Publications by authors named "Giuseppe Maulucci"

57 Publications

Investigation of the Membrane Fluidity Regulation of Fatty Acid Intracellular Distribution by Fluorescence Lifetime Imaging of Novel Polarity Sensitive Fluorescent Derivatives.

Int J Mol Sci 2021 Mar 18;22(6). Epub 2021 Mar 18.

Neuroscience Department, Biophysics Section, Università Cattolica del Sacro Cuore, 00168 Rome, Italy.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.
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http://dx.doi.org/10.3390/ijms22063106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002861PMC
March 2021

Cytoprotective Effects of Punicalagin on Hydrogen-Peroxide-Mediated Oxidative Stress and Mitochondrial Dysfunction in Retinal Pigment Epithelium Cells.

Antioxidants (Basel) 2021 Jan 29;10(2). Epub 2021 Jan 29.

Fondazione Policlinico Universitario A, Gemelli IRCSS, 00168 Rome, Italy.

The retinal pigment epithelium (RPE) is a densely pigmented, monostratified epithelium that provides metabolic and functional support to the outer segments of photoreceptors. Endogenous or exogenous oxidative stimuli determine a switch from physiological to pathological conditions, characterized by an increase of intracellular levels of reactive oxygen species (ROS). Accumulating evidence has elucidated that punicalagin (PUN), the major ellagitannin in pomegranate, is a potent antioxidant in several cell types. The present study aimed to investigate the protective effect of PUN on mitochondrial dysfunction associated with hydrogen peroxide (HO)-induced oxidative stress. For this purpose, we used a human RPE cell line (ARPE-19) exposed to HO for 24 h. The effects of PUN pre-treatment (24 h) were examined on cell viability, mitochondrial ROS levels, mitochondrial membrane potential, and respiratory chain complexes, then finally on caspase-3 enzymatic activity. The results showed that supplementation with PUN: (a) significantly increased cell viability; (b) kept the mitochondrial membrane potential (ΔΨm) at healthy levels and limited ROS production; (c) preserved the activity of respiratory complexes; (d) reduced caspase-3 activity. In conclusion, due to its activity in helping mitochondrial functions, reducing oxidative stress, and subsequent induction of cellular apoptosis, PUN might be considered a useful nutraceutical agent in the treatment of oxidation-associated disorders of RPE.
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http://dx.doi.org/10.3390/antiox10020192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911437PMC
January 2021

Label-free metabolic clustering through unsupervised pixel classification of multiparametric fluorescent images.

Anal Chim Acta 2021 Mar 31;1148:238173. Epub 2020 Dec 31.

Fondazione Policlinico Gemelli IRCSS, Rome, Italy; Department of Neuroscience, Section of Biophysics, Università Cattolica Del Sacro Cuore, Rome, Italy. Electronic address:

Autofluorescence microscopy is a promising label-free approach to characterize NADH and FAD metabolites in live cells, with potential applications in clinical practice. Although spectrally resolved lifetime imaging techniques can acquire multiparametric information about the biophysical and biochemical state of the metabolites, these data are evaluated at the whole-cell level, thus providing only limited insights in the activation of metabolic networks at the microscale. To overcome this issue, here we introduce an artificial intelligence-based analysis that, leveraging the multiparametric content of spectrally resolved lifetime images, allows to detect and classify, through an unsupervised learning approach, metabolic clusters, which are regions having almost uniform metabolic properties. This method contextually detects the cellular mitochondrial turnover and the metabolic activation state of intracellular compartments at the pixel level, described by two functions: the cytosolic activation state (CAF) and the mitochondrial activation state (MAF). This method was applied to investigate metabolic changes elicited in the breast cancer cell line MCF-7 by specific inhibitors of glycolysis and electron transport chain, and by the deregulation of a specific mitochondrial enzyme (ACO2) leading to defective aerobic metabolism associated with tumor growth. In this model, mitochondrial fraction undergoes to a 13% increase upon ACO2 overexpression and the MAF function changes abruptly by altering the metabolic state of about the 25% of the mitochondrial pixels.
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http://dx.doi.org/10.1016/j.aca.2020.12.048DOI Listing
March 2021

Nuclear Localization of PTTG1 Promotes Migration and Invasion of Seminoma Tumor through Activation of MMP-2.

Cancers (Basel) 2021 Jan 8;13(2). Epub 2021 Jan 8.

International Scientific Institute "Paul VI", ISI, Fondazione Policlinico 'A. Gemelli' IRCCS, 00100 Rome, Italy.

(1) Background: PTTG1 sustains the invasiveness of several cancer types. We previously reported that in seminomas, PTTG1 was detected in the peripheral area of the tumor and in the leading infiltrative edge. Here, we investigate the PTTG1 role on the invasive properties of seminoma. (2) Methods: three seminoma cell lines were used as in vitro model. PTTG1 levels and localization were investigated by biochemical and immunofluorescence analyses. Wound-healing, Matrigel invasion assays, and zymography were applied to study migratory and invasive capability of the cell lines. RNA interference and overexpression experiments were performed to address the PTTG1 role in seminoma invasiveness. PTTG1 and its target MMP-2 were analyzed in human testicular tumors using the Atlas database. (3) Results: PTTG1 was highly and differentially expressed in the seminoma cell lines. Nuclear PTTG1 was positively correlated to the aggressive phenotype. Its modulation confirms these results. Atlas database analysis revealed that PTTG1 was localized in the nucleus in seminoma compared with non-seminoma tumors, and that MMP-2 levels were significantly higher in seminomas. (4) Conclusions: nuclear PTTG1 promotes invasiveness of seminoma cell lines. Atlas database supported these results. These data lead to the hypothesis that nuclear PTTG1 is an eligible prognostic factor in seminomas.
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http://dx.doi.org/10.3390/cancers13020212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826632PMC
January 2021

Erythrocyte membrane fluidity as a marker of diabetic retinopathy in type 1 diabetes mellitus.

Eur J Clin Invest 2021 May 1;51(5):e13455. Epub 2020 Dec 1.

