Dual species sphingosine-1-phosphate lyase inhibitors to combine antifungal and anti-inflammatory activities in cystic fibrosis: a feasibility study.

Cystic fibrosis (CF) is an autosomal recessive disorder characterized by respiratory failure due to a vicious cycle of defective Cystic Fibrosis Transmembrane conductance Regulator (CFTR) function, chronic inflammation and recurrent bacterial and fungal infections. Although the recent introduction of CFTR correctors/potentiators has revolutionized the clinical management of CF patients, resurgence of inflammation and persistence of pathogens still posit a major concern and should be targeted contextually. On the background of a network-based selectivity that allows to target the same enzyme in the host and microbes with different outcomes, we focused on sphingosine-1-phosphate (S1P) lyase (SPL) of the sphingolipid metabolism as a potential candidate to uniquely induce anti-inflammatory and antifungal activities in CF. As a feasibility study, herein we show that interfering with S1P metabolism improved the immune response in a murine model of CF with aspergillosis while preventing germination of Aspergillus fumigatus conidia. In addition, in an early drug discovery process, we purified human and A. fumigatus SPL, characterized their biochemical and structural properties, and performed an in silico screening to identify potential dual species SPL inhibitors. We identified two hits behaving as competitive inhibitors of pathogen and host SPL, thus paving the way for hit-to-lead and translational studies for the development of drug candidates capable of restraining fungal growth and increasing antifungal resistance.

Cardiolipin-mediated temporal response to hydroquinone toxicity in human retinal pigmented epithelial cell line.

Hydroquinone, a potent toxic agent of cigarette smoke, damages retinal pigmented epithelial cells by triggering oxidative stress and mitochondrial dysfunction, two events causally related to the development and progression of retinal diseases. The inner mitochondrial membrane is enriched in cardiolipin, a phospholipid susceptible of oxidative modifications which determine cell-fate decision. Using ARPE-19 cell line as a model of retinal pigmented epithelium, we analyzed the potential involvement of cardiolipin in hydroquinone toxicity. Hydroquinone exposure caused an early concentration-dependent increase in mitochondrial reactive oxygen species, decrease in mitochondrial membrane potential, and rise in the rate of oxygen consumption not accompanied by changes in ATP levels. Despite mitochondrial impairment, cell viability was preserved. Hydroquinone induced cardiolipin translocation to the outer mitochondrial membrane, and an increase in the colocalization of the autophagosome adapter protein LC3 with mitochondria, indicating the induction of protective mitophagy. A prolonged hydroquinone treatment induced pyroptotic cell death by cardiolipin-mediated caspase-1 and gasdermin-D activation. Cardiolipin-specific antioxidants counteracted hydroquinone effects pointing out that cardiolipin can act as a mitochondrial "eat-me signal" or as a pyroptotic cell death trigger. Our results indicate that cardiolipin may act as a timer for the mitophagy to pyroptosis switch and propose cardiolipin-targeting compounds as promising approaches for the treatment of oxidative stress-related retinal diseases.

Metastatic Prostate Cancer Cells Secrete Methylglyoxal-Derived MG-H1 to Reprogram Human Osteoblasts into a Dedifferentiated, Malignant-like Phenotype: A Possible Novel Player in Prostate Cancer Bone Metastases.

Bone metastases from prostate cancer (PCa) result from a complex cross-talk between PCa cells and osteoblasts (OB). Thus, targeting this interplay has become an attractive strategy to interfere with PCa bone dissemination. The agents currently used in clinical trials have proved ineffective, boosting research to identify additional mechanisms that may be involved in this two-directional talk. Here, we investigated whether and how 5-hydro-5-methylimidazolone (MG-H1), a specific methylglyoxal (MG)-derived advanced glycation end product (AGE), was a novel player in the dialogue between PCa and OB to drive PCa bone metastases. Conditioned medium from osteotropic PC3 PCa cells, pre-treated or not with a specific MG scavenger, was administrated to human primary OB and cell morphology, mesenchymal trans-differentiation, pro-osteogenic determinants, PCa-specific molecules, and migration/invasion were studied by phase-contrast microscopy, real-time PCR, western blot and specific assays, respectively. We found that PC3 cells were able to release MG-H1 that, by binding to the receptor for AGEs (RAGE) on OB, reprogrammed them into a less-differentiate phenotype, endowed with some PCa-specific molecular features and malignant properties, in a mechanism involving reactive oxidative species (ROS) production and NF-kB pathway activation. These findings provide novel insights into the mechanisms of PCa osteoblastic metastases and foster in vivo research toward new therapeutic strategies interfering with PCa/OB cross-talk.

