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Cytolethal distending toxin (CDT) is produced by a range of Gram-negative pathogenic bacteria such as Campylobacter jejuni. CDT represents an important virulence factor that is a heterotrimeric complex composed of CdtA, CdtB, and CdtC. CdtA and CdtC constitute regulatory subunits whilst CdtB acts as the catalytic subunit exhibiting phosphatase and DNase activities, resulting in cell cycle arrest and cell death. Extracellular vesicle (EV) secretion is an evolutionarily conserved process that is present throughout all kingdoms. Mammalian EVs play important roles in regular cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune responses. Here, we demonstrate that CDT targets the endo-lysosomal compartment, partially evading lysosomal degradation and exploiting unconventional secretion (EV release), which is largely involved in bacterial infections. CDT-like effects are transferred by Caco-2 cells to uninfected heterologous U937 and homologous Caco-2 cells. The journey of EVs derived from CDT-treated Caco-2 cells is associated with both intestinal and myeloid tumour cells. EV release represents the primary route of CDT dissemination, revealing an active toxin as part of the cargo. We demonstrated that bacterial toxins could represent suitable tools in cancer therapy, highlighting both the benefits and limitations. The global cell response involves a moderate induction of apoptosis and autophagic features may play a protective role against toxin-induced cell death. EVs from CDT-treated Caco-2 cells represent reliable CDT carriers, potentially suitable in colorectal cancer treatments. Our data present a potential bacterial-related biotherapeutic supporting a multidrug anticancer protocol.

Cytolethal distending toxin (CDT) is produced by a range of Gram-negative pathogenic bacteria such as

2,2-bis(6-bromo-1H-indol-3-yl) ethanamine, a marine bisindole alkaloid, showed anticancer property in several tumor cell lines thanks to the presence of a 3,3'-diindolylmethane scaffold. Here, the modifications in its chemical structure into alkaloid-like derivatives, have been evaluated, to investigate changes in its biological activities. Three derivatives have been considered and their potential apoptotic action has been evaluated through morpho-functional analyses in a human cancer cell line. Apoptosis appears strongly decreased in the derivatives without the bromine atoms (1) and in those where the bromine atoms have been substituted with fluorine atoms (2). On the contrary, the methylation of indole NH (3) does not alter the alkaloid apoptotic activity that occurs through mitochondria involvement supported by cardiolipin peroxidation and dysfunctional mitochondria presence. This manuscript highlights the alkaloid derivative cytotoxic effect, which is strictly correlated to the presence of N-methylated bisindole alkaloid and bromine atoms, conditions which assure to maintain the pro-apoptotic activity. Since molecular therapies, by targeting mitochondria pathways, have shown positive outcomes against several cancer cells, the alkaloid with bisindole methylated scaffold and the two bromine atoms can be considered a promising candidate to develop new derivatives with strong anticancer property. RESEARCH HIGHLIGHTS: 2,2-bis(6-bromo-1H-indol-3-yl) ethanamine is an alkaloid known for its anticancer properties. Morpho-functional analyses evaluated cytotoxicity of its synthetic derivatives in tumor cells. Anticancer properties depend on the presence of bisindole scaffold and the two bromine units.

Fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, showing a specific subcellular localization. In the present study, we first investigated their preferential mitochondrial targeting in myeloid cells, by flow cytometry, confocal microscopy and TEM on both cells and isolated mitochondria, to acquire knowledge in imaging combined with therapeutic applications. Then, we conjugated SiNPs to one of the most used anticancer drugs, doxorubicin (DOX). As an anticancer agent, DOX has high efficacy but also an elevated systemic toxicity, causing multiple side effects. Nanostructures are usually employed to increase the drug circulation time and accumulation in target tissues, reducing undesired cytotoxicity. We tested these functionalized SiNPs (DOX-NPs) on breast cancer cell line MCF-7. We evaluated DOX-NP cytotoxicity, the effect on the cell cycle and on the expression of CD44 antigen, a molecule involved in adhesion and in tumor invasion, comparing DOX-NP to free DOX and stand-alone SiNPs. We found a specific ability to release a minor amount of CD44+ extracellular vesicles (EVs), from both CD81 negative and CD81 positive pools. Modulating the levels of CD44 at the cell surface in cancer cells is thus of great importance for disrupting the signaling pathways that favor tumor progression.

Introduction Recently, the Food and Drug Administration authorized the marketing of IQOS Tobacco Heating System as a Modified Risk Tobacco Product based on an electronic heat-not-burn technology that purports to reduce the risk. Methods Sprague-Dawley rats were exposed in a whole-body mode to IQOS aerosol for 4 weeks. We performed the chemical characterization of IQOS mainstream and we studied the ultrastructural changes in trachea and lung parenchyma of rats exposed to IQOS stick mainstream and tissue pro-inflammatory markers. We investigated the reactive oxygen species amount along with the markers of tissue and DNA oxidative damage. Moreover, we tested the putative genotoxicity of IQOS mainstream through Ames and alkaline Comet mutagenicity assays. Results Here, we identified irritating and carcinogenic compounds including aldehydes and polycyclic aromatic hydrocarbons in the IQOS mainstream as sign of incomplete combustion and degradation of tobacco, that lead to severe remodelling of smaller and largest rat airways. We demonstrated that IQOS mainstream induces lung enzymes that activate carcinogens, increases tissue reactive radical concentration; promotes oxidative DNA breaks and gene level DNA damage; and stimulates mitogen activated protein kinase pathway which is involved in the conventional tobacco smoke-induced cancer progression. Conclusions Collectively, our findings reveal that IQOS causes grave lung damage and promotes factors that increase cancer risk. Implications IQOS has been proposed as a safer alternative to conventional cigarettes, due to depressed concentration of various harmful constituents typical of traditional tobacco smoke. However, its lower health risks to consumers have yet to be determined. Our findings confirm that IQOS mainstream contains pyrolysis and thermogenic degradation by-products, the same harmful constituents of traditional cigarette smoke, and, for the first time, we show that it causes grave lung damage and promotes factors that increase cancer risk in the animal model.

