Methicillin-susceptible ST398 Staphylococcus aureus responsible for bloodstream infections: an emerging human-adapted subclone?

In the course of an annual 3-month bloodstream infections (BSI) survey conducted during a four-year period in 31 healthcare institutions located in three noncontiguous French regions, we report 18 ST398 Staphylococcus aureus BSI. ST398 BSI incidence showed a seven-fold increase during the study period (0.002 per 1,000 patient days in 2007 vs. 0.014 in 2010). ST398 BSI isolates differed from the pig-borne multiresistant clone: 17/18 BSI isolates were methicillin susceptible and none was of t011, t034 or t108 pig-borne spa-types. ST398 BSI isolates had homogenous resistance patterns (15/18 with only Ery(r)) and prophagic content (all harboured the hlb-converting Sau3int phage). The clustering of BSI and pig-borne isolates by spa-typing and MLVA, the occurrence of Sau3int phage in BSI isolates and the lack of this phage in pig-borne isolates suggest that the emergence of BSI isolates could have arisen from horizontal transfer, at least of the Sau3int phage, in genetically diverse MSSA ST398 isolates. The acquisition of the phage likely plays a role in the increasing ability of the lysogenic ST398 isolates to colonize human. The mode of acquisition of the non pig-borne ST398 isolates by our 18 patients remains unclear. ST398 BSI were diagnosed in patients lacking livestock exposure and were significantly associated with digestive portals of entry (3/18 [16.7%] for ST398 vs. 19/767 [2.5%] for non ST398 BSI; p?=?.012). This raises the question of possible foodborne human infections. We suggest the need for active surveillance to study and control the spread of this human-adapted subclone increasingly isolated in the hospital setting.     

Are ABA,ethylene or their interaction involved in the response of leaf growth to soil water deficit? An analysis using naturally occurring variation or genetic transformation of ABA production in maize

The role of abscisic acid (ABA) and its possible interaction with ethylene in mediating leaf elongation response to soil water deficit are a matter of controversy. To address this question, we used a set of maize genotypes with various levels of ABA either due to natural variability or to genetic transformation targeted on NCED/VP14, a key enzyme of ABA synthesis. The transgenic lines yielded less strong phenotypes than available mutants, making it possible to use them under normal growing conditions. We focused on leaf elongation during night periods in order to avoid the confounding effect of ABA on leaf water status. Our results suggest that over a wide range, internal ABA level (measured in both leaf extracts or xylem sap) has no clear effect on leaf elongation response to soil water deficit, except in the case of an antisense line presenting the strongest reduction in ABA accumulation that showed a slight maintenance of leaf elongation during water deficit. Leaf ethylene production rate was variable and not related to water deficit except in the ABA-deficient transgenic lines where it was increased by water deficit on average but not systematically. Moreover, variability in ethylene production rate was not linked to variability in elongation rate. Our results thus suggest that neither ABA nor ethylene seems to play a major role in the control of leaf elongation response to soil water deficit.     

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.     

Improved agarose gel assay for quantification of growth factor-induced cell motility

Cell chemotaxis is frequently required in normal or pathological situations such as invasion, metastasis, and tumor angiogenesis and may involve many different cell types. At present, no device can simultaneously (i) make morphological observations, (ii) quantify cell migration, (iii) test multiple chemoattracting gradients, and (iv) analyze cell-cell interactions. We developed an agarose-based assay to address these questions. Two glass molds were designed, around which agarose gel could be poured to form specific well shapes. Using a vital nuclear stain (Hoechst 33258), we characterized the migration profile of adherent or suspension cells. Cells could be observed during the entire migration process. We were able to follow cells moving toward chemoattractants or being repulsed by other molecules, and we could estimate average migration speed. Using this inexpensive assay, we were able to obtain precise, reproducible results concerning the chemotactic behavior of different cell types. The resulting data differentiated between chemokinetic and chemotactic movement. Chemotactic potencies could be compared using different criteria, such as the number of attracted cells, induced speed, and morphological aspect. This improved agarose assay appears to be a reliable and inexpensive alternative to other available chemotaxis study tools.     