Fondazione Policlinico Universitario A, Gemelli IRCSS, Rome, Italy.

Background: A high level of glycosylated haemoglobin (HbA1c), which is a nonenzymatic glycosylation product, is correlated with an increased risk of developing microangiopathic complications in Diabetes Mellitus (DM). Erythrocyte membrane fluidity could provide a complementary index to monitor the development of complications since it is influenced by several hyperglycaemia-induced pathways and other independent risk factors.

Materials And Methods: 15 healthy controls and 33 patients with long-duration (≥20 years) type 1 Diabetes Mellitus (T1DM) were recruited. Diabetic subjects were classified into two groups: T1DM, constituted by 14 nonretinopathic patients, and T1DM + RD, constituted by 19 patients in any stage of diabetic retinopathy. Red blood cells (RBC) were incubated with the fluorescent Laurdan probe and median values of Generalized Polarization (GP), representative of membrane fluidity, were compared between the two groups. Baseline characteristics among groups have been compared with Student's t test or ANOVA. Values of P < .05 were considered statistically significant.

Results: All the participants were comparable for age, Body Mass Index (BMI), creatinine and lipid profile. The duration of diabetes was similar for T1DM (34.4 ± 7.8 years) and T1DM + RD (32.8 ± 7.5 years) subjects as well as values of HbA1c: (55.6 ± 8.1) mmol/mol for T1DM and (61.2 ± 11.0) mmol/mol for T1DM + RD, respectively. Erythrocyte plasmatic membranes of RD patients were found to be more fluid (GP: 0.40 ± 0.04) than non-RD patients (GP: 0.43 ± 0.03) with a statistically significant difference (P = .035).

Conclusions: Altered erythrocyte membrane fluidity may therefore represent a marker of retinopathy in T1DM patients as a result of post-translational modifications of multifactorial aetiology (nonenzymatic glycosylation of proteins, generation of reactive oxygen species, lipid peroxidation).
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http://dx.doi.org/10.1111/eci.13455DOI Listing
May 2021

Unsupervised clustering of multiparametric fluorescent images extends the spectrum of detectable cell membrane phases with sub-micrometric resolution.

Biomed Opt Express 2020 Oct 21;11(10):5728-5744. Epub 2020 Sep 21.

Fondazione Policlinico Gemelli IRCSS, 00168 Rome, Italy.

Solvatochromic probes undergo an emission shift when the hydration level of the membrane environment increases and are commonly used to distinguish between solid-ordered and liquid-disordered phases in artificial membrane bilayers. This emission shift is currently limited in unraveling the broad spectrum of membrane phases of natural cell membranes and their spatial organization. Spectrally resolved fluorescence lifetime imaging can provide pixel-resolved multiparametric information about the biophysical state of the membranes, like membrane hydration, microviscosity and the partition coefficient of the probe. Here, we introduce a clustering based analysis that, leveraging the multiparametric content of spectrally resolved lifetime images, allows us to classify through an unsupervised learning approach multiple membrane phases with sub-micrometric resolution. This method extends the spectrum of detectable membrane phases allowing to dissect and characterize up to six different phases, and to study real-time phase transitions in cultured cells and tissues undergoing different treatments. We applied this method to investigate membrane remodeling induced by high glucose on PC-12 neuronal cells, associated with the development of diabetic neuropathy. Due to its wide applicability, this method provides a new paradigm in the analysis of environmentally sensitive fluorescent probes.
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http://dx.doi.org/10.1364/BOE.399655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587257PMC
October 2020

Red blood cells membrane micropolarity as a novel diagnostic indicator of type 1 and type 2 diabetes.

Anal Chim Acta X 2019 Nov 14;3:100030. Epub 2019 Oct 14.

Fondazione Policlinico Universitario A, Gemelli IRCSS, Rome, Italy.

Classification of the category of diabetes is extremely important for clinicians to diagnose and select the correct treatment plan. Glycosylation, oxidation and other post-translational modifications of membrane and transmembrane proteins, as well as impairment in cholesterol homeostasis, can alter lipid density, packing, and interactions of Red blood cells (RBC) plasma membranes in type 1 and type 2 diabetes, thus varying their membrane micropolarity. This can be estimated, at a submicrometric scale, by determining the membrane relative permittivity, which is the factor by which the electric field between the charges is decreased relative to vacuum. Here, we employed a membrane micropolarity sensitive probe to monitor variations in red blood cells of healthy subjects (n=16) and patients affected by type 1 (T1DM, n=10) and type 2 diabetes mellitus (T2DM, n=24) to provide a cost-effective and supplementary indicator for diabetes classification. We find a less polar membrane microenvironment in T2DM patients, and a more polar membrane microenvironment in T1DM patients compared to control healthy patients. The differences in micropolarity are statistically significant among the three groups (p<0.01). The role of serum cholesterol pool in determining these differences was investigated, and other factors potentially altering the response of the probe were considered in view of developing a clinical assay based on RBC membrane micropolarity. These preliminary data pave the way for the development of an innovative assay which could become a tool for diagnosis and progression monitoring of type 1 and type 2 diabetes.
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http://dx.doi.org/10.1016/j.acax.2019.100030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587021PMC
November 2019

Machine-learning assisted confocal imaging of intracellular sites of triglycerides and cholesteryl esters formation and storage.

Anal Chim Acta 2020 Jul 3;1121:57-66. Epub 2020 May 3.

