Metabolic changes associated with sustained 48-Hr shivering thermogenesis in the newborn pig

Metabolic changes associated with sustained 48-hr shivering thermogenesis were studied in piglets maintained at 34 (thermoneutrality) or 25°C (cold) between 6 and 54 hr of life. Despite their high shivering activity and elevated heat production, cold-exposed piglets exhibited a slightly lower rectal temperature than thermoneutral animals (-1.1°C; P < 0.01) at the end of the treatment. The enhancement of heat production and shivering activity were associated with a decrease in muscle glycogen (− 47%; P < 0.05) and total lipid content (− 23%; P < 0.05), a reduction of blood lactate levels (P < 0.05) and an enhancement of muscle cytochrome oxidase activity (+20%;P < 0.05), which suggests that muscle oxidative potential was increased by cold exposure. Potential for capturing lipids (lipoprotein lipase activity) was also higher in the redrhomboideus muscle (+ 71%; P < 0.01) and lower in adipose tissue (−58%; P < 0.01) of the cold-exposed piglets. Measurements performed at the mitochondrial level show no changes inrhomboideus muscle, but respiratory capacities (state IV and FCCP-stimulated respiration) and intermyofibrillar mitochondria oxidative and phosphorylative (creatine kinase activity) capacities were enhanced inlongissimus dorsi muscle (P < 0.05). These changes may contribute to provide muscles with nonlimiting amount of readily oxidable substrates and ATP necessary for shivering thermogenesis. A rise in plasma norepinephrine levels was also observed during the second day of cold exposure (P < 0.05).     

Intracellular nicotinamide adenine dinucleotide promotes TNF-induced necroptosis in a sirtuin-dependent manner