Di recente, la Food and Drug Administration (FDA) ha autorizzato la commercializzazione dei nuovi dispositivi per il rilascio di nicotina (DERN) a riscaldamento del tabacco, come prodotti del tabacco a rischio modificato (MRTP). Si tratta di sigarette elettroniche di ultima generazione che sfruttano la tecnologia “heat-not-burn”, che scalda il tabacco evitando il processo di combustione. Distribuiti con il nome commerciale di IQOS, e concepiti allo scopo di ridurre i rischi per la salute legati al consumo dei prodotti tradizionali del tabacco, tali dispositivi hanno riscosso un notevole successo soprattutto tra i più giovani. Tuttavia, la letteratura è scevra di lavori indipendenti che ne permettano una adeguata valutazione tossicologica. Il presente studio si è posto l’obiettivo di rilevare diverse classi di composti tossici derivanti dalla degradazione termica del tabacco e le modificazioni ultrastrutturali a livello di trachea e del parenchima polmonare in ratti maschi Sprague-Dawley, a seguito di un’esposizione sub-cronica all’ “aerosol” prodotto da IQOS. È stata misurata la concentrazione di specie reattive dell'ossigeno (ROS) nel tessuto polmonare, quindi l’espressione di marcatori di danno ossidativo a livello cellulare e del DNA. Inoltre, sono stati eseguiti i test di mutagenesi di Ames su urine e Comet assay su leucociti. Nell’”aerosol” sono stati identificati composti irritanti e cancerogeni tra cui aldeidi e idrocarburi policiclici aromatici che indicano come il tabacco abbia subito una combustione incompleta. A livello delle vie aeree è stato osservato un marcato rimodellamento tissutale negli animali esposti. Inoltre, è stata riportata una significativa induzione di alcune isoforme del citocromo P450 (CYP1A1, CYP2A1/2, CYP2B1/2, CYP2E1.), una maggiore concentrazione di ROS a livello polmonare a cui segue un aumento dei marcatori di danno ossidativo al DNA, un incremento dell’espressione di alcune citochine pro-infiammatorie e l’attivazione delle principali MAP-chinasi attivate da mediatori dell’infiammazione e stress ossidativo. Infine, i test di mutagenesi eseguiti hanno mostrato come l’esposizione all’aerosol prodotto da IQOS determini un danno al DNA. Nel complesso, i risultati mostrano come IQOS possa indurre danni significativi alle vie aeree e promuovere fattori che aumentano il rischio di cancro.

Background: Designed to deliver nicotine avoiding tobacco combustion, electronic cigarettes (e-cigs) are aggressively promoted as safer alternative to tobacco products and a putative approach to smoking cessation. However, to date the risk-benefit ratio remains largely unknown and recent investigations have questioned their safety. Here we presented our recent in vivo studies on the toxicological aspects associated to the electronic delivery systems. Methods: Male Sprague Dawley rats were whole-body exposed to the vapour generated by the devices. The e-cig was equipped with different coils (1.5 and 0.25 Ohm) and the Voltage applied ranged from 3.5 to 5.5 V in order to explore the role of these parameters on toxicological outcomes. The treatment was carried out loading the device with e-liquid containing or not 18 mg/mL (1 mL/day) of nicotine. E-cig treated animals were subjected to the procedure for 3 h/day for 28 days. The levels of O2, N2, and CO2 were monitored by GC/MS to establish safe O2/N2 and CO2/ O2 ratios in the cages. Furthermore, the experiments were repeated considering also “non-liquid devices” with alternative heating systems able to heat tobacco. Results: When e-cig was run at high voltage (5.5 V; 18 mg/mL nicotine) we recorded the release of hazardous carbonyl compounds (such as formaldehyde, acetaldehyde and acrolein). The animals showed a powerful booster effect on phase-I carcinogen-bioactivating enzymes, an impairment of the antioxidant and phase II catalysts, related to the increment of oxygen free radical (ROS) production, and DNA oxidation through 8-hydroxy-2′-deoxyguanosine yield in lung tissue. Insights into the genotoxicity also emerged from the extensive DNA damage in leukocytes measured by the comet assay, the increment in the percentage of immature micronucleated reticulocytes and the increased number of revertants in the urine. Lowering the voltage at 3.5 V and loading the device with a nicotine-free liquid, we still found aldehydes in the mainstream, and noteworthy, their amount enhanced moving form nicotine to nicotine-free liquids. Interestingly, the aldehydes increased as the resistance decreased from 1.5 to 0.25 Ohm, and the frames from electron microscope confirmed a disorganization of alveolar and bronchial epithelium more marked in the 0.25 Ohm group. Finally, since changes in liver parameters including ALT and hepatocellular vacuolization in rats exposed to the vapour from “non-liquid e-cigs” were reported, we added a breakthrough finding on treated animals showing a disruption of the redox homeostasis, with higher levels of ROS, and increased markers of oxidative injuries such as lipid peroxidation and protein carbonylation, combined with an impairment of the enzymatic and non-enzymatic antioxidant defence systems. Conclusion: As a whole, our studies show how electronic delivery devices can trigger detrimental phenomena typically induced by conventional cigarettes.

Neuro-muscular disorders include a variety of diseases induced by genetic mutations resulting in muscle weakness and waste, swallowing and breathing difficulties. However, muscle alterations and nerve depletions involve specific molecular and cellular mechanisms which lead to the loss of motor-nerve or skeletal-muscle function, often due to an excessive cell death. Morphological and molecular studies demonstrated that a high number of these disorders seem characterized by an upregulated apoptosis which significantly contributes to the pathology. Cell death involvement is the consequence of some cellular processes that occur during diseases, including mitochondrial dysfunction, protein aggregation, free radical generation, excitotoxicity and inflammation. The latter represents an important mediator of disease progression, which, in the central nervous system, is known as neuroinflammation, characterized by reactive microglia and astroglia, as well the infiltration of peripheral monocytes and lymphocytes. Some of the mechanisms underlying inflammation have been linked to reactive oxygen species accumulation, which trigger mitochondrial genomic and respiratory chain instability, autophagy impairment and finally neuron or muscle cell death. This review discusses the main inflammatory pathways contributing to cell death in neuro-muscular disorders by highlighting the main mechanisms, the knowledge of which appears essential in developing therapeutic strategies to prevent the consequent neuron loss and muscle wasting.

