Temporal correlation of morphological and biochemical changes with the recruitment of different mechanisms of reactive oxygen species formation during human SW872 cell adipogenic differentiation

none8noHuman SW872 preadipocyte conversion to mature adipocytes is associated with time-dependent changes in differentiation markers' expression and with morphological changes accompanied by the accumulation of lipid droplets (LDs) as well as by increased mitochondriogenesis and mitochondrial membrane potential. Under identical conditions, the formation of reactive oxygen species (ROS) revealed with a general probe was significant at days 3 and 10 of differentiation and bearly detectable at day 6. NADPH oxidase (NOX)-2 activity determined with an immunocytochemical approach followed a very similar pattern. There was no evidence of mitochondrial ROS (mROS), as detected with a selective fluorescence probe, at days 3 and 6, possibly due to the triggering of the Nrf-2 antioxidant response. mROS were instead clearly detected at day 10, concomitantly with the accumulation of very large LDs, oxidation of both cardiolipin and thioredoxin 2, and decreased mitochondrial glutathione. In conclusion, the morphological and biochemical changes of differentiating SW872 cells are accompanied by the discontinuous formation of ROS derived from NOX-2, increasingly implicated in adipogenesis and adipose tissue dysfunction. In addition, mROS formation was significant only in the late phase of differentiation and was associated with mitochondrial dysfunction.openFiorani, Mara; De Matteis, Rita; Canonico, Barbara; Blandino, Giulia; Mazzoli, Alessandro; Montanari, Mariele; Guidarelli, Andrea; Cantoni, OrazioFiorani, Mara; De Matteis, Rita; Canonico, Barbara; Blandino, Giulia; Mazzoli, Alessandro; Montanari, Mariele; Guidarelli, Andrea; Cantoni, Orazi

Development and in vitro characterization of a humanized scFv against fungal infections

: The resistance and the birth of new intrinsic and multidrug-resistant pathogenic species like C. auris is creating great concern in the antifungal world. Given the limited drug arsenal and the lack of effectiveness of the available compounds, there is an urgent need for innovative approaches. The murine mAb 2G8 was humanized and engineered in silico to develop a single-chain fragment variable (hscFv) antibody against β-1,3-glucans which was then expressed in E. coli. Among the recombinant proteins developed, a soluble candidate with high stability and affinity was obtained. This selected protein is VL-linker-VH oriented, and it is characterized by the presence of two ubiquitin monomers at the N-terminus and a His tag at the C-terminus. This construct, Ub2-hscFv-His, guaranteed stability, solubility, efficient purification and satisfactory recovery of the recombinant product. HscFv can bind β-1,3-glucans both as coated antigens and on C. auris and C. albicans cells similarly to its murine parental and showed long stability and retention of binding ability when stored at 4°, -20° and -80° C. Furthermore, it was efficient in enhancing the antifungal activity of drugs caspofungin and amphotericin B against C. auris. The use of biological drugs as antifungals is limited; here we present a promising hscFv which has the potential to be useful in combination with currently available antifungal drugs