Fondazione Policlinico Universitario A. Gemelli IRCSS, Rome, Italy; Dipartimento di Neuroscienze, Università Cattolica Del Sacro Cuore, Rome, Italy. Electronic address:

All living systems are maintained by a constant flux of metabolic energy and, among the different reactions, the process of lipids storage and lipolysis is of fundamental importance. Current research has focused on the investigation of lipid droplets (LD) as a powerful biomarker for the early detection of metabolic and neurological disorders. Efforts in this field aim at increasing selectivity for LD detection by exploiting existing or newly synthesized probes. However, LD constitute only the final product of a complex series of reactions during which fatty acids are transformed into triglycerides and cholesterol is transformed in cholesteryl esters. These final products can be accumulated in intracellular organelles or deposits other than LD. A complete spatial mapping of the intracellular sites of triglycerides and cholesteryl esters formation and storage is, therefore, crucial to highlight any potential metabolic imbalance, thus predicting and counteracting its progression. Here, we present a machine learning assisted, polarity-driven segmentation which enables to localize and quantify triglycerides and cholesteryl esters biosynthesis sites in all intracellular organelles, thus allowing to monitor in real-time the overall process of the turnover of these non-polar lipids in living cells. This technique is applied to normal and differentiated PC12 cells to test how the level of activation of biosynthetic pathways changes in response to the differentiation process.
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http://dx.doi.org/10.1016/j.aca.2020.04.076DOI Listing
July 2020

Inhibition of Transglutaminase 2 as a Potential Host-Directed Therapy Against .

Front Immunol 2019 24;10:3042. Epub 2020 Jan 24.

Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.

Host-directed therapies (HDTs) are emerging as a potential valid support in the treatment of drug-resistant tuberculosis (TB). Following our recent report indicating that genetic and pharmacological inhibition of transglutaminase 2 (TG2) restricts () replication in macrophages, we aimed to investigate the potentials of the TG2 inhibitors cystamine and cysteamine as HDTs against TB. We showed that both cysteamine and cystamine restricted replication in infected macrophages when provided at equimolar concentrations and did not exert any antibacterial activity when administered directly on cultures. Interestingly, infection of differentiated THP-1 mRFP-GFP-LC3B cells followed by the determination of the autophagic intermediates pH distribution (AIPD) showed that cystamine inhibited the autophagic flux while restricting replication. Moreover, both cystamine and cysteamine had a similar antimicrobial activity in primary macrophages infected with a panel of clinical strains belonging to different phylogeographic lineages. Evaluation of cysteamine and cystamine activity in the human model of granuloma-like structures (GLS) further confirmed the ability of these drugs to restrict replication and to reduce the size of GLS. The antimicrobial activity of the TG2 inhibitors synergized with a second-line anti-TB drug as amikacin in human monocyte-derived macrophages and in the GLS model. Overall, the results of this study support the potential usefulness of the TG2-inhibitors cysteamine and cystamine as HDTs against TB.
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http://dx.doi.org/10.3389/fimmu.2019.03042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992558PMC
November 2020

Altered mitochondrial function in cells carrying a premutation or unmethylated full mutation of the FMR1 gene.

Hum Genet 2020 Feb 9;139(2):227-245. Epub 2020 Jan 9.

Istituto di Medicina Genomica, Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli IRCCS, Largo F. Vito 1, 00168, Roma, Italy.

Fragile X-related disorders are due to a dynamic mutation of the CGG repeat at the 5' UTR of the FMR1 gene, coding for the RNA-binding protein FMRP. As the CGG sequence expands from premutation (PM, 56-200 CGGs) to full mutation (> 200 CGGs), FMRP synthesis decreases until it is practically abolished in fragile X syndrome (FXS) patients, mainly due to FMR1 methylation. Cells from rare individuals with no intellectual disability and carriers of an unmethylated full mutation (UFM) produce slightly elevated levels of FMR1-mRNA and relatively low levels of FMRP, like in PM carriers. With the aim of clarifying how UFM cells differ from CTRL and FXS cells, a comparative proteomic approach was undertaken, from which emerged an overexpression of SOD2 in UFM cells, also confirmed in PM but not in FXS. The SOD2-mRNA bound to FMRP in UFM more than in the other cell types. The high SOD2 levels in UFM and PM cells correlated with lower levels of superoxide and reactive oxygen species (ROS), and with morphological anomalies and depolarization of the mitochondrial membrane detected through confocal microscopy. The same effect was observed in CTRL and FXS after treatment with MC2791, causing SOD2 overexpression. These mitochondrial phenotypes reverted after knock-down with siRNA against SOD2-mRNA and FMR1-mRNA in UFM and PM. Overall, these data suggest that in PM and UFM carriers, which have high levels of FMR1 transcription and may develop FXTAS, SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation.
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http://dx.doi.org/10.1007/s00439-019-02104-7DOI Listing
February 2020

Aconitase 2 inhibits the proliferation of MCF-7 cells promoting mitochondrial oxidative metabolism and ROS/FoxO1-mediated autophagic response.

Br J Cancer 2020 01 10;122(2):182-193. Epub 2019 Dec 10.

Department of Biology, University of Rome "Tor Vergata", Via della Ricerca Scientifica 1, Rome, 00133, Italy.

Background: Deregulation of the tricarboxylic acid cycle (TCA) due to mutations in specific enzymes or defective aerobic metabolism is associated with tumour growth. Aconitase 2 (ACO2) participates in the TCA cycle by converting citrate to isocitrate, but no evident demonstrations of its involvement in cancer metabolism have been provided so far.

Methods: Biochemical assays coupled with molecular biology, in silico, and cellular tools were applied to circumstantiate the impact of ACO2 in the breast cancer cell line MCF-7 metabolism. Fluorescence lifetime imaging microscopy (FLIM) of NADH was used to corroborate the changes in bioenergetics.

Results: We showed that ACO2 levels are decreased in breast cancer cell lines and human tumour biopsies. We generated ACO2- overexpressing MCF-7 cells and employed comparative analyses to identify metabolic adaptations. We found that increased ACO2 expression impairs cell proliferation and commits cells to redirect pyruvate to mitochondria, which weakens Warburg-like bioenergetic features. We also demonstrated that the enhancement of oxidative metabolism was supported by mitochondrial biogenesis and FoxO1-mediated autophagy/mitophagy that sustains the increased ROS burst.

Conclusions: This work identifies ACO2 as a relevant gene in cancer metabolic rewiring of MCF-7 cells, promoting a different utilisation of pyruvate and revealing the potential metabolic vulnerability of ACO2-associated malignancies.
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http://dx.doi.org/10.1038/s41416-019-0641-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051954PMC
January 2020

The Combination of Whole Cell Lipidomics Analysis and Single Cell Confocal Imaging of Fluidity and Micropolarity Provides Insight into Stress-Induced Lipid Turnover in Subcellular Organelles of Pancreatic Beta Cells.

