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The thermodynamic behavior of mixed monolayers of cardiolipin-dipalmitoylphosphatidylcholine (CLP-DPPC) and cardiolipin-dipalmitoylphosphatidylethanolamine (CLP-DPPE) is investigated by studying their Langmuir surface pressure and surface potential isotherms at different cardiolipin molar fractions. The overall thermodynamic behavior is independent of mole fraction for the mixed CLP-DPPC monolayers while a critical fraction exists for the CLP-DPPE monolayers.

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.Cell Death and Differentiation (2005) 12, 78-86. doi:10.1038/sj.cdd.4401522 Published online 29 October 2004 [ABSTRACT FROM AUTHOR]

Studies the influence of phospholipase A2 (PLA2) on sodium/hydrogen antiport and proliferation in rat aortic smooth muscle cells (RASMC). Effects of ammodytin L (AMDL) and pancreatic secretory PLA2 on [3H]thymidine incorporation in RASMC; Role of growth factors in the mitogenic response of RASMC to PLA2 or AMDL stimulation.

Cancer cells may acquire resistance to stress signals and reprogram metabolism to meet the energetic demands to support their high proliferation rate and avoid death. Hence, targeting nutrient dependencies of cancer cells has been suggested as a promising anti-cancer strategy. We explored the possibility of killing breast cancer (BC) cells by modifying nutrient availability. We used in vitro models of BC (MCF7 and MDA-MB-231) that were maintained with a low amount of sulfur amino acids (SAAs) and a high amount of oxidizable polyunsatured fatty acids (PUFAs). Treatment with anti-apoptotic, anti-ferroptotic and antioxidant drugs were used to determine the modality of cell death. We reproduced these conditions in vivo by feeding BC-bearing mice with a diet poor in proteins and SAAs and rich in PUFAs (LSAA/HPUFA). Western blot analysis, qPCR and histological analyses were used to assess the anti-cancer effects and the molecular pathways involved. We found that BC cells underwent oxidative damage to DNA and proteins and both apoptosis and ferroptosis were induced. Along with caspases-mediated PARP1 cleavage, we found a lowering of the GSH-GPX4 system and an increase of lipid peroxides. A LSAA/HPUFA diet reduced tumor mass and its vascularization and immune cell infiltration, and induced apoptosis and ferroptotic hallmarks. Furthermore, mitochondrial mass was found to be increased, and the buffering of mitochondrial reactive oxygen species limited GPX4 reduction and DNA damage. Our results suggest that administration of custom diets, targeting the dependency of cancer cells on certain nutrients, can represent a promising complementary option for anti-cancer therapy.

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT., Competing Interests: Declaration of interests J.R.B. has a pending patent application related to mitochondria transfer. The other authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Radiation therapy is one of the most effective methods of tumor eradication; however, in some forms of neuroblastoma, radiation can increase the risk of secondary neoplasms, due to the ability of irradiated cells to transmit pro-survival signals to non-irradiated cells through vesicle secretion. The aims of this study were to characterize the vesicles released by the human neuroblastoma cell line SH-SY5Y following X-ray radiations and their ability to increase invasiveness in non-irradiated SH-SY5Y cells. We first purified the extracellular vesicles released by the SH-SY5Y cells following X-rays, and then determined their total amount, dimensions, membrane protein composition, and cellular uptake. We also examined the effects of these extracellular vesicles on viability, migration, and DNA damage in recipient SH-SY5Y cells. We found that exposure to X-rays increased the release of extracellular vesicles and altered their protein composition. These vesicles were readily uptaken by non-irradiated cells, inducing an increase in viability, migration, and radio-resistance. The same results were obtained in an MYCN -amplified SK-N-BE cell line. Our study demonstrates that vesicles released from irradiated neuroblastoma cells stimulate proliferation and invasiveness that correlate with the epithelial to mesenchymal transition in non-irradiated cells. Moreover, our results suggest that, at least in neuroblastomas, targeting the extracellular vesicles may represent a novel therapeutic approach to counteract the side effects associated with radiotherapy.