Tm7sf2 gene promotes adipocyte differentiation of mouse embryonic fibroblasts and improves insulin sensitivity.

Adipogenesis is a finely orchestrated program involving a transcriptional cascade coordinated by CEBP and PPAR family members and by hormonally induced signaling pathways. Alterations in any of these factors result into impaired formation of fully differentiated adipocytes. Tm7sf2 gene encodes for a Δ(14)-sterol reductase primarily involved in cholesterol biosynthesis. Furthermore, TM7SF2 modulates the expression of the master gene of adipogenesis PPARγ, suggesting a role in the regulation of adipose tissue homeostasis. We investigated the differentiation of Tm7sf2-/- MEFs into adipocytes, compared to Tm7sf2+/+ MEFs. Tm7sf2 expression was increased at late stage of differentiation in wild type cells, while Tm7sf2-/- MEFs exhibited a reduced capacity to differentiate into mature adipocytes. Indeed, Tm7sf2-/- MEFs had lower neutral lipid accumulation and reduced expression of adipogenic regulators. At early stage, the reduction in C/EBPß expression impaired mitotic clonal expansion, which is needed by preadipocytes for adipogenesis induction. At late stage, the expression and activity of C/EBPα and PPARγ were inhibited in Tm7sf2-/- cells, leading to the reduced expression of adipocyte genes like Srebp-1c, Fasn, Scd-1, Adipoq, Fabp4, and Glut4. Loss of the acquisition of adipocyte phenotype was accompanied by a reduction in the levels of Irs1, and phosphorylated Akt and ERK1/2, indicating a blunted insulin signaling in differentiating Tm7sf2-/- cells. Moreover, throughout the differentiation process, increased expression of the antiadipogenic Mmp3 was observed in MEFs lacking Tm7sf2. These findings indicate Tm7sf2 as a novel factor influencing adipocyte differentiation that could be relevant to adipose tissue development and maintenance of metabolic health.

The efficacy of the anticancer 3-bromopyruvate is potentiated by antimycin and menadione by unbalancing mitochondrial ROS production and disposal in U118 glioblastoma cells.

Metabolic reprogramming of tumour cells sustains cancer progression. Similar to other cancer cells, glioblastoma cells exhibit an increased glycolytic flow, which encourages the use of antiglycolytics as an effective complementary therapy. We used the antiglycolytic 3-bromopyruvate (3BP) as a metabolic modifier to treat U118 glioblastoma cells and investigated the toxic effects and the conditions to increase drug effectiveness at the lowest concentration. Cellular vitality was not affected by 3BP concentrations lower than 40 µM, although p-Akt dephosphorylation, p53 degradation, and ATP reduction occurred already at 30 µM 3BP. ROS generated in mitochondria were enhanced at 30 µM 3BP, possibly by unbalancing their generation and their disposal because of glutathione peroxidase inhibition. ROS triggered JNK and ERK phosphorylation, and cyt c release outside mitochondria, not accompanied by caspases-9 and -3 activation, probably due to 3BP-dependent alkylation of cysteine residues at caspase-9 catalytic site. To explore the possibility of sensitizing cells to 3BP treatment, we exploited 3BP effects on mitochondria by using 30 µM 3BP in association with antimycin A or menadione concentrations that in themselves exhibit poor toxicity. 3BP effect on cyt c release and cell vitality loss was potentiated due the greater oxidative stress induced by antimycin or menadione association with 3BP, supporting a preeminent role of mitochondrial ROS in 3BP toxicity. Indeed, the scavenger of mitochondrial superoxide MitoTEMPO counteracted 3BP-induced cyt c release and weakened the potentiating effect of 3BP/antimycin association. In conclusion, the biochemical mechanisms leading U118 glioblastoma cells to viability loss following 3BP treatment rely on mitochondrial ROS-dependent pathways. Their potentiation at low 3BP concentrations is consistent with the goal to minimize the toxic effect of the drug towards non-cancer cells.

Acetamidine-Based iNOS Inhibitors as Molecular Tools to Counteract Inflammation in BV2 Microglial Cells.