Cellular necrosis has long been regarded as an incidental and uncontrolled form of cell death. However, a regulated form of cell death termed necroptosis has been identified recently. Necroptosis can be induced by extracellular cytokines, pathogens and several pharmacological compounds, which share the property of triggering the formation of a RIPK3-containing molecular complex supporting cell death. Of interest, most ligands known to induce necroptosis (including notably TNF and FASL) can also promote apoptosis, and the mechanisms regulating the decision of cells to commit to one form of cell death or the other are still poorly defined. We demonstrate herein that intracellular nicotinamide adenine dinucleotide (NAD⁺) has an important role in supporting cell progression to necroptosis. Using a panel of pharmacological and genetic approaches, we show that intracellular NAD⁺ promotes necroptosis of the L929 cell line in response to TNF. Use of a pan-sirtuin inhibitor and shRNA-mediated protein knockdown led us to uncover a role for the NAD⁺-dependent family of sirtuins, and in particular for SIRT2 and SIRT5, in the regulation of the necroptotic cell death program. Thus, and in contrast to a generally held view, intracellular NAD⁺ does not represent a universal pro-survival factor, but rather acts as a key metabolite regulating the choice of cell demise in response to both intrinsic and extrinsic factors.Nicotinamide adenine dinucleotide (NAD⁺) has been long recognized as a key intermediate in cellular metabolism. By accepting and donating electrons in reactions catalyzed by dehydrogenases, NAD⁺ has, for example, a central role in the generation of ATP, a molecule required for most energy-consuming cellular reactions. The recognition of NAD⁺ as a substrate in a number of regulatory processes has shed a new light on its role in cell physiology. Indeed, NAD⁺ represents a substrate for a wide range of enzymes including cADP-ribose synthases, poly (ADP-ribose) polymerases (PARPs) and the sirtuin family of NAD⁺-dependent deacylases (SIRTs). In marked contrast to its role in energy metabolism, the involvement of NAD⁺ in these enzymatic reactions is based on its ability to function as a donor of ADP-ribose, a reaction that, if sustained, can lead to the depletion of the intracellular NAD⁺ pool.^{1, 2, 3, 4, 5}The pro-survival role of NAD⁺ has been particularly well described in cells exposed to genotoxic/oxidative stress. In response to DNA damage, PARP1, the founding and most abundant member of the PARP family, binds to DNA strand breaks and initiates a repair response by catalyzing the post-translational modification of several nuclear proteins, including itself. This protective response is characterized by the transfer of successive units of the ADP-ribose moiety (up to 200 units) from NAD⁺ to other proteins, compromising therefore both energy production (slowing the rate of glycolysis, electron transport and ATP formation) and activity of other NAD⁺-dependent enzymes through NAD⁺ depletion.^{6, 7} Moreover, PARP1-synthesized PAR polymers can be degraded into free oligomers, known to translocate to the mitochondria where they can trigger the release of AIF from mitochondria to the nucleus.^{8, 9, 10, 11} The precise molecular steps linking PARP1 activation to this form of stress-induced cell death, termed parthanatos, have not been fully elucidated, and probably depend on the particular metabolic status of the cell examined (i.e., anerobic glycolysis in most in vitro cell lines versus oxidative metabolism of neuronal cells, see Welsby et al.¹² for review). In any instances, and independently of the fine molecular events at work, virtually all studies have identified the brisk loss of intracellular NAD⁺ as the critical step initiating this specific form of cell death. The protective role of NAD⁺ in PARP1-dependent cell death has been indeed amply documented.^{13, 14, 15, 16, 17, 18} In mammals, nicotinamide (Nam) acts as the main precursor for NAD⁺ biosynthesis and nicotinamide phosphoribosyl tranferase (NAMPT) is the rate-limiting enzyme for NAD⁺ biosynthesis from Nam.¹⁹ Nampt deficiency in mice leads to lethality and the heterozygous animals suffer from significant perturbations related to their NAD⁺ metabolism.²⁰ In keeping with the general role of NAD⁺ as a survival factor in cells exposed to genotoxic stress, genetic ablation of Nampt and/or treatment with a specific NAMPT inhibitor (FK866) sensitized cells to the toxic effects of alkylating agents.^{16, 18} Similarly, overexpression of a catalytically active recombinant NAMPT protected the NIH-3T3 cell line from the toxicity of the same DNA alkylating agents,¹⁸ further establishing a functional link between NAD⁺ biosynthesis and sensitivity to stress-induced, PARP1-dependent cell death.While analyzing the influence of NAD metabolism on survival of NIH3T3 cells exposed to genotoxic agents, we observed that overexpression of NAMPT prolonged cell survival of cells exposed to the alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), and unexpectedly, led to increased sensitivity to cell death induced by the pro-inflammatory cytokine TNF.¹⁸ TNF is a pleiotropic cytokine regulating many cellular functions and known to induce several forms of cell death, including apoptosis and the recently uncovered regulated form of necrosis termed necroptosis.^{21, 22} In contrast to apoptosis, necroptosis is largely independent of the so-called executioner caspase (such as caspase-3, 6 and 7) activity and is initiated by the formation of a signaling complex comprising the receptor-interacting serine-threonine kinase 1 (RIPK1), RIPK3 and the recently identified mixed lineage kinase domain-like protein MLKL. Although necroptosis often appears to occur when apoptosis is abortive (such as in situations of caspase inhibition), the cellular factors regulating the choice between these two forms of regulated cell death have not been fully uncovered. Using a model cell line engineered to respond to both apoptosis and necroptosis, we demonstrate herein that intracellular NAD⁺ represents a critical factor in promoting cell death by necroptosis. In keeping with the well-described role of sirtuins as intracellular NAD⁺ sensors, we also demonstrate that sirtuins, and in particular SIRT2 and SIRT5, are required for adequate completion of the necroptotic program in response to TNF. Accordingly, a pan-sirtuin inhibitor was found to attenuate organ damage induced by transient ischemia. Thus, intracellular NAD⁺, rather than acting as a general cell survival factor, appears to promote cell necroptosis in a sirtuin-dependent manner, a finding that may suggest novel therapeutic approaches to attenuate in vivo necrotic insults in several pathological settings.     

Computational Fluid Dynamic Simulations of Maternal Circulation: Wall Shear Stress in the Human Placenta and Its Biological Implications

Introduction

In the human placenta the maternal blood circulates in the intervillous space (IVS). The syncytiotrophoblast (STB) is in direct contact with maternal blood. The wall shear stress (WSS) exerted by the maternal blood flow on the STB has not been evaluated. Our objective was to determine the physiological WSS exerted on the surface of the STB during the third trimester of pregnancy.

Material and Methods

To gain insight into the shear stress levels that the STB is expected to experience in vivo, we have formulated three different computational models of varying levels of complexity that reflect different physical representations of the IVS. Computations of the flow fields in all models were performed using the CFD module of the finite element code COMSOL Multiphysics 4.4. The mean velocity of maternal blood in the IVS during the third trimester was measured in vivo with dynamic MRI (0.94±0.14 mm.s^-1). To investigate if the in silico results are consistent with physiological observations, we studied the cytoadhesion of human parasitized (Plasmodium falciparum) erythrocytes to primary human STB cultures, in flow conditions with different WSS values.

