Lipidomic Analysis Links Mycobactin Synthase K to Iron Uptake and Virulence in M. tuberculosis

The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.     

Insulin Resistance Is Not Associated with an Impaired Mitochondrial Function in Contracting Gastrocnemius Muscle of Goto-Kakizaki Diabetic Rats In Vivo

Insulin resistance, altered lipid metabolism and mitochondrial dysfunction in skeletal muscle would play a major role in type 2 diabetes mellitus (T2DM) development, but the causal relationships between these events remain conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in Goto-Kakizaki (GK) rats, a non-obese T2DM model developing peripheral insulin resistant without abnormal level of plasma non-esterified fatty acids (NEFA). Wistar rats were used as controls. Mechanical performance and energy metabolism were assessed strictly non-invasively using magnetic resonance (MR) imaging and 31-phosphorus MR spectroscopy (³¹P-MRS). Compared with control group, plasma insulin and glucose were respectively lower and higher in GK rats, but plasma NEFA level was normal. In resting GK muscle, phosphocreatine content was reduced whereas glucose content and intracellular pH were both higher. However, there were not differences between both groups for basal oxidative ATP synthesis rate, citrate synthase activity, and intramyocellular contents for lipids, glycogen, ATP and ADP (an important in vivo mitochondrial regulator). During a standardized fatiguing protocol (6 min of maximal repeated isometric contractions electrically induced at a frequency of 1.7 Hz), mechanical performance and glycolytic ATP production rate were reduced in diabetic animals whereas oxidative ATP production rate, maximal mitochondrial capacity and ATP cost of contraction were not changed. These findings provide in vivo evidence that insulin resistance is not caused by an impairment of mitochondrial function in this diabetic model.     

TLR4‐mediated skin carcinogenesis is dependent on immune and radioresistant cells

Skin cancers are the most commonly diagnosed cancers. Understanding what are the factors contributing to skin tumour development can be instrumental to identify preventive therapies. The myeloid differentiation primary response gene (MyD)88, the downstream adaptor protein of most Toll‐like receptors (TLR), has been shown to be involved in several mouse tumourigenesis models. We show here that TLR4, but not TLR2 or TLR9, is upstream of MyD88 in skin tumourigenesis. TLR4 triggering is not dependent on lipopolysaccharide associated to skin‐colonizing bacteria, but on the high mobility group box‐1 protein (HMGB1), an endogenous ligand of TLR4. HMGB1 is released by necrotic keratinocytes and is required for the recruitment of inflammatory cells and for the initiation of inflammation. The expression of TLR4 on both bone marrow‐derived and radioresistant cells is necessary for carcinogenesis. Consistently, a human tissue microarray analysis showed that melanoma and colon cancer display an over‐expression of TLR4 and its downstream adaptor protein MyD88 within tumours. Together, our results suggest that the initial release of HMGB1 triggers a TLR4‐dependent inflammatory response that leads to tumour development.     

Oviposition deterring infochemicals in ladybirds: the role of phylogeny

Faced with an ephemeral prey, aphidophagous ladybirds rely on the hydrocarbons present in the tracks of their larvae to choose an unoccupied patch for egg laying. Although both conspecific and heterospecific larval tracks might deter females from oviposition, the response to the later is often less striking. Several explanations have been suggested to account for this. In this paper we tested the phylogeny hypothesis, which predicts that the chemical composition of the tracks of closely related species of ladybirds will be more similar to one another than to those of more distantly related species. Qualitative and quantitative information on the chemical nature of the larval tracks and a molecular phylogeny of seven species belonging to three different genera are provided, and the congruence between these two sets of results assessed. The results confirm the phylogeny hypothesis and infer a gradual mode of evolution of these infochemicals.     

Antagonistic factors control the unproductive splicing of SC35 terminal intron

Alternative splicing is regulated in part by variations in the relative concentrations of a variety of factors, including serine/arginine-rich (SR) proteins. The SR protein SC35 self-regulates its expression by stimulating unproductive splicing events in the 3′ untranslated region of its own pre-mRNA. Using various minigene constructs containing the terminal retained intron and flanking exons, we identified in the highly conserved last exon a number of exonic splicing enhancer elements responding specifically to SC35, and showed an inverse correlation between affinity of SC35 and enhancer strength. The enhancer region, which is included in a long stem loop, also contains repressor elements, and is recognized by other RNA-binding proteins, notably hnRNP H protein and TAR DNA binding protein (TDP-43). Finally, in vitro and in cellulo experiments indicated that hnRNP H and TDP-43 antagonize the binding of SC35 to the terminal exon and specifically repress the use of SC35 terminal 3′ splice site. Our study provides new information about the molecular mechanisms of SC35-mediated splicing activation. It also highlights the existence of a complex network of self- and cross-regulatory mechanisms between splicing regulators, which controls their homeostasis and offers many ways of modulating their concentration in response to the cellular environment.     