Neurodegenerative diseases are associated with increased levels of nitric oxide (NO) mainly produced by microglial cells through inducible nitric oxide synthase (iNOS) whose expression is induced by inflammatory stimuli. NO can both exert cytotoxic functions and induce a metabolic switch by inhibiting oxidative phosphorylation and upregulating glycolytic flux. Here, we investigated whether two newly synthesized acetamidine based iNOS inhibitors, namely CM292 and CM544, could inhibit lipopolysaccharide (LPS)-induced BV2 microglial cell activation, focusing on both inflammatory and metabolic profiles. We found that CM292 and CM544, without affecting iNOS protein expression, reduced NO production and reverted LPS-induced inflammatory and cytotoxic response. Furthermore, in the presence of the inflammatory stimulus, both the inhibitors increased the expression of glycolytic enzymes. In particular, CM292 significantly reduced nuclear accumulation of pyruvate kinase M2, increased mitochondrial membrane potential and oxygen consumption rate, and augmented the expression of pyruvate dehydrogenase, pointing to a metabolic switch toward oxidative phosphorylation. These data confirm the role played by NO in the connection between cell bioenergetics profile and inflammation, and suggest the potential usefulness of iNOS inhibitors in redirecting microglia from detrimental to pro-regenerative phenotype.

Crosstalk between Long-Term Sublethal Oxidative Stress and Detrimental Inflammation as Potential Drivers for Age-Related Retinal Degeneration.

Age-related retinal degenerations, including age-related macular degeneration (AMD), are caused by the loss of retinal pigmented epithelial (RPE) cells and photoreceptors. The pathogenesis of AMD, deeply linked to the aging process, also involves oxidative stress and inflammatory responses. However, the molecular mechanisms contributing to the shift from healthy aging to AMD are still poorly understood. Since RPE cells in the retina are chronically exposed to a pro-oxidant microenvironment throughout life, we simulated in vivo conditions by growing ARPE-19 cells in the presence of 10 µM H2O2 for several passages. This long-term oxidative insult induced senescence in ARPE-19 cells without affecting cell proliferation. Global proteomic analysis revealed a dysregulated expression in proteins involved in antioxidant response, mitochondrial homeostasis, and extracellular matrix organization. The analyses of mitochondrial functionality showed increased mitochondrial biogenesis and ATP generation and improved response to oxidative stress. The latter, however, was linked to nuclear factor-κB (NF-κB) rather than nuclear factor erythroid 2-related factor 2 (Nrf2) activation. NF-κB hyperactivation also resulted in increased pro-inflammatory cytokines expression and inflammasome activation. Moreover, in response to additional pro-inflammatory insults, senescent ARPE-19 cells underwent an exaggerated inflammatory reaction. Our results indicate senescence as an important link between chronic oxidative insult and detrimental chronic inflammation, with possible future repercussions for therapeutic interventions.

In vitro effect of nerve growth factor on the main traits of rabbit sperm.

BACKGROUND: The nerve growth factor (NGF), a member of the neurotrophins family, plays an important role not only in the nervous but also in other non-nervous systems such as the reproductive system. The aim of the paper is to study the in vitro effect of NGF on rabbit sperm functions. METHODS: Ten adult rabbit bucks were collected five times, and pooled semen samples have been analysed. NGF was quantified in seminal plasma, and the distribution of NGF receptors (TrKA and p75NTR) in sperm was established. Moreover, the dose-effect of NGF on motility rate and track speed was evaluated. Successively, the effect of the neutralisation of NGF receptors was assessed to verify the specific role of each receptor. Untreated sperm were used as control. RESULTS: Our study identified several interesting results: i) We detected NGF in seminal plasma and TrKA and p75NTR in sperm surface. In particular, TrKA is localised in the head and p75NTR in the midpiece and tail of rabbit sperm. ii) Once the optimal dose of NGF (100 ng/mL) was established, its addition affected both kinetics and other physiological traits (capacitation, apoptosis and necrosis) of rabbit sperm. (iii) The neutralisation of TrKA and p75NTR receptors affected sperm traits differently. In particular, sperm speed, apoptosis and capacitation seemed mainly modulated via p75NTR receptor, whereas motile, live cells, necrosis and acrosome reaction were modulated via TrKA. CONCLUSION: For the first time, we showed the presence of p75NTR in rabbit sperm. NGF affects kinetic and other physiological traits of rabbit sperm. Most of these changes are modulated by the receptors involved (TrKA or p75NTR). Considering that some seminal disorders in human have been correlated with a lower NGF concentration and no studies have been done on the possible involvement of NGF receptors, these findings also provide new insights on human fertility.

