Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses

Advances in proteomic techniques have allowed the large-scale identification of phosphorylation sites in complex protein samples, but new biological insight requires an understanding of their in vivo dynamics. Here, we demonstrate the use of a stable isotope-based quantitative approach for pathway discovery and structure-function studies in Arabidopsis cells treated with the bacterial elicitor flagellin. The quantitative comparison identifies individual sites on plasma membrane (PM) proteins that undergo rapid phosphorylation or dephosphorylation. The data reveal both divergent dynamics of different sites within one protein and coordinated regulation of homologous sites in related proteins, as found for the PM H(+)-ATPases AHA1, 2 and 3. Strongly elicitor-responsive phosphorylation sites may reflect direct regulation of protein activity. We confirm this prediction for RbohD, an NADPH oxidase that mediates the rapid production of reactive oxygen species (ROS) in response to elicitors and pathogens. Plant NADPH oxidases are structurally distinct from their mammalian homologues, and regulation of the plant enzymes is poorly understood. On RbohD, we found both unchanging and strongly induced phosphorylation sites. By complementing an RbohD mutant plant with non-phosphorylatable forms of RbohD, we show that only those sites that undergo differential regulation are required for activation of the protein. These experiments demonstrate the potential for use of quantitative phosphoproteomics to determine regulatory mechanisms at the molecular level and provide new insights into innate immune responses.     

Differential cadmium and zinc distribution in relation to their physiological impact in the leaves of the accumulating Zygophyllum fabago L.

Cadmium and zinc share many similar physiochemical properties, but their compartmentation, complexation and impact on other mineral element distribution in plant tissues may drastically differ. In this study, we address the impact of 10 μm Cd or 50 μm Zn treatments on ion distribution in leaves of a metallicolous population of the non‐hyperaccumulating species Zygophyllum fabago at tissue and cell level, and the consequences on the plant response through a combined physiological, proteomic and metabolite approach. Micro‐proton‐induced X‐ray emission and laser ablation inductively coupled mass spectrometry analyses indicated hot spots of Cd concentrations in the vicinity of vascular bundles in response to Cd treatment, essentially bound to S‐containing compounds as revealed by extended X‐ray absorption fine structure and non‐protein thiol compounds analyses. A preferential accumulation of Zn occurred in vascular bundle and spongy mesophyll in response to Zn treatment, and was mainly bound to O/N‐ligands. Leaf proteomics and physiological status evidenced a protection of photosynthetically active tissues and the maintenance of cell turgor through specific distribution and complexation of toxic ions, reallocation of some essential elements, synthesis of proteins involved in photosynthetic apparatus or C‐metabolism, and metabolite synthesis with some specificities regarding the considered heavy metal treatment.     

Granular cell tumour of the soft tissues: a case report and literature review

Granular cell tumours (GCT) of the soft tissues are rare benign tumours but some time may be difficult to distinguish from malignant neoplasms. It is important that clinicians are aware of their existence. We present a new case of GCT of the soft tissues followed by a brief review of literature.     

Can a membrane oxygenator be a model for lung NO and CO transfer?

To model lung nitric oxide (NO) and carbon monoxide (CO) uptake, a membrane oxygenator circuit was primed with horse blood flowing at 2.5 l/min. Its gas channel was ventilated with 5 parts/million NO, 0.02% CO, and 22% O2 at 5 l/min. NO diffusing capacity (Dno) and CO diffusing capacity (Dco) were calculated from inlet and outlet gas concentrations and flow rates: Dno = 13.45 ml.min(-1).Torr(-1) (SD 5.84) and Dco = 1.22 ml.min(-1).Torr(-1) (SD 0.3). Dno and Dco increased (P = 0.002) with blood volume/surface area. 1/Dno (P < 0.001) and 1/Dco (P < 0.001) increased with 1/Hb. Dno (P = 0.01) and Dco (P = 0.004) fell with increasing gas flow. Dno but not Dco increased with hemolysis (P = 0.001), indicating Dno dependence on red cell diffusive resistance. The posthemolysis value for membrane diffusing capacity = 41 ml.min(-1).Torr(-1) is the true membrane diffusing capacity of the system. No change in Dno or Dco occurred with changing blood flow rate. 1/Dco increased (P = 0.009) with increasing Po2. Dno and Dco appear to be diffusion limited, and Dco reaction limited. In this apparatus, the red cell and plasma offer a significant barrier to NO but not CO diffusion. Applying the Roughton-Forster model yields similar specific transfer conductance of blood per milliliter for NO and CO to previous estimates. This approach allows alteration of membrane area/blood volume, blood flow, gas flow, oxygen tension, red cell integrity, and hematocrit (over a larger range than encountered clinically), while keeping other variables constant. Although structurally very different, it offers a functional model of lung NO and CO transfer.     

Drug‐microenvironment perturbations reveal resistance mechanisms and prognostic subgroups in CLL

The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited and systematic analyses are lacking. Using chronic lymphocytic leukaemia (CLL) as a model disease, we investigated the influence of 17 microenvironmental stimuli on 12 drugs in 192 genetically characterised patient samples. Based on microenvironmental response, we identified four subgroups with distinct clinical outcomes beyond known prognostic markers. Response to multiple microenvironmental stimuli was amplified in trisomy 12 samples. Trisomy 12 was associated with a distinct epigenetic signature. Bromodomain inhibition reversed this epigenetic profile and could be used to target microenvironmental signalling in trisomy 12 CLL. We quantified the impact of microenvironmental stimuli on drug response and their dependence on genetic alterations, identifying interleukin 4 (IL4) and Toll‐like receptor (TLR) stimulation as the strongest actuators of drug resistance. IL4 and TLR signalling activity was increased in CLL‐infiltrated lymph nodes compared with healthy samples. High IL4 activity correlated with faster disease progression. The publicly available dataset can facilitate the investigation of cell‐extrinsic mechanisms of drug resistance and disease progression.