The increasing duration of space missions involves a progressively higher exposure of astronauts to cosmic rays, whose most hazardous component is made up of High-Atomic number and High-Energy (HZE) ions. HZE ions interact along their tracks with biological molecules inducing changes on living material qualitatively different from that observed after irradiation for therapeutic purposes or following nuclear accidents. HZE ions trigger in cells different responses initialized by DNA damage and mitochondria dysregulation, which cause a prolonged state of sterile inflammation in the tissues. These cellular phenomena may explain why spending time in space was found to cause the onset of a series of diseases normally related to aging. These changes that mimic aging but take place more quickly make space flights also an opportunity to study the mechanisms underlying aging. In this short review, we describe the biological mechanisms underlying cell senescence and aging; the peculiar characteristics of HZE ions, their interaction with living matter and the effects on the organism; the key role of mitochondria in HZE ion-induced health effects and aging-related phenomena., (Copyright © 2020 Giovanetti, Tortolici and Rufini.)

Decreased expression of mitochondrial frataxin (FXN) causes Friedreich's ataxia (FRDA), a neurodegenerative disease with type 2 diabetes (T2D) as severe comorbidity. Brown adipose tissue (BAT) is a mitochondria-enriched and anti-diabetic tissue that turns excess energy into heat to maintain metabolic homeostasis. Here we report that the FXN knock-in/knock-out (KIKO) mouse shows hyperlipidemia, reduced energy expenditure and insulin sensitivity, and elevated plasma leptin, recapitulating T2D-like signatures. FXN deficiency leads to disrupted mitochondrial ultrastructure and oxygen consumption as well as lipid accumulation in BAT. Transcriptomic data highlights cold intolerance in association with iron-mediated cell death (ferroptosis). Impaired PKA-mediated lipolysis and expression of genes controlling mitochondrial metabolism, lipid catabolism and adipogenesis were observed in BAT of KIKO mice as well as in FXN-deficient T37i brown and primary adipocytes. Significant susceptibility to ferroptosis was observed in adipocyte precursors that showed increased lipid peroxidation and decreased glutathione peroxidase 4. Collectively our data point to BAT dysfunction in FRDA and suggest BAT as promising therapeutic target to overcome T2D in FRDA.

There is a growing interest in therapeutically targeting the inflammatory response that underlies age-related chronic diseases including obesity and type 2 diabetes. Through integrative small RNA sequencing, we show the presence of conserved plant miR159a and miR156c in dried nuts having high complementarity with the mammalian TNF receptor superfamily member 1a (Tnfrsf1a) transcript. We detected both miR159a and miR156c in exosome-like nut nanovesicles (NVs) and demonstrated that such NVs reduce Tnfrsf1a protein and dampen TNF-α signaling pathway in adipocytes. Synthetic single-stranded microRNAs (ss-miRs) modified with 2'- O -methyl group function as miR mimics. In plants, this modification naturally occurs on nearly all small RNAs. 2'- O -methylated ss-miR mimics for miR156c and miR159a decreased Tnfrsf1a protein and inflammatory markers in hypertrophic as well as TNF-α-treated adipocytes and macrophages. miR156c and miR159a mimics effectively suppress inflammation in mice, highlighting a potential role of plant miR-based, single-stranded oligonucleotides in treating inflammatory-associated metabolic diseases., Competing Interests: Competing interestsThe authors declare no competing interests

Crohn's disease is an inflammatory bowel disease showing a high heterogeneity in phenotype and a strong genetic component. The treatment is complex, due to different severity of clinical parameters and to the fact that therapies only permit to control symptoms and to induce remission for short periods. Moreover, all categories of drugs present a great interindividual variability both in terms of efficacy and side effects appearance. For this reason, the identification of specific genomic biomarkers involved in drugs response will be of great clinical utility in order to foresee drug's efficacy and to prevent adverse reactions, permitting a more personalized therapeutic approach. In this review, we focus the attention on the pharmacogenetic studies regarding drugs commonly utilized in Crohn's disease treatment.