Clostridium difficile toxin B induces senescence in enteric glial cells: A potential new mechanism of Clostridium difficile pathogenesis.

Clostridium difficile infection (CDI) causes nosocomial/antibiotic-associated diarrhea and pseudomembranous colitis, with dramatic incidence/mortality worldwide. C. difficile virulence factors are toxin A and toxin B (TcdB) which cause cytopathic/cytotoxic effects and inflammation. Until now studies were focused on molecular effects of C. difficile toxins (Tcds) on different cells while unexplored aspect is the status/fate of cells that survived their cytotoxicity. Recently we demonstrated that enteric glial cells (EGCs) are susceptible to TcdB cytotoxicity, but several EGCs survived and were irreversibly cell-cycle arrested and metabolically active, suggesting that EGCs could became senescent. This is important because allowed us to evaluate the not explored status/fate of cells surviving Tcds cytotoxicity, and particularly if TcdB induces senescence in EGCs. Rat-transformed EGCs were treated with 10 ng/ml TcdB for 6 h-48 h, or for 48 h, followed by incubation for additional 4 or 11 days in absence of TcdB (6 or 13 total days). Senescence markers/effectors were examined by specific assays. TcdB induces senescence in EGCs, as demonstrated by the senescence markers: irreversible cell-cycle arrest, senescence-associated-ß­galactosidase positivity, flat morphology, early and persistent DNA damage (ATM and H2AX phosphorylation), p27 overexpression, pRB hypophosphorylation, c­Myc, cyclin B1, cdc2 and phosphorylated-cdc2 downregulation, Sirtuin­2 and Sirtuin­3 overexpression. TcdB-induced EGC senescence is dependent by JNK and AKT activation but independent by ROS, p16 and p53/p21 pathways. In conclusion, TcdB induces senescence in EGCs. The extrapolation of these results to CDI leads to hypothesize that EGCs that survived TcdB, once they have acquired a senescence state, could cause irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and tumors due to persistent inflammation, transfer of senescence status and stimulation of pre-neoplastic cells.

Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria.

Enteric glial cells (EGCs) are components of the enteric nervous system, an organized structure that controls gut functions. EGCs may be vulnerable to different agents, such as bacterial infections that could alter the intestinal epithelial barrier, allowing bacterial toxins and/or other agents possessing intrinsic toxic effect to access cells. Palmitate, known to exhibit lipotoxicity, is released in the gut during the digestion process. In this study, we investigated the lipotoxic effect of palmitate in cultured EGCs, with particular emphasis on palmitate-dependent intracellular lipid remodeling. Palmitate but not linoleate altered mitochondrial and endoplasmic reticulum lipid composition. In particular, the levels of phosphatidic acid, key precursor of phospholipid synthesis, increased, whereas those of mitochondrial cardiolipin (CL) decreased; in parallel, phospholipid remodeling was induced. CL remodeling (chains shortening and saturation) together with palmitate-triggered mitochondrial burst, caused cytochrome c (cyt c) detachment from its CL anchor and accumulation in the intermembrane space as soluble pool. Palmitate decreased mitochondrial membrane potential and ATP levels, without mPTP opening. Mitochondrial ROS permeation into the cytosol and palmitate-induced ER stress activated JNK and p38, culminating in Bim and Bax overexpression, factors known to increase the outer mitochondrial membrane permeability. Overall, in EGCs palmitate produced weakening of cyt c-CL interactions and favoured the egress of the soluble cyt c pool outside mitochondria to trigger caspase-3-dependent viability loss. Elucidating the mechanisms of palmitate lipotoxicity in EGCs may be relevant in gut pathological conditions occurring in vivo such as those following an insult that may damage the intestinal epithelial barrier.

Microglial activation and the nitric oxide/cGMP/PKG pathway underlie enhanced neuronal vulnerability to mitochondrial dysfunction in experimental multiple sclerosis.