Despite the knowledge gap regarding the risk-benefit ratio of the electronic cigarette (e-cig), its use has grown exponentially, even in teenagers. E-cig vapor contains carcinogenic compounds (eg, formaldehyde, acetaldehyde, and acrolein) and free radicals, especially reactive oxygen species (ROS) that cause toxicological effects, including DNA damage. The role of e-cig voltage customization on molecule generation has been reported, but the effects of the resistance on e-cig emissions and toxicity are unknown. Here, we show that the manipulation of e-cig resistance influences the carbonyls production from nonnicotine vapor and the oxidative and inflammatory status in a rat model. Fixing the voltage at the conventional 3.5 V, we observed that the amount of the selected aldehydes increased as the resistance decreased from 1.5 to 0.25 Ω. Under these conditions, we exposed Sprague Dawley rats to e-cig aerosol for 28 days, and we studied the pulmonary inflammation, oxidative stress, tissue damage, and blood homeostasis. We found a perturbation of the antioxidant and phase II enzymes, probably related to the increased ROS levels due to the enhanced xanthine oxidase and P450-linked monooxygenases. Furthermore, frames from scanning electron microscope showed a disorganization of alveolar and bronchial epithelium in 0.25 Ω group. Overall, various toxicological outcomes, widely recognized as smoke-related injuries, can potentially occur in e-cig consumers who use low-voltage and resistance device. Our study suggests that certain “tips for vaping safety” cannot be established, and encourages further independent investigations to help public health agencies in regulating the e-cig use.

Mitochondrial dysfunction is considered one of the hallmarks of ischemia/reperfusion injury. Mitochondria are plastic organelles that undergo continuous biogenesis, fusion, and fission. They can be transferred between cells through tunneling nanotubes (TNTs), dynamic structures that allow the exchange of proteins, soluble molecules, and organelles. Maintaining mitochondrial dynamics is crucial to cell function and survival. The present study aimed to assess the effects of melatonin on mitochondrial dynamics, TNT formation, and mitochondria transfer in HT22 cells exposed to oxygen/glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin treatment during the reoxygenation phase reduced mitochondrial reactive oxygen species (ROS) production, improved cell viability, and increased the expression of PGC1α and SIRT3. Melatonin also preserved the expression of the membrane translocase proteins TOM20 and TIM23, and of the matrix protein HSP60, which are involved in mitochondrial biogenesis. Moreover, it promoted mitochondrial fusion and enhanced the expression of MFN2 and OPA1. Remarkably, melatonin also fostered mitochondrial transfer between injured HT22 cells through TNT connections. These results provide new insights into the effect of melatonin on mitochondrial network reshaping and cell survival. Fostering TNTs formation represents a novel mechanism mediating the protective effect of melatonin in ischemia/reperfusion injury.

The Na+-dependent Vitamin C transporter 2 (SVCT2) is expressed in the plasma and mitochondrial membranes of various cell types. This notion was also established in proliferating C2C12 myoblasts (Mb), in which the transporter was characterised by a high and low affinity in the plasma and mitochondrial membranes, respectively. In addition, the mitochondrial expression of SVCT2 appeared particularly elevated and, consistently, a brief pre-exposure to low concentrations of Ascorbic Acid (AA) abolished mitochondrial superoxide formation selectively induced by the cocktail arsenite/ATP. Early myotubes (Mt) derived from these cells after 4 days of differentiation presented evidence of slightly increased SVCT2 expression, and were characterised by kinetic parameters for plasma membrane transport of AA in line with those detected in Mb. Confocal microscopy studies indicated that the mitochondrial expression of SVCT2 is well preserved in Mt with one or two nuclei, but progressively reduced in Mt with three or more nuclei. Cellular and mitochondrial expression of SVCT2 was found reduced in day 7 Mt. While the uptake studies were compromised by the poor purity of the mitochondrial preparations obtained from day 4 Mt, we nevertheless obtained evidence of poor transport of the vitamin using the same functional studies successfully employed with Mb. Indeed, even greater concentrations of/longer pre-exposure to AA failed to induce scavenging of mitochondrial superoxide in Mt. These results are therefore indicative of a severely reduced mitochondrial uptake of the vitamin in early Mt, attributable to decreased expression as well as impaired activity of mitochondrial SVCT2.

In this work the role of transmission electron microscopy (TEM) in biology and pathology studies is emphasyzed. A overview on technical procedures is provided and their advantages or disadvantages, with particular attention to possible artifacts, are reported. The importance of the ultrastructural approach in cell and isolated organelle characterization, frequently correlated to functional and molecular analyses, is supported by specific images and by a cospicous number of references. Negative staining, a simple and rapid method, is also described and a number of examples from different types of viruses are illustrated. Cryo-EM is finally mentioned. It is illustrated by means of frozen ultrathin sections, compared to those from epon embedded conventional ones. Finally, freeze-fractured sample replicas are presented, both aimed at membrane detail highlighting and inner cell structure investigation. TEM, in its variety of applications, and in particular if integrated by molecular studies, is a very important tool in the study of life sciences and, in general for nanotechnology.

Holotomographic (HT) microscopy, combines two techniques, holography and tomography, and, in this way, it allows to quantitatively and noninvasively investigate cells and thin tissue slices, by obtaining three-dimensional (3D) images and by monitoring inner morphological changes. HT has indeed two significant advantages: it is label-free and low-energy light passes through the specimen with minimal perturbation. Using quantitative phase imaging with optical diffraction tomography, it can produce 3D images by measuring the refraction index (RI). Therefore, based on RI values, HT can provide structural and chemical cell information, such as dry mass values, morphological changes, or cellular membrane dynamics. In this study, suspended and adherent culture cells have been processed for HT analyses. Some of them have been treated with known apoptotic drugs or pro-oxidant agents and cell response has been investigated both by conventional microscopic approaches and by HT. The ultrastructural and fluorescence images have been compared to those obtained by HT and their congruence has been discussed, with particular attention to apoptotic cell death and on correlated plasma membrane changes. HT appears a valid approach to further characterize well-known apoptotic features such as cell blebbing, chromatin condensation, micronuclei, and apoptotic bodies. Taken together, our data demonstrate that HT appears suitable to highlight suspended or adherent cell behavior under different conditions. In particular, this technique appears an important new tool to distinguish healthy cells from the apoptotic ones, as well as to monitor outer and inner cell changes in a rapid way and with a noninvasive, label-free, approach.

