One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16

Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al.</p

Photocatalytic - anticancer activity of titanium dioxide (TiO2): related mechanisms and applications

Titanium Dioxide (TiO2) is a material with a wide range of common as well as state-of -the-art applications. Actually it is demonstrably used in paints, plastics, cosmetics, artificial bone implants and also in optical coatings on dielectric mirrors. Furthermore, titanium dioxide is widely used in biomedical applications, due to its mechanical properties, biocompatibility and of course for photocatalysis purposes. It is now well established that photoexcited titanium dioxide (TiO2) can drive various chemical reactions due to its strong oxidizing and reducing ability and can also affect cellular activity, including applications in cancer cells treatment, and self-sterilized surfaces. It is crystallized in the form of rutile, anatase and broukite, while at low temperatures, it exists in amorphous condition. It is characterized as n-type semiconductor. Recently, many studies, in the field of cancer treatment research, suggest that the regulatory mechanisms of apoptotic-cell death play as important role in carcinogenesis as control mechanisms of cell proliferation do. Apoptosis is relevant to the majority of malignant tumorigenesis. The apoptotic activity occurs in a more vigorous rate in rapidly growing tumors. The usual applied treatments (chemotherapy, radiotherapy, hormonotherapy), conditionally aim to induce apoptosis. Recent studies demonstrated that TiO2 induces death by apoptosis in different types of cells, such as mesenchymal stem cells, osteoblasts and many others. Furthermore, the photocatalytic properties of TiO2-mediated toxicity have been shown to eradicate cancer cells, upon irradiation with light whose wavelength is 20μM. Σε συγκέντρωση 24μM επιβιώνει το 50% των MDA-MB-468 και ποσοστό ~70% των MCF-7. Σε συγκέντρωση 19 μM φωτο-ενεργοποιημένου (με UV-Α) TiO2 Evonik P25 επιβιώνει το 50% των MDA-MB-468 και ποσοστό ~80% των MCF-7 κυττάρων. Το φωτο-ενεργοποιημένο εναιώρημα TiO2 στα MDA-MB-468, επάγει τη θραύση της PARP, οδηγεί σε μικρή αύξηση της Bax και προκαλεί ασθενές DNA laddering. Η επιβίωση των κυττάρων μειώνεται καθώς αυξάνεται η συγκέντρωση του εναιωρήματος TiO2 (Sigma Aldrich) (ανατάσης 100%) (111.3 nm και ζ δυναμικό: ZP=(-16.7±9) mV). Για συγκέντρωση >20 μM επιβιώνει το 50% των κυττάρων MDA-MB-468 και το 70% των MCF-7. Σε συγκέντρωση 17 μM φωτο-ενεργοποιημένου (με UV–Α) TiO2 Sigma Aldrich επιβιώνει το 50% των MDA-MB-468 και το 75% των MCF-7 κυττάρων. Η επίδραση του φωτο-ενεργοποιημένου ή μη, TiO2 (Sigma Aldrich), στα MDA-MB-468, επάγει τη θραύση της PARP, οδηγεί σε μικρή αύξηση της Bax και προκαλεί DNA laddering. Για συγκεντρώσεις ακόμη και 100μΜ εναιωρήματος τροποποιημένου με άζωτο TiO2 (N-doped TiO2) (129.2 nm) και ζ δυναμικό: ZP=(-11.4±7) mV) επιβιώνει περισσότερο από το 80% των κυττάρων MDA-MB-468 και των MCF-7. Το φωτο-ενεργοποιημένο με ορατή ακτινοβολία, N-doped