranes of the cell body resulting in a pearl-chain-like distribution along the vessels. Retinal pericytes, which were identified by their morphology and NG2 expression, simultaneously express CD146. Furthermore, CD146-positive cells located at small arteriole-to-capillary branching points appear more intensely stained than elsewhere. Evidence for a differential expression of the two markers around capillaries that would hint at a clonal heterogeneity among pericytic cells, however, is lacking. In contrast, the expression of CD105 is exclusively restricted to vascular endothelial cells and Sox2 is detected neither in perivascular nor in endothelial cells. In dissociated retinal cultures, however, simultaneous expression of NG2 and CD105 was observed. Collectively, our data indicate that vascular wall resident retinal pericytes share some phenotypic features (i.e. CD146 expression) with archetypal MSCs, which is even more striking in dissociated retinal cultures (i.e. CD105 expression). These findings might have implications for the treatment of retinal pathologies.

The significance of bioelectricity on all levels of organization of an organism. Part 1: From the subcellular level to cells.

Bioelectricity plays an essential role in the structural and functional organization of biological organisms. In this first article of our three-part series, we summarize the importance of bioelectricity for the basic structural level of biological organization, i.e. from the subcellular level (charges, ion channels, molecules and cell organelles) to cells.

Old, Nonagenarians, and Centenarians in Cilento, Italy and the Association of Lifespan with the Level of Some Physicochemical Elements in Tap Drinking Water.

Longevity, as a complex life-history trait, shares an ontogenetic relationship with other quantitative traits, such as epigenetic and environmental factors. Therefore, it is important to identify environmental factors that may modify the epigenome to establish healthy aging. This study explored the association between tap drinking water and longevity in Cilento, Italy, to understand whether trace elements in local drinking water may have an influence on old, nonagenarian, and centenarian people and promote their health and longevity. Data on population and water sources were collected through the National Demographic Statistics, the Cilento Municipal Archives, and the Cilento Integrated Water Service. Ordinary least squares (OLS) regression and a geographically weight regression (GWR) model were used to study the spatial relationship between the explanatory and outcome variables of longevity. The results of the study showed that the prevalence of longevity is concentrated in the central, northern and southeastern areas of the territory and that some trace elements present in tap water may contribute to local longevity in Cilento. Specifically, all Cilento municipalities had alkaline tap water, and the municipalities with the highest longevity concentrations had higher alkalinity levels than the other municipalities, soft to medium-hard water hardness, an amount of total dissolved solids equivalent to the level of excellent water, lower amounts of sodium, adequate iron concentration, and adequate dietary intake of manganese per day.

Restoring Axonal Organelle Motility and Regeneration in Cultured FUS-ALS Motoneurons through Magnetic Field Stimulation Suggests an Alternative Therapeutic Approach.

Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron disease characterized by sustained loss of neuromuscular junctions, degenerating corticospinal motoneurons and rapidly progressing muscle paralysis. Motoneurons have unique features, essentially a highly polarized, lengthy architecture of axons, posing a considerable challenge for maintaining long-range trafficking routes for organelles, cargo, mRNA and secretion with a high energy effort to serve crucial neuronal functions. Impaired intracellular pathways implicated in ALS pathology comprise RNA metabolism, cytoplasmic protein aggregation, cytoskeletal integrity for organelle trafficking and maintenance of mitochondrial morphology and function, cumulatively leading to neurodegeneration. Current drug treatments only have marginal effects on survival, thereby calling for alternative ALS therapies. Exposure to magnetic fields, e.g., transcranial magnetic stimulations (TMS) on the central nervous system (CNS), has been broadly explored over the past 20 years to investigate and improve physical and mental activities through stimulated excitability as well as neuronal plasticity. However, studies of magnetic treatments on the peripheral nervous system are still scarce. Thus, we investigated the therapeutic potential of low frequency alternating current magnetic fields on cultured spinal motoneurons derived from induced pluripotent stem cells of FUS-ALS patients and healthy persons. We report a remarkable restoration induced by magnetic stimulation on axonal trafficking of mitochondria and lysosomes and axonal regenerative sprouting after axotomy in FUS-ALS in vitro without obvious harmful effects on diseased and healthy neurons. These beneficial effects seem to derive from improved microtubule integrity. Thus, our study suggests the therapeutic potential of magnetic stimulations in ALS, which awaits further exploration and validation in future long-term in vivo studies.

Understanding the Feedback Loops between Energy, Matter and Life.