Objective: Rheumatoid Arthritis (RA) is a progressive autoimmune disease characterized by chronic joint inflammation and structural damage. Remission or at least low disease activity (LDA) represent potentially desirable goals of RA treatment. Single nucleotide polymorphisms (SNPs) in several genes might be useful for prediction of response to therapy. We aimed at exploring 4 SNPs in candidate genes (STAT4, PTPN2, PSORS1C1 and TRAF3IP2) in order to investigate their potential role in the response to therapy with tumor necrosis factor inhibitors (TNF-i) in RA patients., Methods: In 171 RA patients we investigated the following SNPs: rs7574865 (STAT4), rs2233945 (PSORS1C1), rs7234029 (PTPN2) and rs33980500 (TRAF3IP2). Remission, LDA, and EULAR response were registered at 6 months and 2 years after initiation of first line TNF-i [Adalimumab (ADA) and Etanercept (ETN)]., Results: STAT4 variant allele was associated with the absence of a good/moderate EULAR response at 2 years of treatment in the whole RA group and in ETN treated patients. The PTPN2 SNP was associated with no good/moderate EULAR response at 6 months in ADA treated patients. Patients carrying PSORS1C1 variant allele did not reach LDA at 6 months in both the whole RA group and ETN treated patients. TRAF3IP2 variant allele was associated with the lack of LDA and remission achievement at 6 months in all RA cohort while an association with no EULAR response at 2 years of treatment occurred only in ETN treated patients., Conclusions: For the first time, we reported that SNPs in STAT4, PTPN2, PSORS1C1, and TRAF3IP2 are associated with response to TNF-i treatment in RA patients; however, these findings should be validated in a larger population., Competing Interests: Giuseppe Novelli serves as Editor for PLOS ONE. This does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria.

Niemann-Pick type C disease is an autosomal recessive storage disorder, characterized by abnormal sequestration of unesterified cholesterol within the late endolysosomal compartment of cells and accumulation of gangliosides and other sphingolipids. Progressive neurological deterioration and insurgence of symptoms like ataxia, seizure, and cognitive decline until severe dementia are pathognomonic features of the disease. Here, we studied synaptic plasticity phenomena and evaluated ERKs activation in the hippocampus of BALB/c NPC1-/- mice, a well described animal model of the disease. Our results demonstrated an impairment of both induction and maintenance of long term synaptic potentiation in NPC1-/- mouse slices, associated with the lack of ERKs phosphorylation. We then investigated the effects of Miglustat, a recent approved drug for the treatment of NPCD. We found that in vivo Miglustat administration in NPC1-/- mice was able to rescue synaptic plasticity deficits, to restore ERKs activation and to counteract hyperexcitability. Overall, these data indicate that Miglustat may be effective for treating the neurological deficits associated with NPCD, such as seizures and dementia.

Stevens-Johnson syndrome and toxic epidermal necrolysis are severe, life-threatening drug reactions involving skin and membranes mucous, which are associated with significant morbidity and mortality and triggered, especially by drug exposure. Different studies have demonstrated that drug response is a multifactorial character and that the interindividual variability in this response depends on both environmental and genetic factors. The last ones have a relevant significance. In fact, the identification of new specific genetic markers involved in the response to drugs, will be of great utility to establish a more personalized therapeutic approach and to prevent the appearance of these adverse reactions. In this review, we summarize recent progresses in the Pharmacogenetics studies related to Stevens-Johnson syndrome/toxic epidermal necrolysis reporting the major genetic factors identified in the last years as associated with the disease and highlighting the use of some of these genomic variants in the clinical practice.

Steven-Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) are severe adverse drug reactions, characterized by extensive epidermal detachment and erosions of mucous membrane. SJS/TEN is one of the most serious adverse reactions to Nevirapine (NVP) treatment, commonly used in developing countries as first-line treatment of human immunodeficiency virus infection. In the last years TRAF3IP2 gene variants had been described as associated with susceptibility to several diseases such as psoriasis and psoriatic arthritis. We hypothesized that this gene, involved in immune response and in NF-κB activation, could also be implicated in the SJS/TEN susceptibility. We performed a full resequencing of TRAF3IP2 gene in a population of patients treated with NVP. Twenty-seven patients with NVP-induced SJS/TEN and 78 controls, all from Mozambique, were enrolled. We identified eight exonic and three intronic already described variants. The case/control association analysis highlighted an association between the rs76228616 SNP in exon 2 and the SJS/TEN susceptibility. In particular, the variant allele (C) resulted significantly associated with a higher risk to develop SJS/TEN (p = 0.012 and OR = 3.65 (95% CI 1.33-10.01)). A multivariate analysis by logistic regression confirmed its significant contribution (p = 0.027, OR = 4.39 (95% CI 1.19-16.23)). In conclusion, our study suggests that a variant in TRAF3IP2 gene could be involved in susceptibility to SJS/TEN.