Results

The WSS applied to the STB is highly heterogeneous in the IVS. The estimated average values are relatively low (0.5±0.2 to 2.3±1.1 dyn.cm^-2). The increase of WSS from 0.15 to 5 dyn.cm^-2 was associated with a significant decrease of infected erythrocyte cytoadhesion. No cytoadhesion of infected erythrocytes was observed above 5 dyn.cm^-2 applied for one hour.

Conclusion

Our study provides for the first time a WSS estimation in the maternal placental circulation. In spite of high maternal blood flow rates, the average WSS applied at the surface of the chorionic villi is low (<5 dyn.cm^-2). These results provide the basis for future physiologically-relevant in vitro studies of the biological effects of WSS on the STB.     

Regulation cascade of flagellar expression in Gram-negative bacteria

Flagellar motility helps bacteria to reach the most favourable environments and to successfully compete with other micro-organisms. These complex organelles also play an important role in adhesion to substrates, biofilm formation and virulence process. In addition, because their synthesis and functioning are very expensive for the cell (about 2% of biosynthetic energy expenditure in Escherichia coli) and may induce a strong immune response in the host organism, the expression of flagellar genes is highly regulated by environmental conditions. In the past few years, many data have been published about the regulation of motility in polarly and laterally flagellated bacteria. However, the mechanism of motility control by environmental factors and by some regulatory proteins remains largely unknown. In this respect, recent experimental data suggest that the master regulatory protein-encoding genes at the first level of the cascade are the main target for many environmental factors. This mechanism might require DNA topology alterations of their regulatory regions. Finally, despite some differences the polar and lateral flagellar cascades share many functional similarities, including a similar hierarchical organisation of flagellar systems. The remarkable parallelism in the functional organisation of flagellar systems suggests an evolutionary conservation of regulatory mechanisms in Gram-negative bacteria.     

Sexual selection, antennae length and the mating advantage of large males in Asellus aquaticus

In crustacean species with precopulatory mate-guarding, sexual size dimorphism has most often been regarded as the consequence of a large male advantage in contest competition for access to females. However, large body size in males may also be favoured indirectly through scramble competition. This might partly be the case if the actual target of selection is a morphological character, closely correlated with body size, involved in the detection of receptive females. We studied sexual selection on body size and antennae length in natural populations of Asellus aquaticus, an isopod species with precopulatory mate guarding. In this species, males are larger than females and male pairing success is positively related to body size. However, males also have longer antennae, relative to body size, than females, suggesting that this character may also be favoured by sexual selection. We used multivariate analysis of selection to assess the relative influences of body size and antennae length in five different populations in the field. Selection gradients indicated that overall body size was a better predictor of male pairing success than antennae length, although some variation was observed between sites. We then manipulated male antennae length in a series of experiments conducted in the laboratory, and compared the pairing ability of males with short or long antennae. Males with short antennae were less likely to detect, orient to and to pair with a receptive female compared to males with long antennae. We discuss the implications of our results for studies of male body size and sexual dimorphism in relation to sexual selection in crustaceans.     

Functional screening of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-kappaB and caspase-1

PYRIN-containing Apaf-1-like proteins (PYPAFs) are a recently identified family of proteins thought to function in apoptotic and inflammatory signaling pathways. PYPAF1 and PYPAF7 proteins have been found to assemble with the PYRIN–CARD protein ASC and coordinate the activation of NF-κB and pro-caspase-1. To determine if other PYPAF family members function in pro-inflammatory signaling pathways, we screened five other PYPAF proteins (PYPAF2, PYPAF3, PYPAF4, PYPAF5 and PYPAF6) for their ability to activate NF-κB and pro-caspase-1. Co-expression of PYPAF5 with ASC results in a synergistic activation of NF-κB and the recruitment of PYPAF5 to punctate structures in the cytoplasm. The expression of PYPAF5 is highly restricted to granulocytes and T-cells, indicating a role for this protein in inflammatory signaling. In contrast, PYPAF2, PYPAF3, PYPAF4 and PYPAF6 failed to colocalize with ASC and activate NF-κB. PYPAF5 also synergistically activated caspase-1-dependent cytokine processing when co-expressed with ASC. These findings suggest that PYPAF5 functions in immune cells to coordinate the transduction of pro-inflammatory signals to the activation of NF-κB and pro-caspase-1.