Molecules 2019 Oct 17;24(20). Epub 2019 Oct 17.

Institute for Drug Research, Faculty of Medicine, The Hebrew University, 911210 Jerusalem, Israel.

Modern omics techniques reveal molecular structures and cellular networks of tissues and cells in unprecedented detail. Recent advances in single cell analysis have further revolutionized all disciplines in cellular and molecular biology. These methods have also been employed in current investigations on the structure and function of insulin secreting beta cells under normal and pathological conditions that lead to an impaired glucose tolerance and type 2 diabetes. Proteomic and transcriptomic analyses have pointed to significant alterations in protein expression and function in beta cells exposed to diabetes like conditions (e.g., high glucose and/or saturated fatty acids levels). These nutritional overload stressful conditions are often defined as glucolipotoxic due to the progressive damage they cause to the cells. Our recent studies on the rat insulinoma-derived INS-1E beta cell line point to differential effects of such conditions in the phospholipid bilayers in beta cells. This review focuses on confocal microscopy-based detection of these profound alterations in the plasma membrane and membranes of insulin granules and lipid droplets in single beta cells under such nutritional load conditions.
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http://dx.doi.org/10.3390/molecules24203742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6833103PMC
October 2019

Dynamic structural determinants underlie the neurotoxicity of the N-terminal tau 26-44 peptide in Alzheimer's disease and other human tauopathies.

Int J Biol Macromol 2019 Dec 27;141:278-289. Epub 2019 Aug 27.

European Brain Research Institute (EBRI), Viale Regina Elena 295, 00161 Rome, Italy; Institute of Translational Pharmacology (IFT)-CNR, Via Fosso del Cavaliere 100, 00133 Rome, Italy. Electronic address:

The intrinsically disordered tau protein plays a pivotal role in the pathogenesis of Alzheimer's disease (AD) and other human tauopathies. Abnormal post-translational modifications of tau, such as truncation, are causally involved in the onset/development of these neurodegenerative diseases. In this context, the AD-relevant N-terminal fragment mapping between 26 and 44 amino acids of protein (tau26-44) is interesting, being endowed with potent neurotoxic effects in vitro and in vivo. However, the understanding of the mechanism(s) of tau26-44 toxicity is a challenging task because, similarly to the full-length tau, it does not have a unique 3D structure but exists as dynamic ensemble of conformations. Here we use Atomic Force Spectroscopy, Small Angle X-ray Scattering and Molecular Dynamics simulation to gather structural and functional information on the tau26-44. We highlight the presence, the type and the location of its temporary secondary structures and we unveil the occurrence of relevant transient tertiary conformations that could contribute to tau26-44 toxicity. Data are compared with those obtained on the biologically-inactive, reverse-sequence (tau44-26 peptide) which has the same mass, charge, aminoacidic composition as well as the same overall unfolded character of tau26-44.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.08.220DOI Listing
December 2019

Low-Intensity Ultrasound Induces Thermodynamic Phase Separation of Cell Membranes through a Nucleation-Condensation Process.

Ultrasound Med Biol 2019 05 15;45(5):1143-1150. Epub 2019 Feb 15.

Fondazione Policlinico Universitario A. Gemelli IRCSS, Rome, Italy; Istituto di Fisica, Università Cattolica del Sacro Cuore, Rome, Italy. Electronic address:

Membrane fluidity, a broad term adopted to describe the thermodynamic phase state of biological membranes, can be altered by local pressure variations caused by ultrasound exposure. The alterations in lipid spatial configuration and dynamics can modify their interactions with membrane proteins and activate signal transduction pathways, thus regulating several cellular functions. Here fluidity maps of murine fibroblast cells are generated at a sub-micrometric scale during ultrasound stimulation with an intensity and frequency typical of medical applications. Ultrasound induces a phase separation characterized by two-step kinetics leading to a time-dependent decrease in fluidity. First, nucleation of liquid crystallin domains with an average dimension of ∼1 μm occurs. Then, these domains condense into larger clusters with an average dimension of ∼1.5 μm. The induced phase separation could be an important driving force critical for the cellular response connecting the ultrasound-induced mechanical stress and signal transduction.
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http://dx.doi.org/10.1016/j.ultrasmedbio.2019.01.011DOI Listing
May 2019

Hexadecenoic Fatty Acid Positional Isomers and De Novo PUFA Synthesis in Colon Cancer Cells.

Int J Mol Sci 2019 Feb 15;20(4). Epub 2019 Feb 15.

Consiglio Nazionale delle Ricerche, ISOF, Area della Ricerca, 40129 Bologna, Italy.

Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9-16:1; -7 series) and sapienic acid (6-16:1; -10 series), respectively. The corresponding biological consequences and lipidomic research on these positional monounsaturated fatty acid (MUFA) isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the -10 polyunsaturated fatty acid (PUFA) sebaleic acid (5,8-18:2), but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 μM) and provide evidence of the formation of -10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodelling was influenced by the type of fatty acid and positional isomer, with an increase of 8-18:1, -10 PUFA and a decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in cases with sapienic acid. Sapienic acid was the less toxic among the tested fatty acids, showing the highest ECs and inducing death only in 75% of cells at the highest concentration tested. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8-18:1 and the -10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.
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http://dx.doi.org/10.3390/ijms20040832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412212PMC
February 2019

Impact of apolipoprotein A1- or lecithin:cholesterol acyltransferase-deficiency on white adipose tissue metabolic activity and glucose homeostasis in mice.

Biochim Biophys Acta Mol Basis Dis 2019 06 10;1865(6):1351-1360. Epub 2019 Feb 10.