Genetic factors exert an important role in determining Systemic Lupus Erythematosus (SLE) susceptibility, interplaying with environmental factors. Several genetic studies in various SLE populations have identified numerous susceptibility loci. From a clinical point of view, SLE is characterized by a great heterogeneity in terms of clinical and laboratory manifestations. As widely demonstrated, specific laboratory features are associated with clinical disease subset, with different severity degree. Similarly, in the last years, an association between specific phenotypes and genetic variants has been identified, allowing the possibility to elucidate different mechanisms and pathways accountable for disease manifestations. However, except for Lupus Nephritis (LN), no studies have been designed to identify the genetic variants associated with the development of different phenotypes. In this review, we will report data currently known about this specific association.

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease with complex pathogenesis in which genes and environmental factors are involved. We aimed at analyzing previously identified loci associated with SLE or with other autoimmune and/or inflammatory disorders (STAT4, IL10, IL23R, IRAK1, PSORS1C1, HCP5, MIR146a, PTPN2, ERAP1, ATG16L1, IRGM) in a sample of Italian SLE patients in order to verify or confirm their possible involvement and relative contribution in the disease., Materials and Methods: Two hundred thirty-nine consecutive SLE patients and 278 matched healthy controls were enrolled. Study protocol included complete physical examination, and clinical and laboratory data collection. Nineteen polymorphisms were genotyped by allelic discrimination assays. A case-control association study and a genotype-phenotype correlation were performed., Results: STAT4 was the most associated gene [P = 3 × 10(-7), OR = 2.13 (95% CI: 1.59-2.85)]. IL10 confirmed its association with SLE [rs3024505: P = 0.02, OR = 1.52 (95% CI: 1.07-2.16)]. We describe a novel significant association between HCP5 locus and SLE susceptibility [rs3099844: P = 0.01, OR = 2.06 (95% CI: 1.18-3.6)]. The genotype/phenotype correlation analysis showed several associations including a higher risk to develop pericarditis with STAT4, and an association between HCP5 rs3099844 and anti-Ro/SSA antibodies., Conclusions: STAT4 and IL10 confirm their association with SLE. We found that some SNPs in PSORS1C1, ATG16L1, IL23R, PTPN2 and MIR146a genes can determine particular disease phenotypes. HCP5 rs3099844 is associated with SLE and with anti-Ro/SSA. This polymorphism has been previously found associated with cardiac manifestations of SLE, a condition related with anti-Ro/SSA antibodies. Thus, our results may provide new insights into SLE pathogenesis.

Mutants of Kluyveromyces lactis denominated vga (vanadate glycosylation affected) bear various combinations of glycosylation and cell-wall defects. The vga3 mutation of K. lactis was mapped in the KlOCH1 gene, encoding the functional homologue of the Saccharomyces cerevisiaealpha1,6-mannosyltransferase. Quantitative analysis of cell-wall components indicated a noticeable increase of chitin and beta1,6-glucans and a severe decrease of mannoproteins in the mutant cells as compared with the wild-type counterparts. Fine-structure determination of the beta1,6-glucan polymer indicated that, in the vga3-1 strain, the beta1,6-glucans are shorter and have more branches than in the wild-type strain. This suggests that cell-wall remodelling changes take place in K. lactis in the presence of glycosylation defects. Moreover, the vga3 cells showed a significantly improved capability of secreting heterologous proteins. Such a capability, accompanied by the highly reduced N-glycosylation, may be of biotechnological interest, especially when hyper-glycosylation of recombinant products must be avoided.