TiO2, μειώνει βαθμιαία τον πληθυσμό των κυττάρων και των δύο κυτταρικών σειρών, με ελαφρά εντονότερη δράση στα MDA-MB-468. Σε συγκέντρωση 40μΜ επιβιώνει το 70% του πληθυσμού των MDA-MB-468 και το 90% των MCF-7 κυττάρων. Επάγεται η θραύση της PARP, σε πολύ μικρό ποσοστό, οδηγεί σε πολύ μικρή αύξηση της Bax και προκαλεί ασθενές DNA laddering. Η συγκέντρωση 21μΜ εναιωρήματος τροποποιημένου με Άργυρο TiO2 (Ag-doped TiO2) (144.5 nm) και ζ δυναμικό: ZP=(-14.8±8) mV), επάγει το θάνατο στο 50% των κυττάρων MDA-MB-468, αφήνοντας το 80% των κυττάρων MCF-7 σχεδόν ανεπηρέαστο. Σε συγκέντρωση 18 μΜ φωτο-ενεργοποιημένου (με UV–Α) Ag-doped TiO2 επιβιώνει τελικά περίπου το 50% των κυττάρων MDA-MB-468 και το 70% των MCF-7. Το φωτο-ενεργοποιημένο με UV-A ή μη, Ag-doped TiO2 επάγει τη θραύση της PARP, οδηγεί σε μείωση της Bcl-2, σε αύξηση της Bax και προκαλεί DNA laddering. Συμπερασματικά, τα σημαντικότερα αποτελέσματα της διδακτορικής διατριβής συνοψίζονται ως εξής:•Το sol-gel TiO2, είναι πιο δραστικό από όλα τα εναιωρήματα που δοκιμάστηκαν. Είναι περισσότερο ομοιογενές από τα εναιωρήματα, επιτρέποντας ακριβέστερη εκτίμηση της τελικής συγκέντρωσης TiO2 στα δείγματα. Επιπλέον, η κατανομή του μεγέθους των νανοσωματιδίων σε μορφή sol-gel, δείχνει ότι κατά μέσο όρο είναι πολύ μικρότερα στο τελικό διάλυμα, συγκρινόμενα με τα εναιωρήματα, αφού σε μορφή εναιωρήματος ευνοείται ο σχηματισμός συσσωματωμάτων, που αυξάνει το μέγεθος των σωματιδίων. •Το εναιώρημα TiO2 Sigma Aldrich είναι δραστικότερο από το Degussa P25, λόγω της διαφορετικής φάσης κρυστάλλωσης. Το Sigma Aldrich, είναι 100% ανατάσης, ενώ στο Degussa P25, συνυπάρχουν ανατάσης (75%) και ρουτίλιο (25%). Η ύπαρξη φάσης ρουτιλίου μειώνει τη δραστικότητα. Ο καθαρός ανατάσης αυξάνει την ποσότητα των ROS. Η δραστικότητα του TiO2 μειώνεται μεταβαίνοντας από το άμορφο TiO2, στον καθαρό ανατάση, στον ανατάση/ρουτίλιο και τέλος στο καθαρό ρουτίλιο.•Το N-doped TiO2 παρ’ όλο που δεν έδειξε θεαματική κυτταροτοξική δράση, εν τούτοις προσφέρει αποτελέσματα ιδιαίτερα ενθαρρυντικά, αφού επιτρέπει την ενεργοποίηση με ορατή ακτινοβολία. Το Ag-doped TiO2 είναι αρκετά δραστικό.•Η κυτταροτοξικότητα του φωτο-ενεργοποιημένου TiO2 σχετίζεται με το μηχανισμό της απόπτωσης. Το φωτο-ενεργοποιημένο TiO2 επάγει τη θραύση της PARP στα κύτταρα MDA-MB-468, αλλά όχι και στα MCF-7, ενώ η μείωση της Bcl-2 και η αύξηση της Bax, επιβεβαιώνουν κατά περίπτωση, το συμπέρασμα αυτό, σε συνδυασμό με την εικόνα του κατακερματισμένου DNA. • Η επιλεκτική τοξικότητα των νανοσωματιδίων TiO2 μεταξύ των δύο κυτταρικών σειρών, πιθανώς σχετίζεται με τις ιδιότητες της επιφάνειας των κυττάρων. Η διαφορετική σύσταση της κυτταρικής μεμβράνης ίσως διαφοροποιεί και τις αλληλεπιδράσεις μεμβρανικών πρωτεϊνών με το TiO2, που για τα MCF-7 είναι μάλλον ασθενέστερες, με αποτέλεσμα τον επιλεκτικό θάνατο των MDA-MB-468. •Η βελτιστοποίηση της μεθόδου θα μπορούσε να περιλαμβάνει την προσπάθεια αντικατάστασης ης ακτινοβολίας UV-A, με ορατό φως. Αυτό επιτυγχάνεται με τροποποίηση του TiO2 με άζωτο, αλλά με μέθοδο που να συμβάλλει στη δραστικότητά του