The phosphoinositide kinase PIKfyve/Fab1p regulates terminal lysosome maturation in Caenorhabditis elegans

Membrane dynamics is necessary for cell homeostasis and signal transduction and is in part regulated by phosphoinositides. Pikfyve/Fab1p is a phosphoinositide kinase that phosphorylates phosphatidylinositol 3-monophosphate into phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and is implicated in membrane homeostasis in yeast and in mammalian cells. These two phosphoinositides are substrates of myotubularin phosphatases found mutated in neuromuscular diseases. We studied the roles of phosphatidylinositol phosphate kinase 3 (PPK-3), the orthologue of PIKfyve/Fab1p, in a multicellular organism, Caenorhabditis elegans. Complete loss of ppk-3 function induces developmental defects characterized by embryonic lethality, whereas partial loss of function leads to growth retardation. At the cellular level, ppk-3 mutants display a striking enlargement of vacuoles positive for lysosome-associated membrane protein 1 in different tissues. In the intestine, RAB-7-positive late endosomes are also enlarged. Membranes of the enlarged lysosomes originate at least in part from smaller lysosomes, and functional and genetic analyses show that the terminal maturation of lysosomes is defective. Protein degradation is not affected in the hypomorphic ppk-3 mutant and is thus uncoupled from membrane retrieval. We measured the level of PtdIns(3,5)P2 and showed that its production is impaired in this mutant. This work strongly suggests that the main function of PPK-3 is to mediate membrane retrieval from matured lysosomes through regulation of PtdIns(3,5)P2.     

The NLRP3 inflammasome is activated by nanoparticles through ATP,ADP and adenosine

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO₂) and titanium dioxide (nano-TiO₂) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO₂ or nano-TiO₂ caused a significant increase in P2Y1, P2Y2, A2_A and/or A2_B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2_A or A2_B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.The inflammasome is a major factor of the innate immune system acting as a multiprotein platform to activate caspase-1. We showed recently that nanoparticles of TiO₂ (nano-TiO₂) and SiO₂ (nano-SiO₂) are sensed by the NLRP3 inflammasome to induce the release of mature IL-1β,¹ as observed previously with the environmental irritants asbestos or silica.² Despite the identification and characterisation of numerous sterile or microbial activators, the precise mechanisms mediating NLRP3 inflammasome activation remain to be determined. Here, we investigated whether ATP release and purinergic signalling through ATP, ADP and adenosine may be involved in inflammasome activation by nanoparticles. Intracellular ATP is released after cellular stress and/or activation, and purinergic signalling has been shown to modulate inflammation and immunity.^{3, 4} In the extracellular space, ATP is rapidly hydrolysed in a stepwise manner to ADP, AMP (adenosine monophosphate) and adenosine by ectoenzymes.⁴ Adenosine is then irreversibly hydrolysed to inosine by adenosine deaminase (ADA). Extracellular ATP (eATP) signals through both ATP-gated ion channels P2X and G protein-coupled receptor (GPCR) P2Y membrane receptors, whereas ADP signals through P2Y receptors and adenosine through P1 receptors (or A receptors).⁵ P2Y receptors and A receptors may be coupled to the G_q protein, which activates phospholipase C-beta (PLC-β), to the stimulatory G (G_s) protein, which stimulates adenylate cyclase inducing an increase in cyclic AMP (cAMP) levels, or to the G inhibitory (G_i) protein, which inhibits adenylate cyclase. Extracellular adenosine level is the result of adenosine production from extracellular ATP and ADP, its degradation into inosine and its reuptake by cells. Both ATP and adenosine can be transported outside of the cell via diffusion or active transport, whereas only adenosine can enter the cells through adenosine transporters.⁶ Most cells possess equilibrative and concentrative adenosine transporters (respectively, ENTs and CNTs), which allow adenosine to quickly cross the plasma membrane.⁷ Intracellular adenosine is converted to ATP via phosphorylation steps mediated by adenosine kinase (AK) and AMP kinase (AMPK). The basal physiological level of extracellular adenosine has been estimated to be in the range of 30–200 nM.⁸ ATP-derived adenosine and its subsequent signalling through P1 receptors have beneficial roles in acute disease states.^{4, 9} However, during tissue injury, elevated adenosine levels participate in the progression to chronic diseases by promoting aberrant wound healing leading to fibrosis in different organs including the lungs, liver, skin and kidney. In these conditions the blockade of adenosine signalling is beneficial.^{10, 11, 12, 13, 14, 15, 16} In murine models, ADA-knockout mice present high persistent adenosine levels, which lead to airspace enlargement and fibrosis, cardinal signs of COPD and IPF.^{14, 17, 18}Here we investigate in more detail the critical contribution of purinergic signalling in driving NLRP3 inflammasome activation in response to nanoparticles pointing out the effect of ATP, ADP, as well as adenosine and its receptors. We also identify ATP-derived adenosine as a potential activator of the inflammasome.     