This review gathers recent findings in biophysics that shed light on the biological principle of self-organization, spanning from molecules to more complicated systems with higher information processing capacity. The focus is on "feedback loops" from information and matter to an exchange component with a more fundamental meaning than "cybernetic regulation" and "maintenance of homeostasis". This article proposes that electric and electromagnetic forces are the most important mediators over large distances. Field-like mediation is distinguished from cell-to-cell communication by special electric- or ion-guiding mechanisms that create additional pathways to the "classical" mediators such as nerve conduction or blood flow. Resonance phenomena from phonons and photons in the visible range will be discussed in relation to organelles, cytoskeletal elements and molecules. In this context, the aqueous surrounding of molecules and cells is an important aspect. Many of these phenomena are caused by quantum physics, such as the tunneling of electrons in enzymes or in other coherent working systems. This suggests that quantum information processing is also spread over large-scale areas of an organism.

Extreme Longevity: Analysis of the Direct or Indirect Influence of Environmental Factors on Old, Nonagenarians, and Centenarians in Cilento, Italy.

Longevity is rightly considered one of the greatest achievements of modern society, an achievement understood as the possibility of increasing the healthy part of life and not only its full duration. This study investigated the phenomenon of regional longevity in Cilento by analyzing the association between longevity indicators and some environmental factors, in order to understand if factors such as altitude, climate, UNESCO protected areas, and hinterland can directly or indirectly influence the measure of healthy living and lead to longevity. Demographic and environmental data were collected through the Archives of the Cilento municipalities, the National Institute of Statistics, the Italian Institute for Environmental Protection and Research, and the Italian National Commission for UNESCO. The Geographically Weighted Regression were used to determine the association between longevity indicators and environmental factors. Correlation analysis between the longevity indicators was investigated in order to have a complete picture of longevity in Cilento. It was discovered that Cilento longevity is mainly found in the central area of the territory and from there, by age groups, it widens towards the south-eastern area. This designated area is part of the hilly area of the Cilento, at an altitude between 400 and 700 m above sea level. The towns of this area are part of the UNESCO heritage and are characterized by a transitional climate between Mediterranean and temperate climate. Moreover, the correlation analysis between the six indicators of longevity has confirmed a linear relationship between the six variables and this indicates that in the years to come there will be the probabilities of a generational turn over between the old, great old, nonagenarians and current centenarians, provided that the SARS-CoV-2 pandemic subsides.

Effect of Direct Current Electric Fields on Cone Like Retinal Photoreceptor Cells.

INTRODUCTION: Studies show that electric fields are used as therapy during nerve and tissue injuries along with trans-retinal stimulation. However, cellular and molecular changes induced by such treatments remain largely unknown especially in retinal photoreceptor cells. In vitro studies show that direct current electric fields (dcEF) were known to influence cell division, polarity, shape, and motility. Here we could characterize for the first time the reactions of 661W, a retinal cone photoreceptor especially regarding organelle polarization, membrane polarization of mitochondria, O2 consumption, ATP/ADP ratio and gene expression. METHODS: The 661W cells were stimulated with a constant dcEF of field strength 5 V/cm during 30 min or 5 h depending on the parameters studied. RESULTS: In response to dcEF, the cells aligned perpendicular to the field by forming a leading edge with extended membrane protrusions towards the cathode. Using immunofluorescence and live cell imaging, we show that the cell membrane depolarized at the cathodal side. The microtubules spread into the direction of migration. Also, the microtubule organization center re-oriented into this direction. Concomitantly with the microtubules, actin filaments reorganized in an asymmetrical fashion mainly at the cathodal side. The Golgi apparatus, which is involved in many steps of actin synthesis, moved to the cathodal side. In the last 2 h of the 5 h experiment, microtubules positioned themselves at the rear (anodal side), like the nucleus. The averaged displacement of the whole cells under dcEF was 155% of control for 3 V/cm and 235% for 5 V/cm. The average speed increased by 142% and 243% respectively. Inside the cells mitochondria moved to the cathodal side, where the energy consuming producing processes take place. In this line, we measured an increase in ATP production and O2 consumption. Mitochondrial calcium was found more on the anodal side, at the site of the nucleus with its calcium delivering endoplasmic reticulum. In addition, oxymetry studies reveal an increased ATP synthesis by 115.2% and oxygen consumption by 113.3% 3 h after dcEF stimulation. An analysis of differentially expressed genes by RNA sequencing revealed an upregulation of genes involved in cellular movement, cell to cell and intracellular signaling, molecular transport, assembly and organization. CONCLUSIONS: The mechanisms found can enhance our understanding regarding the beneficial effects of EF treatment in retinal diseases.