Breath Analysis: A Promising Tool for Disease Diagnosis&mdash;The Role of Sensors

Early-stage disease diagnosis is of particular importance for effective patient identification as well as their treatment. Lack of patient compliance for the existing diagnostic methods, however, limits prompt diagnosis, rendering the development of non-invasive diagnostic tools mandatory. One of the most promising non-invasive diagnostic methods that has also attracted great research interest during the last years is breath analysis; the method detects gas-analytes such as exhaled volatile organic compounds (VOCs) and inorganic gases that are considered to be important biomarkers for various disease-types. The diagnostic ability of gas-pattern detection using analytical techniques and especially sensors has been widely discussed in the literature; however, the incorporation of novel nanomaterials in sensor-development has also proved to enhance sensor performance, for both selective and cross-reactive applications. The aim of the first part of this review is to provide an up-to-date overview of the main categories of sensors studied for disease diagnosis applications via the detection of exhaled gas-analytes and to highlight the role of nanomaterials. The second and most novel part of this review concentrates on the remarkable applicability of breath analysis in differential diagnosis, phenotyping, and the staging of several disease-types, which are currently amongst the most pressing challenges in the field

Non-Canonical Functions of the ARF Tumor Suppressor in Development and Tumorigenesis

P14ARF (ARF; Alternative Reading Frame) is an extensively characterized tumor suppressor which, in response to oncogenic stimuli, mediates cell cycle arrest and apoptosis via p53-dependent and independent routes. ARF has been shown to be frequently lost through CpG island promoter methylation in a wide spectrum of human malignancies, such as colorectal, prostate, breast, and gastric cancers, while point mutations and deletions in the p14ARF locus have been linked with various forms of melanomas and glioblastomas. Although ARF has been mostly studied in the context of tumorigenesis, it has been also implicated in purely developmental processes, such as spermatogenesis, and mammary gland and ocular development, while it has been additionally involved in the regulation of angiogenesis. Moreover, ARF has been found to hold important roles in stem cell self-renewal and differentiation. As is often the case with tumor suppressors, ARF functions as a pleiotropic protein regulating a number of different mechanisms at the crossroad of development and tumorigenesis. Here, we provide an overview of the non-canonical functions of ARF in cancer and developmental biology, by dissecting the crosstalk of ARF signaling with key oncogenic and developmental pathways

Marine-Originated Materials and Their Potential Use in Biomedicine

Aquatic habitats cover almost 70% of the Earth, containing several species contributing to marine biodiversity. Marine and aquatic organisms are rich in chemical compounds that can be widely used in biomedicine (dentistry, pharmacy, cosmetology, etc.) as alternative raw biomaterials or in food supplements. Their structural characteristics make them promising candidates for tissue engineering approaches in regenerative medicine. Thus, seaweeds, marine sponges, arthropods, cnidaria, mollusks, and the biomaterials provided by them, such as alginate, vitamins, laminarin, collagen, chitin, chitosan, gelatin, hydroxyapatite, biosilica, etc., are going to be discussed focusing on the biomedical applications of these marine-originated biomaterials. The ultimate goal is to highlight the sustainability of the use of these biomaterials instead of conventional ones, mainly due to the antimicrobial, anti-inflammatory, anti-aging and anticancer effect