OBJECTIVES: Autophagy is a physiological and highly regulated mechanism, crucial for cell homeostasis maintenance. Its impairment seems to be involved in the onset of several diseases, including muscular dystrophies, myopathies and sarcopenia. According to few papers, chemotherapeutic drug treatment is able to trigger side effects on skeletal muscle tissue and, among these, a defective autophagic activation, which leads to the persistence of abnormal organelles within cells and, finally, to myofiber degeneration. The aim of this work is to find a strategy, based on diet modulation, to prevent etoposide-induced damage, in a model of in vitro skeletal muscle cells. METHODS: Glutamine supplementation and nutrient deprivation have been chosen as pre-treatments to counteract etoposide effect, a chemotherapeutic drug known to induce oxidative stress and cell death. Cell response has been evaluated by means of morpho-functional, cytofluorimetric and molecular analyses. RESULTS: Etoposide treated cells, if compared to control, showed dysfunctional mitochondria presence, ER stress and lysosomal compartment damage, confirmed by molecular investigations. CONCLUSIONS: Interestingly, both dietary approaches were able to rescue myofiber from etoposide-induced damage. Glutamine supplementation, in particular, seemed to be a good strategy to preserve cell ultrastructure and functionality, by preventing the autophagic impairment and partially restoring the normal lysosomal activity, thus maintaining skeletal muscle homeostasis. ispartof: JOURNAL OF NUTRITION HEALTH & AGING vol:23 issue:8 pages:739-745 ispartof: location:France status: published

It is known that, besides a wide range of functions, melatonin provides protection against oxidative stress, thanks to its ability to act, directly, as a free radical scavenger and, indirectly, by stimulating antioxidant enzymes production and mitochondrial electron transport chain efficiency. Oxidative stress is one of the major players in initiating apoptotic cell death in skeletal muscle, as well as in other tissues. Apoptosis is essential for skeletal muscle development and homeostasis; nevertheless, its misregulation has been frequently observed in several myopathies, in sarcopenia, as well as in denervation and disuse. Melatonin activity was investigated in undifferentiated C2C12 skeletal muscle cells, after exposure to various apoptotic chemical triggers, chosen for their different mechanisms of action. Cells were pretreated with melatonin and then exposed to hydrogen peroxide, etoposide and staurosporine. Morphofunctional and molecular analyses show that in myoblasts melatonin prevents oxidative stress and apoptosis induced by chemicals following, at least in part, the mitochondria pathway. These results confirm melatonin ability to act as an antioxidant and antiapoptotic molecule in skeletal muscle cells, thus suggesting a possible therapeutic strategy for myopathies involving apoptosis misregulation. Microsc. Res. Tech. 79:532-540, 2016. © 2016 Wiley Periodicals, Inc.

Marine organisms such as corals, sponges and tunicates produce active molecules which could represent a valid starting point for new drug development processes. Among the various structural classes, the attention has been focused on 2,2-bis(6-bromo-3-indolyl) ethylamine, a marine alkaloid which showed a good anticancer activity against several tumor cell lines. Here, for the first time, the mechanisms of action of 2,2-bis(6-bromo-3-indolyl) ethylamine have been evaluated in a U937 tumor cell model. Morpho-functional and molecular analyses, highlighting its preferred signaling pathway, demonstrated that apoptosis is the major death response induced by this marine compund. Chromatin condensation, micronuclei formation, blebbing and in situ DNA fragmentation, occurring through caspase activation (extrinsic and intrinsic pathways), were observed. In particular, the bisindole alkaloid induces a mitochondrial involvement in apoptosis machinery activation with Blc-2/Bcl-x down-regulation and Bax up-regulation. These findings demonstrated that 2,2-bis(6-bromo-3-indolyl) ethylamine alkaloid-induced apoptosis is regulated by the Bcl-2 protein family upstream of caspase activation.

Melatonin is a small indole produced by the pineal gland and other tissues, and has numerous functions that aid in the maintenance of the whole body homeostasis, ranging from the regulation of circadian rhythms and sleep to protection from oxidative stress. Melatonin has also been reported to counteract cell growth and chemoresistance in different types of cancer. In the present study, we investigated the effects of exogenous melatonin administration on different human cell lines and primary mouse tumor cultures of rhabdomyosarcoma (RMS), the most frequent soft tissue sarcoma affecting childhood. The results showed that melatonin significantly affected the behavior of RMS cells, leading to inhibition of cell proliferation and impairment of myogenic differentiation followed by increased apoptotic cell death, as observed by immunoblotting analysis of apoptosis-related markers including Bax, Bcl-2 and caspase-3. Similar findings were observed using a combination of microscopy techniques, including scanning/transmission electron and confocal microscopy. Furthermore, melatonin in combination with doxorubicin or cisplatin, two compounds commonly used for the treatment of solid tumors, increased the sensitivity of RMS cells to apoptosis. These data indicated that melatonin may be effective in counteracting RMS tumor growth and chemoresistance.

Skin cells can respond to UVB-induced damage either by tolerating it, or restoring it through antioxidant activation and DNA repair mechanisms or, ultimately, undergoing programmed cell death, when damage is massive. Nutritional factors, in particular, food antioxidants, have attracted much interest because of their potential use in new preventive, protective, and therapeutic strategies for chronic degenerative diseases, including skin inflammation and cancer. Some polyphenols, present in virgin olive oil, well tolerated by organism after oral administration, show a variety of pharmacological and clinical benefits such as anti-oxidant, anti-cancer, anti-inflammatory, and neuro-protective activities. Here, the protective effects of antioxidant compounds against UV-induced apoptosis have been described in HaCat cell line. Human keratinocytes were pre-treated with antioxidants before UVB exposure and their effects have been evaluated by means of ultrastructural analyses. After UVB radiation, a known cell death trigger, typical apoptotic features, absent in control condition and in antioxidant alone-treated cells, appear. An evident numerical decrease of ultrastructural apoptotic patterns and TUNEL positive nuclei can be observed when natural antioxidants were supplied before cell death induction. These data have been confirmed by molecular investigation of caspase activity. In conclusion, this paper highlights antioxidant compound ability to prevent apoptotic cell death in human keratinocytes exposed to UVB, suggesting, for these molecules, a potential role in preventing skin damage.