Myotube vs myoblast sensitivity to apoptosis induction by chemical triggers

Apoptosis is necessary for skeletal muscle tissue development and homeostasis, where it plays a multifaceted role. Muscle cell death increase is associated to disuse, denervation and to several muscle myopathies (Siu et al., Life Sci, 2009), while apoptosis resistance is typical of muscle differentiation (Xiao et al., Apoptosis, 2011) The aim of this study is to evaluate in vitro apoptotic response in differentiated myotubes exposed to chemical triggers and to compare apoptosis susceptibility to that of proliferant myoblasts. (Salucci et al., Micron, 2010). C2C12 myotubes, differentiated as previously described (Curci et al, Micron, 2008), were exposed to etoposide (ETO), cisplatin (CP), hydrogen peroxide (H2O2) and staurosporine (SP) and apoptosis was evaluated by flow cytometry and transmission electron microscopy. Flow cytometry analysis revealed a certain subdiploid peak after ETO and CP exposure, lower, however, than that evidenced in myoblasts. Differently, DNA cleavage after H2O2 or SP treatments, was absent, in contrast with undifferentiated cells. At ultrastructural analysis, characteristic chromatin condensation was observed after ETO and CP treatments, but few apoptotic cells were detected, differently from what evidenced at undifferentiated stage. Moreover, autophagic cell death appeared. Cells exposed to ETO presented a particular behaviour: myonuclei with condensed chromatin coexisted with normal nuclei, in the same myotube, as previously demonstrated in UVB-induced apoptosis (D’Emilio et al., Histol Histopathol, 2010). On the contrary, and differently from myoblasts, apoptosis was absent in myotubes after H2O2 exposure, being necrosis and autophagy the most common cell death processes. After SP treatment all cells showed features of secondary necrosis, a response present also in myoblasts. In addition, autophagic vacuoles were observed, exclusively in differentiated cells. These results show a certain resistance to apoptosis in myotubes, if compared to myoblasts, that could be associated to an increase in autophagic cell death and in a probable upregulation of anti-apoptotic pathways. Particularly intriguing appears the response to ETO treatment where apoptosis could be correlated to a localized gene expression within each myonuclear domain, suggesting that this process works to selectively eliminate the designated nuclei in multinucleated skeletal muscle fibers

Melatonin reshapes the mitochondrial network and promotes intercellular mitochondrial transfer via tunneling nanotubes after ischemic-like injury in hippocampal HT22 cells

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

Melatonin Attenuates Ischemic-like Cell Injury by Promoting Autophagosome Maturation via the Sirt1/FoxO1/Rab7 Axis in Hippocampal HT22 Cells and in Organotypic Cultures

Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis

Erythrocyte-mediated delivery of phenylalanine ammonia lyase for the treatment of phenylketonuria in BTBR-Pahenu2 mice

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Synthesis and biological characterization of a new fluorescent probe for vesicular trafficking based on polyazamacrocycle derivative

The fluorescent probes represent an important tool in the biological study, in fact characterization of cellular structures and organelles are an important tool-target for understanding the mechanisms regulating most biological processes. Recently, a series of polyamino-macrocycles based on 1,4,7,10-tetraazacyclododecane was synthesized, bearing one or two NBD units (AJ2NBD·4HCl) useful as sensors for metal cations and halides able to target and to detect apolar environment, as lipid membranes. In this paper, we firstly illustrate the chemical synthesis of the AJ2NBD probe, its electronic absorption spectra and its behavior regarding pH of the environment. Lack of any cellular toxicity and an efficient labelling on fresh, living cells was demonstrated, allowing the use of AJ2NBD in biological studies. In particular, this green fluorescent probe may represent a potential dye for the compartments involved in the endosomal/autophagic pathway. This research's field should benefit from the use of AJ2NBD as a vesicular tracer, however, to ensure the precise nature of vesicles/vacuoles traced by this new probe, other more specific tests are needed

A new humanized antibody is effective against pathogenic fungi in vitro

Invasive fungal infections mainly affect patients undergoing transplantation, surgery, neoplastic disease, immunocompromised subjects and premature infants, and cause over 1.5 million deaths every year. The most common fungi isolated in invasive diseases are Candida spp., Cryptococcus spp., and Aspergillus spp. and even if four classes of antifungals are available (Azoles, Echinocandins, Polyenes and Pyrimidine analogues), the side effects of drugs and fungal acquired and innate resistance represent the major hurdles to be overcome. Monoclonal antibodies are powerful tools currently used as diagnostic and therapeutic agents in different clinical contexts but not yet developed for the treatment of invasive fungal infections. In this paper we report the development of the first humanized monoclonal antibody specific for β-1,3 glucans, a vital component of several pathogenic fungi. H5K1 has been tested on C. auris, one of the most urgent threats and resulted efficient both alone and in combination with Caspofungin and Amphotericin B showing an enhancement effect. Our results support further preclinical and clinical developments for the use of H5K1 in the treatment of patients in need