Cellular senescence as a source of SARS-CoV-2 quasispecies

In-depth analysis of SARS-CoV-2 biology and pathogenesis is rapidly unraveling the mechanisms through which the virus induces all aspects of COVID-19 pathology. Emergence of hundreds of variants and several important variants of concern has focused research on the mechanistic elucidation of virus mutagenesis. RNA viruses evolve quickly either through the error-prone polymerase or the RNA-editing machinery of the cell. In this review, we are discussing the links between cellular senescence, a natural aging process that has been recently linked to SARS-CoV-2 infection, and virus mutagenesis through the RNA-editing enzymes APOBEC. The action of APOBEC, enhanced by cellular senescence, is hypothesized to assist the emergence of novel variants, called quasispecies, within a cell or organism. These variants when introduced to the community may lead to the generation of a variant of concern, depending on fitness and transmissibility of the new genome. Such a mechanism of virus evolution may highlight the importance of inhibitors of cellular senescence during SARS-CoV-2 clinical treatment

Surfactant and Block Copolymer Nanostructures: From Design and Development to Nanomedicine Preclinical Studies

The medical application of nanotechnology in the field of drug delivery has so far exhibited many efforts in treating simple to extremely complicated and life-threatening human conditions, with multiple products already existing in the market. A plethora of innovative drug delivery carriers, using polymers, surfactants and the combination of the above, have been developed and tested pre-clinically, offering great advantages in terms of targeted drug delivery, low toxicity and immune system activation, cellular biomimicry and enhanced pharmacokinetic properties. Furthermore, such artificial systems can be tailor-made with respect to each therapeutic protocol and disease type falling under the scope of personalized medicine. The simultaneous delivery of multiple therapeutic entities of different nature, such as genes and drugs, can be achieved, while novel technologies can offer systems with multiple modalities often combining therapy with diagnosis. In this review, we present prominent, innovative and state-of-the-art scientific efforts on the applications of surfactant-based, polymer-based, and mixed surfactant-polymer nanoparticle drug formulations intended for use in the medical field and in drug delivery. The materials used, formulation steps, nature, properties, physicochemical characteristics, characterization techniques and pharmacokinetic behavior of those systems, are presented extensively in the length of this work. The material presented is focused on research projects that are currently in the developmental, pre-clinical stage

Composite Nanoarchitectonics of Photoactivated Titania-Based Materials with Anticancer Properties

The synthesis of titania-based composite materials with anticancer potential under visible-light irradiation is the aim of this study. In specific, titanium dioxide (TiO2) nanoparticles (NPs) chemically modified with silver were embedded in a stimuli-responsive microgel (a crosslinked interpenetrating network (IP) network that was synthesized by poly (N-Isopropylacrylamide) and linear chains of polyacrylic acid sodium salt, forming composite particles. The ultimate goal of this research, and for our future plans, is to develop a drug-delivery system that uses optical fibers that could efficiently photoactivate NPs, targeting cancer cells. The produced Ag-TiO2 NPs, the microgel and the composite materials were characterized through X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), micro-Raman spectroscopy, ultraviolet-visible spectroscopy (UV-Vis), dynamic light scattering (DLS) and transmission electron microscopy (TEM). Our results indicated that Ag-TiO2 NPs were successfully embedded within the thermoresponsive microgel. Either Ag-TiO2 NPs or the composite materials exhibited high photocatalytic degradation efficiency on the pollutant rhodamine B and significant anticancer potential under visible-light irradiation

Therapeutic Approaches in Locked-in Syndrome

Locked-in syndrome (LIS) is a neuropsychological condition, in which patients present with quadriplegia, lower cranial nerve paralysis, and mutism. Diagnosis of LIS is difficult because of the similarities with other related syndromes, but it is of crucial importance to establish precise and early diagnosis in order to make the appropriate decisions according to the intervention and treatment planning. Access to a multidisciplinary, specialized team provides opportunity for continued improvement. Individualized treatment improves long-term management. Assistive technology and advanced communication aids may help people with disabilities to regain more independence and take part in everyday life. Technological achievements, such as brain-computer interfaces (BCIs) may potentially be of significant practical value to patients with LIS. Advancements in medical care, rehabilitation, and communication technology have focused on leading LIS patients to live meaningful lives in the society with the involvement of their families