The Cytosolic Tail Dipeptide Ile-Met of the Pea Receptor BP80 Is Required for Recycling from the Prevacuole and for Endocytosis

Pea (Pisum sativum) BP80 is a vacuolar sorting receptor for soluble proteins and has a cytosolic domain essential for its intracellular trafficking between the trans-Golgi network and the prevacuole. Based on mammalian knowledge, we introduced point mutations in the cytosolic region of the receptor and produced chimeras of green fluorescent protein fused to the transmembrane domain of pea BP80 along with the modified cytosolic tails. By analyzing the subcellular location of these chimera, we found that mutating Glu-604, Asp-616, or Glu-620 had mild effects, whereas mutating the Tyr motif partially redistributed the chimera to the plasma membrane. Replacing both Ile-608 and Met-609 by Ala (IMAA) led to a massive redistribution of fluorescence to the vacuole, indicating that recycling is impaired. When the chimera uses the alternative route, the IMAA mutation led to a massive accumulation at the plasma membrane. Using Arabidopsis thaliana plants expressing a fluorescent reporter with the full-length sequence of At VSR4, we demonstrated that the receptor undergoes brefeldin A–sensitive endocytosis. We conclude that the receptors use two pathways, one leading directly to the lytic vacuole and the other going via the plasma membrane, and that the Ileu-608 Met-609 motif has a role in the retrieval step in both pathways.     

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch,Perca fluviatilis

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated.Plasma cortisol and glucose levels increased markedly 1 h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1–72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses.Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.     

Treatment failure with antagonists of TNF-α: mechanisms and implications for the care of patients

The use of TNF-α antagonists has substantially improved the care of many patients with inflammatory and autoimmune diseases. However, approximately one third of such patients fail to respond well to treatment, regardless of the antagonist used or of the underlying disease. The mechanisms underlying these failures are analyzed in this review, and proposals made concerning how best to adapt therapeutic decisions in these instances.     

Reduced oxygen tension results in reduced human T cell proliferation and increased intracellular oxidative damage and susceptibility to apoptosis upon activation

Cell culture and in vitro models are the basis for much biological research, especially in human immunology. Ex vivo studies of T cell physiology employ conditions attempting to mimic the in vivo situation as closely as possible. Despite improvements in controlling the cellular milieu in vitro, most of what is known about T cell behavior in vitro is derived from experiments on T cells exposed to much higher oxygen levels than are normal in vivo. In this study, we report a reduced proliferative response and increased apoptosis susceptibility after T cell activation at 2% oxygen compared to in air. To explain this observation, we tested the hypothesis of an impaired efficacy of intracellular protective mechanisms including antioxidant levels, oxidized protein repair (methionine sulfoxide reductases), and degradation (proteasome) activities. Indeed, after activation, there was a significant accumulation of intracellular oxidized proteins at more physiological oxygen levels concomitant with a reduced GSH:GSSG ratio. Proteasome and methionine sulfoxide reductase activities were also reduced. These data may explain the increased apoptotic rate observed at more physiological oxygen levels. Altogether, this study highlights the importance of controlling oxygen levels in culture when investigating oxygen-dependent phenomena such as oxidative stress.     

Pollen and non-pollen palynomorph evidence of medieval farming activities in southwestern Greenland

Radiocarbon dating, pollen and non-pollen palynomorph analyses from a lake core were used to establish the timing and effects of farming activities around Lake Igaliku, Eastern Settlement, Greenland. The absence of agro-pastoral impact before the medieval colonization by Europeans provides an opportunity to understand the development of farming activity in a pristine landscape. The results show that the first phase of clearance and grazing pressure, without the expansion of the Norse apophyte (native plant, in habitats created by humans) Rumex acetosa type, could have occurred in the 9–10th century a.d. The presence of Norse settlers and livestock is clearly recorded from the 11–12th century a.d. with increasing frequencies of the Norse apophytes Rumex acetosa type and Ranunculus acris type, and coprophilous fungi. This colonization phase is followed by a period of decreasing human impact at the beginning of the 14th century, with a decrease in weeds, apophytes and coprophilous fungi suggesting a reduced grazing pressure. The regrowth of Salix and Betula and the disappearance of anthropogenic indicators except Rumex acetosa type between the 15th and 18th century demonstrate the abandonment of the settlement, until the development of contemporary agriculture in the 20th century.