University of Patras, School of Medicine, Department of Pharmacology, Rio, Achaias, TK. 26500, Greece. Electronic address:

High density lipoprotein (HDL) has attracted the attention of biomedical community due to its well-documented role in atheroprotection. HDL has also been recently implicated in the regulation of islets of Langerhans secretory function and in the etiology of peripheral insulin sensitivity. Indeed, data from numerous studies strongly indicate that the functions of pancreatic β-cells, skeletal muscles and adipose tissue could benefit from improved HDL functionality. To better understand how changes in HDL structure may affect diet-induced obesity and type 2 diabetes we aimed at investigating the impact of Apoa1 or Lcat deficiency, two key proteins of peripheral HDL metabolic pathway, on these pathological conditions in mouse models. We report that universal deletion of apoa1 or lcat expression in mice fed western-type diet results in increased sensitivity to body-weight gain compared to control C57BL/6 group. These changes in mouse genome correlate with discrete effects on white adipose tissue (WAT) metabolic activation and plasma glucose homeostasis. Apoa1-deficiency results in reduced WAT mitochondrial non-shivering thermogenesis. Lcat-deficiency causes a concerted reduction in both WAT oxidative phosphorylation and non-shivering thermogenesis, rendering lcat mice the most sensitive to weight gain out of the three strains tested, followed by apoa1 mice. Nevertheless, only apoa1 mice show disturbed plasma glucose homeostasis due to dysfunctional glucose-stimulated insulin secretion in pancreatic β-islets and insulin resistant skeletal muscles. Our analyses show that both apoa1 and lcat mice fed high-fat diet have no measurable Apoa1 levels in their plasma, suggesting no direct involvement of Apoa1 in the observed phenotypic differences among groups.
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http://dx.doi.org/10.1016/j.bbadis.2019.02.003DOI Listing
June 2019

Quantitative imaging of membrane micropolarity in living cells and tissues by spectral phasors analysis.

MethodsX 2018 29;5:1399-1412. Epub 2018 Oct 29.

Università Cattolica del Sacro Cuore, largo Francesco Vito 1, 00168, Rome, Italy.

Intracellular micropolarity is essential in several metabolic processes, as it controls membrane permeability, regulating the fluxes of molecules and energy. Here we describe a method for the determination of the micropolarity in living cells using spectral confocal microscopy. The method is based on a phasor analysis of spectrally resolved images of live cells, labelled with the solvatochromic probe Nile Red. An application is provided to extract a polarity profile from the acquired Spectral datasets, which represent the contribution of hyperpolar, polar and non-polar lipids, and to generate a micropolarity map at submicrometric spatial resolution. A metabolic parameter, representing a quantitative index of the fatty acid-triacylglycerol turnover, is also furnished. This method allows a functional profiling of cells and tissues and the detection of metabolic imbalances between lipid storage and usage. •Use of spectral resolved confocal microscopy of Nile Red labelled cells for pixel resolved determination of the membranes micropolarity.•Spectral acquisition increases the specificity and sensitivity of the detection to provide a polarity profile and a metabolic index for fatty acid-TG turnover.•Use of spectral resolved confocal microscopy of Nile Red labelled cells for pixel resolved determination of the membranes micropolarity.
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http://dx.doi.org/10.1016/j.mex.2018.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232651PMC
October 2018

Nanoscale mechanics of brain abscess: An atomic force microscopy study.

Micron 2018 10 20;113:34-40. Epub 2018 Jun 20.

Physics Institute, Catholic University of Sacred Heart, Largo F. Vito, 1, Rome, 00168, Italy.

Mechanical stimuli are a fundamental player in the pathophysiology of the brain influencing its physiological development and contributing to the onset and progression of many diseases. In some pathological states, the involvement of mechanical and physical stimuli might be extremely subtle; in others, it is more evident and particularly relevant. Among the latter pathologies, one of the most serious life-threatening condition is the brain abscess (BA), a focal infection localized in the brain parenchyma, which causes large brain mechanical deformations, giving rise to a wide range of neurological impairments. In this paper, we present the first nano-mechanical characterization of surgically removed human brain abscess tissues by means of atomic force microscopy (AFM) in the spectroscopy mode. Consistently with previous histological findings, we modeled the brain abscess as a multilayered structure, composed of three main layers: the cerebritis layer, the collagen capsule, and the internal inflammatory border. We probed the viscoelastic behavior of each layer separately through the measure of the apparent Young's modulus (E), that gives information about the sample stiffness, and the AFM hysteresis (H), that estimates the contribution of viscous and dissipative forces. Our experimental findings provide a full mechanical characterization of the abscess, showing an average E of (94 ± 5) kPa and H of 0.37 ± 0.01 for the cerebritis layer, an average E = (1.04 ± 0.05) MPa and H = 0.10 ± 0.01 for the collagen capsule and an average E = (9.8 ± 0.4) kPa and H = 0.57 ± 0.01 for the internal border. The results here presented have the potential to contribute to the development of novel surgical instruments dedicated to the treatment of the pathology and to stimulate the implementation of novel constitutive mechanical models for the estimation of brain compression and damage during BA progression.
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http://dx.doi.org/10.1016/j.micron.2018.06.012DOI Listing
October 2018

A decision support system for type 1 diabetes mellitus diagnostics based on dual channel analysis of red blood cell membrane fluidity.

Comput Methods Programs Biomed 2018 Aug 17;162:263-271. Epub 2018 May 17.

Unit of Computer Systems and Bioinformatics, Department of Engineering, Università Campus Bio-Medico di Roma, Rome, Italy.

Background And Objective: Investigation of membrane fluidity by metabolic functional imaging opens up a new and important area of translational research in type 1 diabetes mellitus, being a useful and sensitive biomarker for disease monitoring and treatment. We investigate here how data on membrane fluidity can be used for diabetes monitoring.

Methods: We present a decision support system that distinguishes between healthy subjects, type 1 diabetes mellitus patients, and type 1 diabetes mellitus patients with complications. It leverages on dual channel data computed from the physical state of human red blood cells membranes by means of features based on first- and second-order statistical measures as well as on rotation invariant co-occurrence local binary patterns. The experiments were carried out on a dataset of more than 1000 images belonging to 27 subjects.