During multiple sclerosis (MS), a close link has been demonstrated to occur between inflammation and neuro-axonal degeneration, leading to the hypothesis that immune mechanisms may promote neurodegeneration, leading to irreversible disease progression. Energy deficits and inflammation-driven mitochondrial dysfunction seem to be involved in this process. In this work we investigated, by the use of striatal electrophysiological field-potential recordings, if the inflammatory process associated with experimental autoimmune encephalomyelitis (EAE) is able to influence neuronal vulnerability to the blockade of mitochondrial complex IV, a crucial component for mitochondrial activity responsible of about 90% of total cellular oxygen consumption. We showed that during the acute relapsing phase of EAE, neuronal susceptibility to mitochondrial complex IV inhibition is markedly enhanced. This detrimental effect was counteracted by the pharmacological inhibition of microglia, of nitric oxide (NO) synthesis and its intracellular pathway (involving soluble guanylyl cyclase, sGC, and protein kinase G, PKG). The obtained results suggest that mitochondrial complex IV exerts an important role in maintaining neuronal energetic homeostasis during EAE. The pathological processes associated with experimental MS, and in particular the activation of microglia and of the NO pathway, lead to an increased neuronal vulnerability to mitochondrial complex IV inhibition, representing promising pharmacological targets.

Clostridium difficile-related postinfectious IBS: a case of enteroglial microbiological stalking and/or the solution of a conundrum?

Post-infectious irritable bowel syndrome is a well-defined pathological entity that develops in about one-third of subjects after an acute infection (bacterial, viral) or parasitic infestation. Only recently it has been documented that an high incidence of post-infectious irritable bowel syndrome occurs after Clostridium difficile infection. However, until now it is not known why in some patients recovered from this infection the gastrointestinal disturbances persist for months or years. Based on our in vitro studies on enteric glial cells exposed to the effects of C. difficile toxin B, we hypothesize that persistence of symptoms up to the development of irritable bowel syndrome might be due to a disturbance/impairment of the correct functions of the enteroglial intestinal network.

Lethal digenic mutations in the K+ channels Kir4.1 (KCNJ10) and SLACK (KCNT1) associated with severe-disabling seizures and neurodevelopmental delay.

A 2-yr-old boy presented profound developmental delay, failure to thrive, ataxia, hypotonia, and tonic-clonic seizures that caused the death of the patient. Targeted and whole exome sequencing revealed two heterozygous missense variants: a novel mutation in the KCNJ10 gene that encodes for the inward-rectifying K+ channel Kir4.1 and another previously characterized mutation in KCNT1 that encodes for the Na+-activated K+ channel known as Slo2.2 or SLACK. The objectives of this study were to perform the clinical and genetic characterization of the proband and his family and to examine the functional consequence of the Kir4.1 mutation. The mutant and wild-type KCNJ10 constructs were generated and heterologously expressed in Xenopus laevis oocytes, and whole cell K+ currents were measured using the two-electrode voltage-clamp technique. The KCNJ10 mutation c.652C>T resulted in a p.L218F substitution at a highly conserved residue site. Wild-type KCNJ10 expression yielded robust Kir current, whereas currents from oocytes expressing the mutation were reduced, remarkably. Western Blot analysis revealed reduced protein expression by the mutation. Kir5.1 subunits display selective heteromultimerization with Kir4.1 constituting channels with unique kinetics. The effect of the mutation on Kir4.1/5.1 channel activity was twofold: a reduction in current amplitudes and an increase in the pH-dependent inhibition. We thus report a novel loss-of-function mutation in Kir4.1 found in a patient with a coexisting mutation in SLACK channels that results in a fatal disease.NEW & NOTEWORTHY We present and characterize a novel mutation in KCNJ10 Unlike previously reported EAST/SeSAME patients, our patient was heterozygous, and contrary to previous studies, mimicking the heterozygous state by coexpression resulted in loss of channel function. We report in the same patient co-occurrence of a KCNT1 mutation resulting in a more severe phenotype. This study provides new insights into the phenotypic spectrum and to the genotype-phenotype correlations associated with EAST/SeSAME and MMFSI.

A channelopathy mutation in the voltage-sensor discloses contributions of a conserved phenylalanine to gating properties of Kv1.1 channels and ataxia.

Channelopathy mutations prove informative on disease causing mechanisms and channel gating dynamics. We have identified a novel heterozygous mutation in the KCNA1 gene of a young proband displaying typical signs and symptoms of Episodic Ataxia type 1 (EA1). This mutation is in the S4 helix of the voltage-sensing domain and results in the substitution of the highly conserved phenylalanine 303 by valine (p.F303V). The contributions of F303 towards K+ channel voltage gating are unclear and here have been assessed biophysically and by performing structural analysis using rat Kv1.2 coordinates. We observed significant positive shifts of voltage-dependence, changes in the activation, deactivation and slow inactivation kinetics, reduced window currents, and decreased current amplitudes of both Kv1.1 and Kv1.1/1.2 channels. Structural analysis revealed altered interactions between F303V and L339 and I335 of the S5 helix of a neighboring subunit. The substitution of an aromatic phenylalanine with an aliphatic valine within the voltage-sensor destabilizes the open state of the channel. Thus, F303 fine-tunes the Kv1.1 gating properties and contributes to the interactions between the S4 segment and neighboring alpha helices. The resulting channel's loss of function validates the clinical relevance of the mutation for EA1 pathogenesis.