GDP-mannose is the mannosyl donor for the glycosylation reactions and is synthesized by GDP-mannose pyrophosphorylase from GTP and d-mannose-1-phosphate; in Saccharomyces cerevisiae this enzyme is encoded by the PSA1/VIG9/SRB1 gene. We isolated the Kluyveromyces lactis KlPSA1 gene by complementing the osmotic growth defects of S. cerevisiae srb1/psa1 mutants. KlPsa1p displayed a high degree of similarity with other GDP-mannose pyrophosphorylases and was demonstrated to be the functional homologue of S. cerevisiae Psa1p. Phenotypic analysis of a K. lactis strain overexpressing the KlPSA1 gene revealed changes in the cell wall assembly. Increasing the KlPSA1 copy number restored the defects in O-glycosylation, but not those in N-glycosylation, that occur in K. lactis cells depleted for the hexokinase Rag5p. Overexpression of GDP-mannose pyrophosphorylase also enhanced heterologous protein secretion in K. lactis as assayed by using the recombinant human serum albumin and the glucoamylase from Arxula adeninivorans.

The present study was carried out to investigate the potential involvement of cholesterol-rich membrane microdomains in the mobilization of calcium induced by NMDA-receptors (NMDA-R). We herein provide evidence that agents interfering with plasma membrane cholesterol (namely, filipin and methyl-beta-cyclodextrin (Cdex)) inhibit the NMDA-stimulated influx of calcium in hippocampal cells in culture. Filipin-treated cells maintained their morphology and were able to respond with a calcium influx to high K(+) challenge, whereas Cdex altered both cellular parameters. These results suggest that the NMDA-R can be located in cholesterol-rich membrane microdomains or alternatively that the mechanisms coupling their dynamics in the post-synaptic membrane are dependent on the integrity of the microdomains.

We report a study on the regulation by 2-chloro adenosine (2CA) of a glutamate (Glu) transporter in myogenic C2C12 cells. Long-term 2CA exposition significantly increased the V(max) of the Glu transporter. Moreover, 2CA-treated cells responded to Glu challenge by a rapid and transient increase in their intracellular calcium level. The above reported effects were totally abolished by treating C2C12 cells with the Na(+)-dependent Glu transporter inhibitors DL-threo-b-hydroxyaspartic acid and L-trans-pyrrolidine-2,4-dicarboxylic acid. We propose that the possible link between the Glu uptake increase and the Glu induction of calcium rise could be the depolarizing currents carried by Na(+) coupled with transporter activity.

Ammodytin L, purified from the venom of Vipera ammodytes, triggers a rapid and dramatic lytic process in myotubes in vitro, as well as in differentiated muscle cells in vivo, through a mechanism that is not well understood. Despite its great sequence similarity to phospholipase A2, it is devoid of any enzyme activity. Data on artificial membranes demonstrating a direct interaction between this toxin and the hydrophobic core of the lipid bilayer suggest that the toxin also acts on the lipid microenvironment in cell membranes. Recent experiments on living cells do not confirm this hypothesis, and a more intricate mechanism is proposed. In vitro, ammodytin L has necrotic effects only in well-differentiated myogenic cells, whereas other cell types such as platelets, red blood cells and lymphocytes show neither morphological nor functional alterations. In this work we demonstrate that rat 208F fibroblasts in culture after ammodytin L challenge increase [3H]thymidine incorporation, indicating that this toxin has a myogenic effect. Moreover, ammodytin L increases intracellular Ca2+ by acting on intracellular stores probably by activating a phosphatidylinositol-specific phospholipase C. Preincubation of the cells with ammodytin L did not prevent the massive Ca2+ release evoked by bradykinin, a phenomenon observed when fibroblasts were incubated with both thapsigargin and ionomycin. Heparin, an agent that inhibits the necrotic effect of the myotoxin in myotubes, also reduces the effect of ammodytin L on DNA synthesis. Heparin inhibits only the late sustained increase in intracellular Ca2+ induced by the toxin.

The temperature-dependence of both the lipid order parameter (SDPH) and acetylcholinesterase (AChE) activity from native and cholesterol-enriched human erythrocyte membranes was investigated. Cholesterol enrichment abolishes an inflection observed around 30 degrees C in the temperature-dependence of native membrane lipid order parameter, whereas the Arrhenius plot of the enzymic activity is substantially unaffected. These results support the view that the breaks in the Arrhenius plot of the enzyme activity are not related to sudden changes of bulk membrane physical state, but arise from a direct effect of temperature on enzyme conformation.