Results: Our method shows a global accuracy of 100%, outperforming also the state-of-the-art approach based on the glycosylated hemoglobin.

Conclusions: The proposed recognition approach permits to achieve promising results.
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http://dx.doi.org/10.1016/j.cmpb.2018.05.025DOI Listing
August 2018

Real time quantitative analysis of lipid storage and lipolysis pathways by confocal spectral imaging of intracellular micropolarity.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 07 11;1863(7):783-793. Epub 2018 Apr 11.

Institute for Drug Research, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9112001, Israel.

Organisms store fatty acids in triacylglycerols in the form of lipid droplets, or hydrolyze triacylglycerols in response to energetic demands via activation of lipolytic or storage pathways. These pathways are complex sets of sequential reactions that are finely regulated in different cell types. Here we present a high spatial and temporal resolution-based method for the quantification of the turnover of fatty acids into triglycerides in live cells without introducing sample preparation artifacts. We performed confocal spectral imaging of intracellular micropolarity in cultured insulin secreting beta cells to detect micropolarity variations as they occur in time and at different pixels of microscope images. Acquired data are then analyzed in the framework of the spectral phasors technique. The method furnishes a metabolic parameter, which quantitatively assesses fatty acids - triacylglycerols turnover and the activation of lipolysis and storage pathways. Moreover, it provides a polarity profile, which represents the contribution of hyperpolar, polar and non-polar classes of lipids. These three different classes can be visualized on the image at a submicrometer resolution, revealing the spatial localization of lipids in cells under physiological and pathological settings. This new method allows for a fine-tuned, real-time visualization of the turnover of fatty acids into triglycerides in live cells with submicrometric resolution. It also detects imbalances between lipid storage and usage, which may lead to metabolic disorders within living cells and organisms.
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http://dx.doi.org/10.1016/j.bbalip.2018.04.004DOI Listing
July 2018

Systemic profiling of ectopic fat deposits in the reproductive tract of dairy cows.

Theriogenology 2018 Jul 17;114:46-53. Epub 2018 Mar 17.

Istituto di Fisica, Università Cattolica del Sacro cuore, Largo Francesco Vito 1, Rome, Italy.

During the transition period, high-yielding dairy cows suffer from negative energy balance, intense lipomobilization and impaired lipid metabolism; this metabolic condition can lead to overburdened triglycerides accumulation in the liver, known as liver lipidosis, which has been associated to impaired fertility in dairy cows. The mechanisms of this impairment can be in principle correlated with the presence and the extent of ectopic fat depots. However, current methods for evaluating fat accumulation in liver and in the reproductive tract suffer from low resolution, sensitivity, and specificity. Confocal microscopes are equipped with Gallium arsenide phosphide detectors, thus enabling the acquisition of intense signals from tissue biopsies. This method could differentiate whether fat deposition occurred without requiring sample sectioning. Here, we examined with this technique liver, uterine and ovarian samples of heifers and regularly slaughtered repeat breeder and overconditioned dairy cows, to quantify lipid droplets and depots at a submicrometer scale with high specificity. With the aid of this technique, we found lipid depots in uterine and ovarian specimens. Moreover, we found that the size and number of depots increased with the degree of liver lipidosis. Further studies are needed to elucidate the relationship between the severity and extent of these deposits and the fertility of lactating dairy cows. Since tissues other than liver display different characteristic lipid droplet distributions, this technique can be potentially employed to shed new light on the pathogenesis of lipidosis and to assess new risk factors for infertility.
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http://dx.doi.org/10.1016/j.theriogenology.2018.03.026DOI Listing
July 2018

Phase separation of the plasma membrane in human red blood cells as a potential tool for diagnosis and progression monitoring of type 1 diabetes mellitus.

PLoS One 2017 7;12(9):e0184109. Epub 2017 Sep 7.

Istituto di Fisica, Università Cattolica del Sacro Cuore, Rome, Italy.

Glycosylation, oxidation and other post-translational modifications of membrane and transmembrane proteins can alter lipid density, packing and interactions, and are considered an important factor that affects fluidity variation in membranes. Red blood cells (RBC) membrane physical state, showing pronounced alterations in Type 1 diabetes mellitus (T1DM), could be the ideal candidate for monitoring the disease progression and the effects of therapies. On these grounds, the measurement of RBC membrane fluidity alterations can furnish a more sensitive index in T1DM diagnosis and disease progression than Glycosylated hemoglobin (HbA1c), which reflects only the information related to glycosylation processes. Here, through a functional two-photon microscopy approach we retrieved fluidity maps at submicrometric scale in RBC of T1DM patients with and without complications, detecting an altered membrane equilibrium. We found that a phase separation between fluid and rigid domains occurs, triggered by systemic effects on membranes fluidity of glycation and oxidation. The phase separation patterns are different among healthy, T1DM and T1DM with complications patients. Blood cholesterol and LDL content are positively correlated with the extent of the phase separation patterns. To quantify this extent a machine learning approach is employed to develop a Decision-Support-System (DSS) able to recognize different fluidity patterns in RBC. Preliminary analysis shows significant differences(p<0.001) among healthy, T1DM and T1DM with complications patients. The development of an assay based on Phase separation of the plasma membrane of the Red Blood cells is a potential tool for diagnosis and progression monitoring of type 1 diabetes mellitus, and could allow customization and the selection of medical treatments in T1DM in clinical settings, and enable the early detection of complications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184109PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589169PMC
October 2017

The future development of bacteria fighting medical devices: the role of graphene oxide.

Expert Rev Med Devices 2016 Nov;13(11):1013-1019

a Physics Institute , Catholic University of Sacred Hearth , Rome , Italy.

