Rhizosphere carbon flow measurement and implications: from isotopes to reporter genes

Despite the fundamental importance of rhizosphere C-flow in managed and natural systems, reliable measurement/resolution of C-flow and assessment of its consequences have largely remained elusive to soil biologists. Techniques involving both radioactive (¹⁴C) and stable (¹³C) isotopes of carbon have made some progress in terms of studying rhizosphere C-flow. Pulse-chase techniques have been used effectively to study dynamics of C-transfer to the rhizosphere and rhizosphere microbial biomass. The information obtained through pulse-chase is strongly dependent on the chase period following the labelling event. Continuous labelling is primarily used to determine plant inputs to soil over an extended time period and includes all kinds of C input – from root turnover, root respiration, root exudation, production of mucilage, etc. One of the main constraints to both approaches is that distinguishing root from microbial respiration is difficult, if not impossible. ¹³C techniques have gone some way towards resolving this difficulty, although ¹³C signatures in the plant–soil system are not easy to interpret and detailed resolution of carbon flow through different components of the rhizosphere biomass is unlikely to be achieved in such an inherently `noisy' system. Recent developments in molecular biology now provide a new opportunity to resolve rhizosphere C-flow and its implications. Reporter gene systems where, for example, rhizobacteria are marked with lux and unstable gfp reporters, overcome the difficulty of distinguishing root and microbial C fluxes and complement the isotopic and more traditional approaches. Reporter systems have now begun to resolve the competitive C sink strengths of different components of the rhizosphere microbial community and assess how a rhizobacterial inoculum may change C-flow in applications such as disease control and rhizoremediation of contaminated land. Fusion of reporter genes to nutrient (N and P) starvation genes in rhizobacteria has also enabled in situ characterisation of nutrient depletion around the root and assessment of the impact of changes in C-flow (such as those induced by climate change) on nutrient depletion dynamics. The availability of an integrated approach involving isotopic, molecular biological and other techniques now offers an exciting new era where reliable measurement and resolution of rhizosphere C-flow (and its consequences) can contribute to our understanding of ecosystem function and to management of crop-microbe interactions.     

TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo–N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.     

初夏渤海湾初级生产力分布特征及影响因素

根据2016年初夏在渤海湾周边重要经济开发区外海域所进行的调查和研究数据进行分析,利用稳定同位素¹³C示踪技术估算海域内初级生产力（Primary Productivity,PP）等,同时结合叶绿素a（Chlorophyll-a,Chl a）,营养盐以及透明度等水文环境参数,深入分析和探讨初夏渤海湾环境特征对浮游植物生物量（Chl a）和初级生产力的影响。结果表明：受陆地径流和渤海中部冷流输入等因素的影响,调查海域呈现3个温盐特征差异显著的海区,即近岸高温低盐海区、中部高温高盐海区和湾口低温高盐海区。Chl a受温度和营养盐等因素影响整体呈现近岸高湾口低、表层高底层低的分布特征,含量变化范围为1.27-20.82 mg/m³;在近岸营养盐含量充足,温度适宜,浮游植物生产旺盛,Chl a平均含量达（8.37±2.90）mg/m³,其中表层近27.5%水样中含量超10 mg/m³,存在发生赤潮的风险。而在中部和湾口区域受营养盐限制和温度的影响,Chl a含量远低于近岸。PP整体水平在44.79-792.73 mg C m^-3 d^-1之间,平均为（144.13±137.79）mg C m^-3 d^-1。其中近岸营养盐含量充足浮游植物生长旺盛,初级生产力水平较高,而中部和湾口海域受营养盐和温度的限制,初级生产力水平较低。初级生产力指数I（同化系数）变化范围在0.79-5.90 mg C/（mg Chl a·h）之间,平均为（3.40±1.33）mg C/（mg Chl a·h）。利用标准深度积分模型对水柱初级生产力（Depth-integrated primary productivity,∑PP）进行估算,结果表明其范围在56.88-772.31 mg C m^-2 d^-1之间,平均为（232.26±126.47）mg C m^-2 d^-1,近岸受陆源输入影响Chl a较高,在天津海河口和黄骅市排污河外出现高值点,受透明度的影响,中部和湾口部分站点出现高生产力区。     

1-Deoxysphingolipid synthesis compromises anchorage-independent growth and plasma membrane endocytosis in cancer cells

Serine palmitoyltransferase (SPT) predominantly incorporates serine and fatty acyl-CoAs into diverse sphingolipids (SLs) that serve as structural components of membranes and signaling molecules within or amongst cells. However, SPT also uses alanine as a substrate in the contexts of low serine availability, alanine accumulation, or disease-causing mutations in hereditary sensory neuropathy type I, resulting in the synthesis and accumulation of 1-deoxysphingolipids (deoxySLs). These species promote cytotoxicity in neurons and impact diverse cellular phenotypes, including suppression of anchorage-independent cancer cell growth. While altered serine and alanine levels can promote 1-deoxySL synthesis, they impact numerous other metabolic pathways important for cancer cells. Here, we combined isotope tracing, quantitative metabolomics, and functional studies to better understand the mechanistic drivers of 1-deoxySL toxicity in cancer cells. We determined that both alanine treatment and SPTLC1^C133W expression induce 1-deoxy(dihydro)ceramide synthesis and accumulation but fail to broadly impact intermediary metabolism, abundances of other lipids, or growth of adherent cells. However, we found that spheroid culture and soft agar colony formation were compromised when endogenous 1-deoxySL synthesis was induced via SPTLC1^C133W expression. Consistent with these impacts on anchorage-independent cell growth, we observed that 1-deoxySL synthesis reduced plasma membrane endocytosis. These results highlight a potential role for SPT promiscuity in linking altered amino acid metabolism to plasma membrane endocytosis.     

La TEP en gynéco-oncologie,au-delà du [18F]FDG ?

Understanding the mechanisms of carcinogenesis led to the development of new therapeutic approaches over the last decades, according to the specificities of tumor phenotypes, particularly in gynecological malignancies. [18F]FDG PET does not specifically assess these potential therapeutic targets. This update details the innovative PET tracers studied for this purpose, according to three families of oncological characteristics: tumor receptors, oncological mechanisms and tumor microenvironment.     

Phosphoglucoisomerase-catalyzed interconversion of hexose-phosphates

Summary The rate of conversion of D-glucose 6-phosphate to D-fructose 6-phosphate as catalyzed by yeast phosphoglucoisomerase is about fourfold lower when ³H, rather than ¹H, is present on the C2 of D-glucose 6-phosphate. This difference appears to be due mainly to a change in maximal velocity, rather than affinity. Phosphoglucoisomerase also distinguishes between ¹H and ³H in terms of either their intramolecular transfer from C2 to C1 or their incorporation from water on the C1 of D-fructose 6-phosphate.     

Multi-site-specific isotopic labeling accelerates high-resolution structural investigations of pathogenic huntingtin exon-1

1. Download : Download high-res image (181KB)
2. Download : Download full-size image

     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.