Selected cholesterol biosynthesis inhibitors produce accumulation of the intermediate FF-MAS that targets nucleus and activates LXRα in HepG2 cells.

Sterol intermediates of the cholesterol biosynthetic pathway have drawn attention for novel biological activities. Follicular fluid meiosis activating sterol (FF-MAS) is a LXRα ligand and a potential modulator of physiologic processes regulated by nuclear receptors, such as lipid homeostasis and cell proliferation. In this work, we established a model to selectively accumulate FF-MAS in HepG2 cells, by using a combination of the inhibitors AY9944 and 17-hydroxyprogesterone to block C14-sterol reductases and the downstream C4-demethylase complex. We investigated the effects produced by altered levels of cholesterol biosynthesis intermediates, in order to dissect their influence on LXRα signaling. In particular, endogenously accumulated FF-MAS was able to modulate the expression of key genes in cholesterol metabolism, to activate LXRα nuclear signaling resulting in increased lipogenesis, and to inhibit HepG2 cells proliferation. Moreover, a fluorescent ester derivative of FF-MAS localized in nuclear lipid droplets, suggesting a role for these organelles in the storage of signaling lipids interacting with nuclear partners.

Enteric glial cells counteract Clostridium difficile Toxin B through a NADPH oxidase/ROS/JNK/caspase-3 axis, without involving mitochondrial pathways.

Enteric glial cells (EGCs) are components of the intestinal epithelial barrier essential for regulating the enteric nervous system. Clostridium difficile is the most common cause of antibiotic-associated colitis, toxin B (TcdB) being the major virulence factor, due to its ability to breach the intestinal epithelial barrier and to act on other cell types. Here we investigated TcdB effects on EGCs and the activated molecular mechanisms. Already at 2 hours, TcdB triggered ROS formation originating from NADPH-oxidase, as demonstrated by their reduction in the presence of the NADPH-oxidase inhibitor ML171. Although EGCs mitochondria support almost completely the cellular ATP need, TcdB exerted weak effects on EGCs in terms of ATP and mitochondrial functionality, mitochondrial ROS production occurring as a late event. ROS activated the JNK signalling and overexpression of the proapoptotic Bim not followed by cytochrome c or AIF release to activate the downstream apoptotic cascade. EGCs underwent DNA fragmentation through activation of the ROS/JNK/caspase-3 axis, evidenced by the ability of ML171, N-acetylcysteine, and the JNK inhibitor SP600125 to inhibit caspase-3 or to contrast apoptosis. Therefore, TcdB aggressiveness towards EGCs is mainly restricted to the cytosolic compartment, which represents a peculiar feature, since TcdB primarily influences mitochondria in other cellular types.

3-Bromopyruvate treatment induces alterations of metabolic and stress-related pathways in glioblastoma cells.

Glioblastoma (GBM) is the most common and aggressive brain tumour of adults. The metabolic phenotype of GBM cells is highly dependent on glycolysis; therefore, therapeutic strategies aimed at interfering with glycolytic pathways are under consideration. 3-Bromopyruvate (3BP) is a potent antiglycolytic agent, with a variety of targets and possible effects on global cell metabolism. Here we analyzed the changes in protein expression on a GBM cell line (GL15 cells) caused by 3BP treatment using a global proteomic approach. Validation of differential protein expression was performed with immunoblotting and enzyme activity assays in GL15 and U251 cell lines. The results show that treatment of GL15 cells with 3BP leads to extensive changes in the expression of glycolytic enzymes and stress related proteins. Importantly, other metabolisms were also affected, including pentose phosphate pathway, aminoacid synthesis, and glucose derivatives production. 3BP elicited the activation of stress response proteins, as shown by the phosphorylation of HSPB1 at serine 82, caused by the concomitant activation of the p38 pathway. Our results show that inhibition of glycolysis in GL15 cells by 3BP influences different but interconnected pathways. Proteome analysis may help in the molecular characterization of the glioblastoma response induced by pharmacological treatment with antiglycolytic agents. SIGNIFICANCE: Alteration of the glycolytic pathway characterizes glioblastoma (GBM), one of the most common brain tumours. Metabolic reprogramming with agents able to inhibit carbohydrate metabolism might be a viable strategy to complement the treatment of these tumours. The antiglycolytic agent 3-bromopyruvate (3BP) is able to strongly inhibit glycolysis but it may affect also other cellular pathways and its precise cellular targets are currently unknown. To understand the protein expression changes induced by 3BP, we performed a global proteomic analysis of a GBM cell line (GL15) treated with 3BP. We found that 3BP affected not only the glycolytic pathway, but also pathways sharing metabolic intermediates with glycolysis, such as the pentose phosphate pathway and aminoacid metabolism. Furthermore, changes in the expression of proteins linked to resistance to cell death and stress response were found. Our work is the first analysis on a global scale of the proteome changes induced by 3BP in a GBM model and may contribute to clarifying the anticancer potential of this drug.