Introduction: The clinical challenge that research on antibacterial coatings faces nowadays is the need of reduction of resistant bacterial infections, major source of implant rejection and repeated surgery. In order to avoid microorganisms attachment and biofilm formation, coating materials on medical devices have been developed with shortcomings represented by short-term durability and induction of new mechanisms of bacterial resistance. Graphene-based films and hydrogel could represent the next generation protective coatings due to their excellent mechanical, chemical and thermal properties, high nanoparticle adsorption and antibacterial action. Areas covered: In this short commentary, we will report the recent developments of graphene oxide based coatings. Graphene oxide is a water-soluble derivative of graphene that allows high drug loading and miscibility with polymers, making it mouldable in any desired shape. Recent applications in wound healing and tissue engineering will be discussed as well as critical issues prior to clinical use of graphene oxide coatings. Expert commentary: The current evidence is insufficient to establish the efficacy of Graphene Oxide against bacteria and the durability of coatings. Further studies should clarify how to control Graphene Oxide antibacterial mechanism.
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http://dx.doi.org/10.1080/17434440.2016.1245612DOI Listing
November 2016

Placental Chemokine Receptor D6 Is Functionally Impaired in Pre-Eclampsia.

PLoS One 2016 25;11(10):e0164747. Epub 2016 Oct 25.

Department of Obstetrics and Gynaecology, Fondazione Policlinico Agostino Gemelli, Università Cattolica Del Sacro Cuore, Rome, Italy.

Background: Pre-eclampsia (PE) is a major cause of maternal and perinatal morbidity and mortality worldwide. It is defined by new onset of hypertension and proteinuria after the 20th week of gestation and characterized by systemic exaggerated inflammatory response. D6 is a chemokines scavenger receptor that binds with high affinity CC chemokines, internalizes and targets the ligands for degradation. It is expressed in trophoblast-derived tissues and prevents excessive placenta leukocyte infiltration.The aim of this study was to investigate the expression and function of D6 in human placentae from pre-eclamptic and healthy pregnant women.

Methods And Results: Plasma levels of D6-binding CC chemokines (CCL-2, CCL-3, CCL-4, CCL-7, CCL-11) and pro-inflammatory cytokines (IL-6, TNF-α, CRP) were analyzed in 37 healthy pregnant women and 38 patients with PE by multiplex bead assay. Higher circulating levels of CCL7, CCL11, IL-6, (p<0.0001) and CRP (p<0.05) were observed in PE women compared to controls. Levels of circulating CCL4 were decreased in PE (p<0.001), while no significant differences of CCL2, CCL3 or TNF-α levels were detected. Immunofluorescent staining of placental sections showed higher expression of D6 receptor in the PE syncytiotrophoblast. Confocal and Western blot (WB) analyses revealed a prevalent distribution of D6 in trophoblast cells membranes in PE. Increased activation of D6 intracellular pathway was observed by Western blot analyses of p-LIMK and p-cofilin in trophoblast cell lysates. D6 functional assays showed reduced scavenging of CCL2 in PE cells compared to controls. Since actin filaments spatial assembling is essential for D6 intracellular trafficking and scavenging activity, we investigated by confocal microscopy trophoblast cytoskeleton organization and we observed a dramatic disarrangement in PE compared to controls.

Conclusions: our results suggest membrane distribution of D6 receptor on trophoblast cell membranes in PE, together with reduced functionality, probably due to cytoskeleton impairment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164747PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079655PMC
June 2017

Imaging Reactive Oxygen Species-Induced Modifications in Living Systems.

Antioxid Redox Signal 2016 06;24(16):939-58

1 Institute of Physics, Catholic University of Sacred Heart , Roma, Italy .

Significance: Reactive Oxygen Species (ROS) may regulate signaling, ion channels, transcription factors, and biosynthetic processes. ROS-related diseases can be due to either a shortage or an excess of ROS.

Recent Advances: Since the biological activity of ROS depends on not only concentration but also spatiotemporal distribution, real-time imaging of ROS, possibly in vivo, has become a need for scientists, with potential for clinical translation. New imaging techniques as well as new contrast agents in clinically established modalities were developed in the previous decade.

Critical Issues: An ideal imaging technique should determine ROS changes with high spatio-temporal resolution, detect physiologically relevant variations in ROS concentration, and provide specificity toward different redox couples. Furthermore, for in vivo applications, bioavailability of sensors, tissue penetration, and a high signal-to-noise ratio are additional requirements to be satisfied.

Future Directions: None of the presented techniques fulfill all requirements for clinical translation. The obvious way forward is to incorporate anatomical and functional imaging into a common hybrid-imaging platform. Antioxid. Redox Signal. 24, 939-958.
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http://dx.doi.org/10.1089/ars.2015.6415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900226PMC
June 2016

Hormetic and regulatory effects of lipid peroxidation mediators in pancreatic beta cells.

Mol Aspects Med 2016 06 21;49:49-77. Epub 2016 Mar 21.

Department of Pharmacology, Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel. Electronic address:

Nutrient sensing mechanisms of carbohydrates, amino acids and lipids operate distinct pathways that are essential for the adaptation to varying metabolic conditions. The role of nutrient-induced biosynthesis of hormones is paramount for attaining metabolic homeostasis in the organism. Nutrient overload attenuate key metabolic cellular functions and interfere with hormonal-regulated inter- and intra-organ communication, which may ultimately lead to metabolic derangements. Hyperglycemia and high levels of saturated free fatty acids induce excessive production of oxygen free radicals in tissues and cells. This phenomenon, which is accentuated in both type-1 and type-2 diabetic patients, has been associated with the development of impaired glucose tolerance and the etiology of peripheral complications. However, low levels of the same free radicals also induce hormetic responses that protect cells against deleterious effects of the same radicals. Of interest is the role of hydroxyl radicals in initiating peroxidation of polyunsaturated fatty acids (PUFA) and generation of α,β-unsaturated reactive 4-hydroxyalkenals that avidly form covalent adducts with nucleophilic moieties in proteins, phospholipids and nucleic acids. Numerous studies have linked the lipid peroxidation product 4-hydroxy-2E-nonenal (4-HNE) to different pathological and cytotoxic processes. Similarly, two other members of the family, 4-hydroxyl-2E-hexenal (4-HHE) and 4-hydroxy-2E,6Z-dodecadienal (4-HDDE), have also been identified as potential cytotoxic agents. It has been suggested that 4-HNE-induced modifications in macromolecules in cells may alter their cellular functions and modify signaling properties. Yet, it has also been acknowledged that these bioactive aldehydes also function as signaling molecules that directly modify cell functions in a hormetic fashion to enable cells adapt to various stressful stimuli. Recent studies have shown that 4-HNE and 4-HDDE, which activate peroxisome proliferator-activated receptor δ (PPARδ) in vascular endothelial cells and insulin secreting beta cells, promote such adaptive responses to ameliorate detrimental effects of high glucose and diabetes-like conditions. In addition, due to the electrophilic nature of these reactive aldehydes they form covalent adducts with electronegative moieties in proteins, phosphatidylethanolamine and nucleotides. Normally these non-enzymatic modifications are maintained below the cytotoxic range due to efficient cellular neutralization processes of 4-hydroxyalkenals. The major neutralizing enzymes include fatty aldehyde dehydrogenase (FALDH), aldose reductase (AR) and alcohol dehydrogenase (ADH), which transform the aldehyde to the corresponding carboxylic acid or alcohols, respectively, or by biding to the thiol group in glutathione (GSH) by the action of glutathione-S-transferase (GST). This review describes the hormetic and cytotoxic roles of oxygen free radicals and 4-hydroxyalkenals in beta cells exposed to nutritional challenges and the cellular mechanisms they employ to maintain their level at functional range below the cytotoxic threshold.
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http://dx.doi.org/10.1016/j.mam.2016.03.001DOI Listing
June 2016