Iron exopolysaccharide nanoparticles were biogenerated during ferric citrate fermentation by Klebsiella oxytoca DSM 29614. Before investigating their effects on Tuber borchii (“bianchetto” truffle) mycelium growth and morphology, they were tested on human K562 cell line and Lentinula edodes pure culture and shown to be non-toxic. Using these nanoparticles as iron supplement, the truffles showed extremely efficient iron uptake of over 300 times that of a commercial product. This avoided morphological changes in T. borchii due to lack of iron during growth and, with optimum nanoparticle dosage, increased growth without cell wall disruption or alteration of protoplasmatic hyphal content, the nuclei, mitochondria, and rough endoplasmic reticula being preserved. No significant modifications in gene expression were observed. These advantages derive from the completely different mechanism of iron delivery to mycelia compared to commercial iron supplements. The present data, in fact, show the nanoparticles attached to the cell wall, then penetrating it non-destructively without damage to cell membrane, mitochondria, chromatin, or ribosome. Low dosage significantly improved mycelium growth, without affecting hyphal morphology. Increases in hyphal diameter and septal distance indicated a healthier state of the mycelia compared to those grown in the absence of iron or with a commercial iron supplement. These positive effects were confirmed by measuring fungal biomass as mycelium dry weight, total protein, and ergosterol content. This “green” method for biogenerating iron exopolysaccharide nanoparticles offers many advantages, including significant economic savings, without toxic effects on the ectomycorrhizal fungus, opening the possibility of using them as iron supplements in truffle plantations.

Wine contains various polyphenols such as flavonoids, anthocyanins, and tannins. These molecules are responsible for the quality of wines, influencing their astringency, bitterness, and color and they are considered to have antioxidant activity. Polyphenols, extracted from grapes during the processes of vinification, could protect the body cells against reactive oxygen species level increase and could be useful to rescue several pathologies where oxidative stress represents the main cause. For that, in this study, red and white wine, provided by an Italian vinery (Marche region), have been analyzed. Chromatographic and morphofunctional analyses have been carried out for polyphenol extraction and to evaluate their protective effect on human myeloid U937 cells exposed to hydrogen peroxide. Both types of wines contained a mix of phenolic compounds with antioxidant properties and their content decreased, as expected, in white wine. Ultrastructural observations evidenced that wines, in particular red wine, strongly prevent mitochondrial damage and apoptotic cell death. In conclusion, the considered extracts show a relevant polyphenol content with strong antioxidant properties and abilities to prevent apoptosis. These findings suggest, for these compounds, a potential role in all pathological conditions where the body antioxidant system is overwhelmed.

Oxidative stress increase induces cellular damage and apoptosis activation, a mechanism believed to represent a final common pathway correlated to sarcopenia and many skeletal muscle disorders. The goal of this study is to evaluate if melatonin, a ROS scavenger molecule, is able to counteract or modulate myotube death. Here, differentiated C2C12 skeletal muscle cells have been treated with melatonin before chemicals known to induce apoptotic death and oxidative stress, and its effect has been investigated by means of morpho-functional analyses. Ultrastructural observations show melatonin protection against triggers by the reducing of membrane blebbing, chromatin condensation, myonuclei loss and in situ DNA cleavage. Moreover, melatonin is able to prevent mitochondrial dysfunctions which occur in myotubes exposed to the trigger alone. These findings demonstrate melatonin ability in preventing apoptotic cell death in skeletal muscle fibers in vitro, suggesting for this molecule a potential therapeutic role in the treatment of various muscle disorders.

Myotendinous junction (MTJ) is the highly specialized complex which connects the skeletal muscle to the tendon for transmitting the contractile force between the two tissues. The purpose of this study was to investigate the MTJ development and rat EDL was chosen as a model. 1, 15, 30 day animals were considered and the junctions were analyzed by light and electron microscopy. The MTJ interface architecture increased during the development, extending the interaction between muscle and tendon. 1-day-old rats showed disorganized myofibril bundles, spread cytosol and incomplete rough endoplasmic reticulum, features partially improved in 15-day-old rats, and completely developed in 30-day-old animals. These findings indicate that muscle-tendon interface displays, during rat lifetime, numerically increased and longer tendon interdigitations, correlated with an improved organization of both tissues and with a progressive acquirement of full functionality.

Hydroxytyrosol (HyT) is a polyphenol primarily released in olive mill wastewater and in olive oil. In animal and cell model studies, HyT and its metabolites have strong antioxidant and antimicrobial activities, as well as beneficial effects on the cardiovascular system and in several human diseases. Differently, many researchers reported that HyT down-regulates tumor cell viability and cell cycle progression, and induces reactive oxygen species (ROS) production and apoptosis.In this study we have investigated the effects of HyT and the corresponding ester hydroxytyrosyl laurate in U937 cells, a human monocytoid cell line, and in C2C12 myoblasts, a murine proliferating muscle cell model, after apoptotic death induction. Inverted, light and transmission electron microscopy have been utilized to characterize cell death patterns. H2O2, at the concentrations known to induce apoptosis, was utilized as cell death trigger. The results obtained show that laur-HyT has a protective antioxidant effect against H2O2 treatment, greater than HyT, so having a role in the prevention of apoptotic death in normal and tumor cells. These data suggest these compounds as good candidate for novel therapeutic strategies. © 2013 Elsevier Ltd.

It is known that, besides a wide range of functions, melatonin provides protection against oxidative stress, thanks to its ability to act, directly, as a free radical scavenger and, indirectly, by stimulating antioxidant enzymes production and mitochondrial electron transport chain efficiency. Oxidative stress is one of the major players in initiating apoptotic cell death in skeletal muscle, as well as in other tissues. Apoptosis is essential for skeletal muscle development and homeostasis; nevertheless, its misregulation has been frequently observed in several myopathies, in sarcopenia, as well as in denervation and disuse. Melatonin activity was investigated in undifferentiated C2C12 skeletal muscle cells, after exposure to various apoptotic chemical triggers, chosen for their different mechanisms of action. Cells were pretreated with melatonin and then exposed to hydrogen peroxide, etoposide and staurosporine. Morphofunctional and molecular analyses show that in myoblasts melatonin prevents oxidative stress and apoptosis induced by chemicals following, at least in part, the mitochondria pathway. These results confirm melatonin ability to act as an antioxidant and antiapoptotic molecule in skeletal muscle cells, thus suggesting a possible therapeutic strategy for myopathies involving apoptosis misregulation. Microsc. Res. Tech. 79:532-540, 2016. © 2016 Wiley Periodicals, Inc.