Enteric glial cells are susceptible to Clostridium difficile toxin B.

Clostridium difficile causes nosocomial/antibiotic-associated diarrhoea and pseudomembranous colitis. The major virulence factors are toxin A and toxin B (TcdB), which inactivate GTPases by monoglucosylation, leading to cytopathic (cytoskeleton alteration, cell rounding) and cytotoxic effects (cell-cycle arrest, apoptosis). C. difficile toxins breaching the intestinal epithelial barrier can act on underlying cells, enterocytes, colonocytes, and enteric neurons, as described in vitro and in vivo, but until now no data have been available on enteric glial cell (EGC) susceptibility. EGCs are crucial for regulating the enteric nervous system, gut homeostasis, the immune and inflammatory responses, and digestive and extradigestive diseases. Therefore, we evaluated the effects of C. difficile TcdB in EGCs. Rat-transformed EGCs were treated with TcdB at 0.1-10 ng/ml for 1.5-48 h, and several parameters were analysed. TcdB induces the following in EGCs: (1) early cell rounding with Rac1 glucosylation; (2) early G2/M cell-cycle arrest by cyclin B1/Cdc2 complex inactivation caused by p27 upregulation, the downregulation of cyclin B1 and Cdc2 phosphorylated at Thr161 and Tyr15; and (3) apoptosis by a caspase-dependent but mitochondria-independent pathway. Most importantly, the stimulation of EGCs with TNF-α plus IFN-γ before, concomitantly or after TcdB treatment strongly increased TcdB-induced apoptosis. Furthermore, EGCs that survived the cytotoxic effect of TcdB did not recover completely and showed not only persistent Rac1 glucosylation, cell-cycle arrest and low apoptosis but also increased production of glial cell-derived neurotrophic factor, suggesting self-rescuing mechanisms. In conclusion, the high susceptibility of EGCs to TcdB in vitro, the increased sensitivity to inflammatory cytokines related to apoptosis and the persistence of altered functions in surviving cells suggest an important in vivo role of EGCs in the pathogenesis of C. difficile infection.

A Calsequestrin-1 Mutation Associated with a Skeletal Muscle Disease Alters Sarcoplasmic Ca2+ Release.

An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and calsequestrin-1 (CASQ1) accumulation in skeletal muscle, has been recently associated with a missense mutation in CASQ1 gene. The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as "CASQ1-couplonopathy".

Activity, Expression, and Substrate Preference of the Δ(6)-Desaturase in Slow- or Fast-Growing Rabbit Genotypes.

In the present paper liver fatty acid Δ(6) desaturation (fads2) activity was analyzed in two rabbit strains with slow- (S, 27.5 g/day) or fast-growing (F, 48.5 g/day) rate. The fatty acid profile of the liver showed a different PUFA profile in the two strains with a lower n-6/n-3 ratio in the S rabbits. The expression of fads2 was 2-fold higher in S than in F rabbits, whereas enzyme activity was higher in F and more oriented toward the desaturation of linoleic acid (90%). In contrast, S showed a higher preference for linolenic acid (38.9 vs 10%). This study identified a single difference in the fads2 amino acid sequence between these two strains. Such a difference consists in the substitution of Gly104 to Ser104 in the sequence of F fads2. These results indicate for the first time that genetic selection for performance may affect the preference for PUFA toward desaturation of linoleic/linolenic acid.