A CREB-Sirt1-Hes1 Circuitry Mediates Neural Stem Cell Response to Glucose Availability.

Cell Rep 2016 Feb 21;14(5):1195-1205. Epub 2016 Jan 21.

Institute of Human Physiology, Università Cattolica Medical School, 00168 Rome, Italy; San Raffaele Pisana Scientific Institute for Research, Hospitalization and Health Care, 00163 Rome, Italy.

Adult neurogenesis plays increasingly recognized roles in brain homeostasis and repair and is profoundly affected by energy balance and nutrients. We found that the expression of Hes-1 (hairy and enhancer of split 1) is modulated in neural stem and progenitor cells (NSCs) by extracellular glucose through the coordinated action of CREB (cyclic AMP responsive element binding protein) and Sirt-1 (Sirtuin 1), two cellular nutrient sensors. Excess glucose reduced CREB-activated Hes-1 expression and results in impaired cell proliferation. CREB-deficient NSCs expanded poorly in vitro and did not respond to glucose availability. Elevated glucose also promoted Sirt-1-dependent repression of the Hes-1 promoter. Conversely, in low glucose, CREB replaced Sirt-1 on the chromatin associated with the Hes-1 promoter enhancing Hes-1 expression and cell proliferation. Thus, the glucose-regulated antagonism between CREB and Sirt-1 for Hes-1 transcription participates in the metabolic regulation of neurogenesis.
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http://dx.doi.org/10.1016/j.celrep.2015.12.092DOI Listing
February 2016

Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates.

Autophagy 2015 ;11(10):1905-16

a Istituto di Fisica; Università Cattolica del Sacro Cuore ; Rome , Italy.

Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5-6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates.
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http://dx.doi.org/10.1080/15548627.2015.1084455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824579PMC
September 2016

Human IgG Antinuclear Antibodies Induce Pregnancy Loss in Mice by Increasing Immune Complex Deposition in Placental Tissue: In Vivo Study.

Am J Reprod Immunol 2015 Dec 21;74(6):542-52. Epub 2015 Sep 21.

Department of Obstetrics and Gynecology, Policlinico A. Gemelli, Università Cattolica Del Sacro Cuore, Rome, Italy.

Problem: A threefold higher prevalence of antinuclear antibodies (ANA) has been reported in patients with recurrent pregnancy loss (RPL). Nevertheless, the role of ANA in reproductive failure is still unclear. The aim of this study was to investigate the role of ANA during early pregnancy in vivo.

Method Of Study: We used pregnant mice treated with immunoglobulin G (IgG) obtained from normal healthy subjects (NHS); ANA(+) sera of patients with RPL; and ANA(+) sera from women with uncomplicated pregnancies (HW). Placental immunohistochemical/immunofluorescence staining was performed to detect complement and immune complex deposition. ELISA was performed to evaluate complement levels.

Results: ANA(+) IgG from RPL women significantly increased embryo resorption rate, reduced C3, and increased C3a serum levels compared to NHS IgG or ANA(+) -HW IgG. Increased C3 deposition and increased immune complex staining in placental tissues from mice treated with ANA(+) -RPL IgG fraction compared to NHS- and ANA(+) -HW-IgG-treated mice were found.

Conclusion: ANA(+) IgG injection in mice is able to induce fetal resorption and complement activation. The presence on placental tissues of immune complexes and complement fragments suggests the complement activation as a possible mechanism of placental damage.
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http://dx.doi.org/10.1111/aji.12429DOI Listing
December 2015

The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing.

Sci Rep 2015 Mar 17;5:9149. Epub 2015 Mar 17.

Institute of Internal Medicine and Gastroenterology, Catholic University of the Sacred Heart School of Medicine.

Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expression was performed by Affymetrix technology. SFB cytotoxicity was evaluated by multiple assays in the presence or absence of metabolic inhibitors, or in cells genetically depleted of mitochondria. We found that low concentrations (2.5-5 μM) of SFB had a relatively modest effect on LCSC-2 or 293 T cell growth, but damaged mitochondria and increased intracellular ROS. Gene expression profiling of SFB-treated cells was consistent with a shift toward aerobic glycolysis and, accordingly, SFB cytotoxicity was dramatically increased by glucose withdrawal or the glycolytic inhibitor 2-DG. Under metabolic stress, activation of the AMP dependent Protein Kinase (AMPK), but not ROS blockade, protected cells from death. We conclude that mitochondrial damage and ROS drive cell killing by SFB, while glycolytic cell reprogramming may represent a resistance strategy potentially targetable by combination therapies.
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http://dx.doi.org/10.1038/srep09149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361992PMC
March 2015