Melatonin (Mel), or N-acetyl-5-methoxytryptamine, is a circadian hormone that can diffuse through all the biological membranes thanks to its amphiphilic structure, also overcoming the blood-brain barrier and placenta. Although Mel has been reported to exhibit strong antioxidant properties in healthy tissues, studies carried out on tumor cultures gave a different picture of its action, often describing Mel as effective to trigger the cell death of tumor cells by enhancing oxidative stress. Based on this premise, here Mel effect was investigated using a tumor cell line representative of the human alveolar rhabdomyosarcoma (ARMS), the most frequent soft tissue sarcoma affecting childhood. For this purpose, Mel was given either dissolved in ethanol (EtOH) or dimethyl sulfoxide (DMSO) at different concentrations and time exposures. Cell viability assays and ultrastructural observations demonstrated that Mel was able to induce a dose- and time-dependent cell death independently on the dissolution solvent. Microscopy analyses highlighted the presence of various apoptotic and necrotic patterns correlating with the increasing Mel dose and time of exposure. These findings suggest that Mel, triggering apoptosis in ARMS cells, could be considered as a promising drug for future multitargeted therapies.

Skeletal muscle cell differentiation is a multistage process extensively studied over the years. Even if great improvements have been achieved in defining biological process underlying myogenesis, many molecular mechanisms need still to be clarified. To further highlight this process, we studied cells at undifferentiated, intermediate and highly differentiated stages, and we analyzed, for each condition, morphological and proteomic changes. We also identified the proteins that showed statistical significant changes by a ESI-Q-TOF mass spectrometer. This work provides further evidence of the involvement of particular proteins in skeletal muscle development. Furthermore, the high level of expression of many heat shock proteins, suggests a relationship between differentiation and cellular stress. Intriguingly, the discovery of myogenesis-correlated proteins, known to play a role in apoptosis, suggests a link between differentiation and this type of cell death.

During muscle tissue differentiation, in particular in the formation of myotubes from the myoblasts, plasma membrane changes its morpho-functional characteristics. In this study, muscle cell membrane behaviour has been studied along the differentiation of C2C12, a mouse myoblastic adherent cell line. Flat undifferentiated cells, cultured for 3-4 days in the differentiation medium, progressively become thick, long and multinucleated myotubes covered with microvilli. They lose stress fibers and adhesion to the underlying substrate evidentiating an actin redistribution, followed by the spatial organization of thick and thin myofilaments. Sarcomeres and myofibrils occasionally appear, even if a certain percentage of "myosacs" containing randomly oriented filaments can be identified all along the differentiation. M-cadherin, a molecule involved in cell-cell adhesion, also appears in the early differentiation stage, during myoblast fusion. Occasional focal contractions can also be observed in myotubes, which prompt an electrophysiological membrane analysis. When studied by means of patch clamp technique, resting membrane potential appears to undergo a transient depolarization, while input resistance increases until day 5 after differentiation induction, then successively decreases. Capacitance declines until day 5, later appearing enhanced. Moreover, with the induction of differentiation, the pattern of functional voltage-dependent ion channels changes. Therefore, during myogenesis, cell maturation is coupled with changes in cell membrane morphological features and functional characteristics.

Background. Exposure to intermittent hypoxia (IH) may enhance cardiac function and protects heart against ischemia-reperfusion (I/R) injury. To elucidate the underlying mechanisms, we developed a cardioprotective IH model that was characterized at hemodynamic, biochemical and molecular levels. Methods. Mice were exposed to 4 daily IH cycles (each composed of 2-min at 6-8% O2 followed by 3-min reoxygenation for 5 times) for 14 days, with normoxic mice as controls. Mice were then anesthetized and subdivided in various subgroups for analysis of contractility (pressure-volume loop), morphology, biochemistry or resistance to I/R (30-min occlusion of the left anterior descending coronary artery (LAD) followed by reperfusion and measurement of the area at risk and infarct size). In some mice, the phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin was administered (24 μg/kg ip) 15 min before LAD. Results. We found that IH did not induce myocardial hypertrophy; rather both contractility and cardiac function improved with greater number of capillaries per unit volume and greater expression of VEGF-R2, but not of VEGF. Besides increasing the phosphorylation of protein kinase B (Akt) and the endothelial isoform of NO synthase with respect to control, IH reduced the infarct size and post-LAD proteins carbonylation, index of oxidative damage. Administration of wortmannin reduced the level of Akt phosphorylation and worsened the infarct size. Conclusion. We conclude that the PI3K/Akt pathway is crucial for IH-induced cardioprotection and may represent a viable target to reduce myocardial I/R injury.

Background Phenolic compounds, the biggest group of natural antioxidants, have attracted much attention due to their known and wide-ranging biological activities, as well as to their health effects. In particular, regardless their antioxidant activity, they play a key role in the control of several inflammation-associated processes as well as in improving antioxidant defense system. In an our previous work we have demonstrated the ability of Hydroxytyrosol, an ortho-diphenolic compound, essential component of oleuropein, in preventing apoptotic cell death induced by UVB radiation in HaCaT cell lines in vitro. In olive oil, besides Hydroxytyrosol, there are appreciable amounts of Tyrosol and its secoiridoid derivatives. Objective It has been well established that Tyrosol has a significantly lower antioxidant activity than Hydroxytyrosol, but despite this, recent studies suggest that Tyrosol exerts a powerful protective effect against oxidative injuries in cell systems and that it is able to improve the intracellular antioxidant defenses. Materials and methods Here, Tyrosol effect has been evaluated in HaCaT cells exposed to UVB radiation by means of morphological and molecular analyses. Results Our study revealed the polyphenol ability in reducing apoptotic markers and in protecting HaCaT cells from damage. Conclusion These findings suggest an important role of Tyrosol in protecting cells from apoptotic cell death and encourage the use of this phytochemical as biological ingredient in topical preparations as possible tool to prevent skin damage.