How skeletal muscle cells die after chemical treatments

Apoptosis deregulation is pathogenetic in several skeletal muscle disorders characterised by muscle mass loss, myofiber number decrease, apoptotic myonuclei increase and an elevated DNA fragmentation (Dupont-Versteegden, 2006). The aim of this work is to study in vitro, in C2C12 myoblasts and myotubes, the apoptotic behavior induced byetoposide, staurosporine and H2O2. Cell response was investigated by means of cytofluorimetric and morphological analyses, as well as by confocal microscopy of TUNEL reaction. Myotubes appeared more resistant than myoblasts to apoptotic induction in all experimental conditions. In particular, in myoblasts treated with etoposide and staurosporine apoptotic nuclei with chromatin margination and condensation were observed, in the presence of a diffuse DNA fragmentation evidenced after TUNEL reaction. The latter was observed also in myotubes, where apoptotic and normal nuclei inside the same syncytium appeared (D’Emilio et al., 2010). After H2O2 exposure, myotubes, differently from myoblasts, showed a poor cell sensitivity to cell death even if a certain DNA cleavage was observed. Intriguingly, autophagic granules diffusely appeared in myotubes after each treatment. In myoblasts all chemicals induced ROS increase. When their production exceeds cellular antioxidant capability, oxidative stress results and apoptosis is triggered. On the other hand, in myotubes mitochondria appeared better preserved than myoblasts, and, if damaged, they are probably degradated by autophagic processes. Finally, myotube resistance to apoptotic stimuli could be correlated to the fact that myogenic cells acquire an apoptosis-resistant phenotype during differentiation (Xiao et al., 2011)

LFA-1 antigen identifies immature stages of human NK cell differentiation

Background. Human Natural killer (NK) cells are characterized by NK cell receptors (NKRs) with inhibitory and activatory function that finely control their functional activities. In particular, they express inhibitory receptors for MHC class I molecules, named killer cell immunoglobulin (Ig)-like receptors (KIRs) and C-type lectin CD94-CD159a, and many triggering molecules like NKp30, NKp44, NKp46, (called natural cytotoxicity receptors, NCRs), NKG2D, CD161, and CD244. The majority of peripheral blood human NK cells are characterized by a phenotype with a low density expression of CD56 (CD56dim) and a high expression of CD16 (CD16bright), whereas a minority (approximately 5–10%) shows a bright expression of CD56 (CD56bright). This latter NK subset presents relatively high expression of some cytokine receptors (CD117 and CD25) and the CD94-CD159a heterodimeric inhibitory receptor. CD56bright NK cells are widely expressed in lymphoid tissues and can be generated from CD34+ cells when cultured with combinations of flt-3 ligand (FL) or stem cell factor plus IL-15 or IL-2. During their development, NK cells sequentially acquire many different antigens but there is still limited knowledge on differentiation antigens able to identify immature human NK cells and the specific sequence through which developing NK cells acquire the expression of NKR.Methods. NK cells obtained from human CD34+ hematopoietic progenitor cells after 30-day culture with FL plus IL-15, or from peripheral and umbilical cord blood samples were characterized.Results. Virtually, all CD56 NK cells differentiated in vitro expressed CD117, CD25, NCRs, NKG2D, CD161, and CD244, while only a subset expressed CD18-CD11a (LFA-1), and CD94 molecule, defining an immature CD56bright/NCRs+/NKG2D+/LFA-1-/CD94- subset. Another small subset of cells expressing CD94 but not LFA-1 integrin was also identified, suggesting that during NK differentiation LFA-1 might be upregulated later than CD94. To verify this hypothesis in vivo, we evaluated the NK cell expression of LFA-1 in both peripheral and umbilical cord blood samples. Interestingly, in these blood fluids, we have identified a lineage negative CD34-/LFA-1low/NKp46dim/NKG2Ddim/CD94- subset that resembled an immature stage of NK cells present in lymph nodes. Conclusions. Altogether, the results indicate that CD18-CD11a integrin, as well as CD11b in mice, may be a useful marker to identify immature stages of human NK cell differentiation