New SUCLG1 patients expanding the phenotypic spectrum of this rare cause of mild methylmalonic aciduria

Deficiencies in two subunits of the succinyl-coenzyme A synthetase (SCS) have been involved in patients with encephalomyopathy and mild methylmalonic aciduria (MMA). In this study, we described three new SUCLG1 patients and performed a meta-analysis of the literature. Our report enlarges the phenotypic spectrum of SUCLG1 mutations and confirms that a characteristic metabolic profile (presence of MMA and C4-DC carnitine in urines) and basal ganglia MRI lesions are the hallmarks of SCS defects. As mitochondrial DNA depletion in muscle is not a constant finding in SUCLG1 patients, this may suggest that diagnosis should not be based on it, but also that alternative physiopathological mechanisms may be considered to explain the combined respiratory chain deficiency observed in SCS patients.     

Inhibition of stearoyl-CoA desaturase 1 expression induces CHOP-dependent cell death in human cancer cells

Background

Cancer cells present a sustained de novo fatty acid synthesis with an increase of saturated and monounsaturated fatty acid (MUFA) production. This change in fatty acid metabolism is associated with overexpression of stearoyl-CoA desaturase 1 (Scd1), which catalyses the transformation of saturated fatty acids into monounsaturated fatty acids (e.g., oleic acid). Several reports demonstrated that inhibition of Scd1 led to the blocking of proliferation and induction of apoptosis in cancer cells. Nevertheless, mechanisms of cell death activation remain to be better understood.

Principal Findings

In this study, we demonstrated that Scd1 extinction by siRNA triggered abolition of de novo MUFA synthesis in cancer and non-cancer cells. Scd1 inhibition-activated cell death was only observed in cancer cells with induction of caspase 3 activity and PARP-cleavage. Exogenous supplementation with oleic acid did not reverse the Scd1 ablation-mediated cell death. In addition, Scd1 depletion induced unfolded protein response (UPR) hallmarks such as Xbp1 mRNA splicing, phosphorylation of eIF2α and increase of CHOP expression. However, the chaperone GRP78 expression, another UPR hallmark, was not affected by Scd1 knockdown in these cancer cells indicating a peculiar UPR activation. Finally, we showed that CHOP induction participated to cell death activation by Scd1 extinction. Indeed, overexpression of dominant negative CHOP construct and extinction of CHOP partially restored viability in Scd1-depleted cancer cells.

Conclusion

These results suggest that inhibition of de novo MUFA synthesis by Scd1 extinction could be a promising anti-cancer target by inducing cell death through UPR and CHOP activation.     

Variability in the bioluminescence response of the dinoflagellate Pyrocystis lunula

Light emission in dinoflagellates is induced by water motions. But although it is known that mechanical stimulations of these organisms trigger the bioluminescent response, the exact mechanism that involves some cell membrane excitations by fluid motions is not yet fully understood and is still controversial. We show in this experimental study that the accelerated shear flow, created by abrupt rotations of one or both co-axial cylinders of a Couette shearing chamber excites the light emission from cultured dinoflagellates Pyrocystis lunula. Following our first results published earlier that state that pure laminar shear does not excite the main bioluminescent response in dinoflagellates, our present experiments show that both shear and acceleration in the flow are needed to trigger the bioluminescent response. Besides, the probability to stimulate this bioluminescent response under acceleration and shear is deduced from the response curves. This response follows a Gaussian distribution that traduces a heterogeneity in individual cell thresholds for the stimulation of bioluminescence in a dinoflagellate population. All these results will have a repercussion in the possible applications of dinoflagellate bioluminescence in flow visualizations and measurements. Moreover, this study opens a new way in studying mechanically-induced stimulus thresholds at the cell level.