Apoptosis is an essential biological function required during embryogenesis, tissue homeostasis, organ development and immune system regulation. It is an active cell death pathway involved in a variety of pathological conditions. During this process cytoskeletal proteins appear damaged and undergo an enzymatic disassembling, leading to formation of apoptotic features. This study was designed to examine the three-dimensional chromatin behavior and cytoskeleton involvement, in particular actin re-modeling. HL-60 cells, exposed to hyperthermia, a known apoptotic trigger, were examined by means of a Field Emission in Lens Scanning Electron Microscope (FEISEM). Ultrastructural observations revealed in treated cells the presence of apoptotic patterns after hyperthermia trigger. In particular, three-dimensional apoptotic chromatin rearrangements appeared involving the translocation of filamentous actin from cytoplasm to the nucleus. FEISEM immunogold techniques showed actin labeling and its precise three-dimensional localization in the diffuse chromatin, well separated from the condensed one. The actin presence in dispersed chromatin inside the apoptotic nucleus can be considered an important feature, indispensable to permit the apoptotic machinery evolution.

a b s t r a c t -Actinin is involved in the assembly and maintenance of muscle fibers. -Actinin is required to cross- link actin filaments and to connect the actin cytoskeleton to the cell membrane and it is necessary for the attachment of actin filaments to Z-disks in skeletal muscle fibers and to dense bodies in smooth muscle ones. In addition to its mechanical role, sarcomeric -actinin interacts with proteins involved in a variety of signaling and metabolic pathways. The aim of this work is to monitor Z-disk formation, in order to clear up the role of sarcomeric -actinin in undifferentiated stage, after 4 days of differentiation (intermediate differentiation stage) and after 7 days of differentiation (fully differentiated stage). For this purpose, C2C12 murine skeletal muscle cells, grown in vitro, were analyzed at three time points of differentiation. Confocal laser scanner microscopy and transmission electron microscopy have been utilized for - actinin immunolocalization. Both techniques reveal that in undifferentiated cells labeling appears uniformly distributed in the cytoplasm with punctate -actinin Z-bodies. Moreover, we found that when differentiation is induced, -actinin links at first membrane-associated proteins, then it aligns longitudinally across the cytoplasm and finally binds actin, giving rise to Z-disks. These findings evidence -actinin involvement in sarcomeric development, suggesting for this protein an important role in stabilizing the muscle contractile apparatus.

CXCR2 ligands contribute to chondrocyte hypertrophy and apoptosis, important determinants in cartilage pathophysiology. We unraveled the kinetics of signaling, biochemical, transcriptional, and morphological events triggered by GROα in human osteoarthritic chondrocytes kept in three-dimensional culture. p38 MAPK activation was assessed with a highly sensitive ELISA. Effector caspase activation was evaluated by cleavage of a fluorogenic substrate. Gene expression of key markers of hypertrophy (MMP-13, Runx-2) and matrix synthesis (aggrecan), and of cathepsin B isoform CB(-2,3) was evaluated by real time PCR. Occurrence of the morphological markers of apoptosis was investigated by transmission electron microscopy (TEM). GROα led to p38 MAPK activation in passaged chondrocytes cultured in micromass but not as a high-density monolayer. This caused the downstream triggering of chondrocyte hypertrophy (MMP-13 and Runx-2 upregulation, and calcium deposition) and apoptosis/anoikis following concurrence of matrix degrading activity, and inhibition of matrix synthesis which also involved the induction of CB(-2,3). These phenomena proved to be dependent on the co-receptor role of sulfated glycosaminoglycans (sGAG) and the activation of p38 MAPK, since they were abrogated either by preincubation with soluble chondroitin-4 sulfate or p38 MAPK inhibitors. The co-receptor role of sGAG was further demonstrated by colocalization experiments of these molecules with GROα in the stimulated micromasses. These findings suggest that extracellular matrix exerts a regulatory role in chondrocytes differentiation, and that meaningful investigation of the effects of chemokines on chondrocyte biology requires culture conditions respectful of both the differentiated status of the chondrocytes and of their three-dimensional interaction with the extracellular matrix. J. Cell. Physiol. 210: 417–427, 2007. © 2006 Wiley-Liss, Inc.

In this report, we have evaluated the effects of a TransFixR-based stabilisation technique on leukocyte scatter characteristics, immunophenotyping, membrane permeability, absolute cell counting and morphology to extend previously reported flow cytometric data focused on the lymphocyte population. We show that scatter characteristics, immunophenotyping and absolute cell counting are well preserved, particularly in the lymphocyte population. Nevertheless, a general increase in membrane permeability, evaluated by propidium iodide (PI) uptake, was observed in TransFixR-treated leukocyte subsets. Ultrastructural observations show selective morphological preservation (up to 10 days of storage) of lymphocytes and, to a lesser extent, of monocytes. In contrast, granulocytes have necrosis-like features, although the plasma membrane seems well preserved. Therefore, electron microscopy observations reflect modifications induced in different cell populations as evidenced by flow cytometry (FC). The data indicate that this short-term stabilisation method is particularly suitable for the analysis of human lymphocytes and it is a good procedure for quality control programmes for inter- and intra-laboratory performance evaluation; good results are obtained with respect to antigen definition and absolute cell counting procedures. Any apoptotic pathways in leukocyte subsets are blocked for at least 10 days.

A morphological study was performed on 27 human placentas from normal gestations (Group 1) and compared with those from eight cases of intrauterine growth restriction (IUGR) (Group 2). Semithin section light microscopy, transmission, and scanning electron microscopy were carried out on trophoblastic terminal villi, carefully identified under the stereomicroscope. In growth retardation cases, villi appear longer, thinner, and less vascularized, compared to the normal condition. Fibrinoid, an extracellular material of hematic origin, frequently fills villar stroma. The density of apical microvilli appears considerably reduced and occasional microvilli-free areas are observed in growth retardation cases. Moreover, the underlying basal membrane appears significantly thicker than that of normal syncytiotrophoblast. Recently, particular attention has been paid to apoptosis as a possible cell deletion mechanism in growth restriction. In our study, a majority of typical apoptotic features appear indifferently in both IUGR and normal pregnancy. Our data hints that growth retardation might be correlated with a complex of structural changes, suggestive of maternofetal traffic downregulation, but further studies are required to understand the underlying functional mechanisms.

During apoptosis, cell chromatin undergoes characteristic morphological changes, which have been long described in a variety of experimental models but are mostly not yet understood. The aim of the present study was to investigate the mechanisms underlying this phenomenon and the possible role of cytoskeleton, in particular actin. The chosen apoptotic model were HL60 hemopoietic cells undergoing hyperthermia and the starting point was the observation of thin filament bundles in decondensed chromatin of their early apoptotic nuclei. The characterization of these structures was undertaken by cytochemical, fluorescent and immunogold techniques, directed to actin identification. Taken together, our results suggest, in apoptotic cells, a deep actin rearrangement. Moreover, this cytoskeletal component, never present in normal nucleus, appears in the early apoptotic one, where it can be found in polymerized form, promptly recognizable both by conventional and immunogold electron microscopy. We suggest that, similarly to the role played by nuclear matrix in interphase and mitotic nucleus, actin could be directly involved in chromatin rearrangement occurring in apoptosis.

Skin cells can respond to UVB-induced damage by counteracting it through antioxidant activation and DNA repair mechanisms or, when damage is massive by undergoing programmed cell death. Antioxidant factors, and, in particular, food compounds, have attracted much interest because of their potential use in new protective strategies for degenerative skin disorders. Melatonin, creatine and hydroxytyrosol show a variety of pharmacological and clinical benefits including anti-oxidant and anti-inflammatory activities. Here, the potential protective actions of antioxidant compounds against UVB-induced apoptosis were investigated in human keratinocytes. The cells were pre-treated with antioxidants before UVB exposure and their effect evaluated by means of ultrastructural and molecular analyses. After UVB radiation typical morphological apoptotic features and in situ DNA fragmentation after TUNEL reaction, appeared. A significant numerical decrease of apoptotic patterns could be observed when antioxidants were administrated before cell death induction. Moreover, both the intrinsic and extrinsic apoptotic pathways appeared activated after UVB radiation, and their down-regulation has been shown when antioxidants were added to cells before death induction. In conclusion, these compounds are able to prevent apoptotic cell death in human keratinocytes exposed to UVB, suggesting, for these molecules, an important role in preventing skin damage.

Melatonin (MEL), a methoxyindole synthesized by the pineal gland, is a powerful antioxidant in tissues as well as within cells, with a fundamental role in ameliorating homeostasis in a number of specific pathologies. It acts both as a direct radical scavenger and by stimulating production/activity of intracellular antioxidant enzymes. In this work, some chemical triggers, with different mechanisms of action, have been chosen to induce cell death in U937 hematopoietic cell line. Cells were pre-treated with 100 μM MEL and then exposed to hydrogen peroxide or staurosporine. Morphological analyses, TUNEL reaction and Orange/PI double staining have been used to recognize ultrastructural apoptotic patterns and to evaluate DNA behavior. Chemical damage and potential MEL anti-apoptotic effects were quantified by means of Tali® Image-Based Cytometer, able to monitor cell viability and apoptotic events. After trigger exposure, chromatin condensation, micronuclei formation and DNA fragmentation have been observed, all suggesting apoptotic cell death. These events underwent a statistically significant decrease in samples pre-treated with MEL. After caspase inhibition and subsequent assessment of cell viability, we demonstrated that apoptosis occurs, at least in part, through the mitochondrial pathway and that MEL interacts at this level to rescue U937 cells from death. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

The present review discusses the apoptotic behavior induced by chemical and physical triggers in C2C12 skeletal muscle cells, comparing myoblast to myotube sensitivity, and investigating it by means of morphological, biochemical and cytofluorimetric analyses. After all treatments, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, in cells exposed to staurosporine, etoposide and UVB radiation, apoptotic and normal nuclei within the same fibercould be revealed. The presence of nuclear-dependent “territorial” death domains in the syncytium could explain a delayed cell death of myotubes compared to mononucleated cells. Moreover, autophagic granules abundantly appeared in myotubes after each treatment. Autophagy could protect muscle cell integrity against chemical and physical stimuli, making C2C12 myotubes, more resistant to cell death induction

In this study the role of hyperthermia as an apoptotic trigger was analyzed in four human tumor cell lines: HL60, U937, DOHH2, and K562. These cell lines were chosen because of their well known and different expression of bcl-2 and bcr-abl genes, the expression of which is known to be an antiapoptotic condition. HL60 and U937 cells were strongly susceptible to heat exposure, while DOHH2 cells were weakly sensitive and K562 cells were resistant, thus suggesting a possible gene involvement in this type of programmed cell death. The mechanisms underlying this apoptosis were investigated by flow cytometry, agarose gel electrophoresis, and light and electron microscopy. A subdiploid peak and DNA laddering, both of which are parameters specifically correlated to programmed cell death, were present in HL60 and U937 and, even if less evident, in DOHH2 cells undergoing hyperthermic treatment, and were absent in K562 cells. In addition, DNA single-strand cleavage was revealed by in situ nick translation, observed by confocal microscopy. Morphological analysis confirmed these results and revealed the typical chromatin changes, followed by the appearance of micronuclei and apoptotic bodies.

Human promyelocytic leukemia HL-60 cells have been used as a model to study both the expression of matrix-metalloproteinases and the mechanisms of programmed cell death. In the present study we examined the expression of these proteases in HL-60 cells stimulated by different apoptotic triggers. As shown by zymography, HL-60 cells released three major isofroms of the matrix-degrading proteases; when the leukemic cells were grown in serum-free conditions, as well as after hyperthermia and methotrexate treatment, we found a significant loss of the constitutive production of the 92 kDa matrix-metalloprotease, with an unequivocable molecular and ultrastructural evidence of programmed cell death. These results suggest that in HL-60 cells the expression/release of matrix metalloproteases can be down-regulated in the presence of the apoptotic-induced alterations, and that the decreased matrix-degrading capacity of this leukemic cell line during apoptosis may reduce its invasive potential.