Growth line deposition and variability in growth of two circumpolar bivalves (Serripes groenlandicus, and Clinocardium ciliatum)

Growth patterns of two common circumpolar bivalves, the Greenland cockle (Serripes groenlandicus), and the hairy cockle (Clinocardium ciliatum) have been used in previous studies to reconstruct environmental conditions in the arctic. To date, there has been no direct determination that growth lines in either species are deposited periodically, and there has been no examination of factors affecting growth. We placed calcein-marked individuals of both species on oceanographic moorings in two fjords (Rijpfjord and Kongsfjord) in the Svalbard archipelago for one and two (Kongsfjord only) years. Growth patterns were compared with concurrent in situ temperature and fluorescence data in order to assess environmental controls on growth. Dark growth lines are evident on the outer shell surface and internally in shell cross section in both S. groenlandicus and C. ciliatum, and both species deposited only one line per year, unequivocally confirming that internal lines are deposited annually. Growth line deposition in both species began in late summer to early fall, before the seasonal decline in temperature. There was no difference in growth of S. groenlandicus between the two fjords despite differences in water temperature (3°C), fluorescence (nearly threefold) and the onset and duration of the winter season. C. ciliatum, however, grew approximately 2.8 times faster in the warmer, more food-rich Kongsfjord than in Rijpfjord. Subannual lines were counted in two individuals of each species from each fjord, but deposition of these lines was not clearly related to number of growing days estimated by temperature and fluorescence.     

Conserving the functional and phylogenetic trees of life of European tetrapods

Protected areas (PAs) are pivotal tools for biodiversity conservation on the Earth. Europe has had an extensive protection system since Natura 2000 areas were created in parallel with traditional parks and reserves. However, the extent to which this system covers not only taxonomic diversity but also other biodiversity facets, such as evolutionary history and functional diversity, has never been evaluated. Using high-resolution distribution data of all European tetrapods together with dated molecular phylogenies and detailed trait information, we first tested whether the existing European protection system effectively covers all species and in particular, those with the highest evolutionary or functional distinctiveness. We then tested the ability of PAs to protect the entire tetrapod phylogenetic and functional trees of life by mapping species'' target achievements along the internal branches of these two trees. We found that the current system is adequately representative in terms of the evolutionary history of amphibians while it fails for the rest. However, the most functionally distinct species were better represented than they would be under random conservation efforts. These results imply better protection of the tetrapod functional tree of life, which could help to ensure long-term functioning of the ecosystem, potentially at the expense of conserving evolutionary history.     

Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes

Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants.     

Mascot file parsing and quantification (MFPaQ), a new software to parse, validate, and quantify proteomics data generated by ICAT and SILAC mass spectrometric analyses: application to the proteomics study of membrane proteins from primary human endothelial cells

Proteomics strategies based on nanoflow (nano-) LC-MS/MS allow the identification of hundreds to thousands of proteins in complex mixtures. When combined with protein isotopic labeling, quantitative comparison of the proteome from different samples can be achieved using these approaches. However, bioinformatics analysis of the data remains a bottleneck in large scale quantitative proteomics studies. Here we present a new software named Mascot File Parsing and Quantification (MFPaQ) that easily processes the results of the Mascot search engine and performs protein quantification in the case of isotopic labeling experiments using either the ICAT or SILAC (stable isotope labeling with amino acids in cell culture) method. This new tool provides a convenient interface to retrieve Mascot protein lists; sort them according to Mascot scoring or to user-defined criteria based on the number, the score, and the rank of identified peptides; and to validate the results. Moreover the software extracts quantitative data from raw files obtained by nano-LC-MS/MS, calculates peptide ratios, and generates a non-redundant list of proteins identified in a multisearch experiment with their calculated averaged and normalized ratio. Here we apply this software to the proteomics analysis of membrane proteins from primary human endothelial cells (ECs), a cell type involved in many physiological and pathological processes including chronic inflammatory diseases such as rheumatoid arthritis. We analyzed the EC membrane proteome and set up methods for quantitative analysis of this proteome by ICAT labeling. EC microsomal proteins were fractionated and analyzed by nano-LC-MS/MS, and database searches were performed with Mascot. Data validation and clustering of proteins were performed with MFPaQ, which allowed identification of more than 600 unique proteins. The software was also successfully used in a quantitative differential proteomics analysis of the EC membrane proteome after stimulation with a combination of proinflammatory mediators (tumor necrosis factor-alpha, interferon-gamma, and lymphotoxin alpha/beta) that resulted in the identification of a full spectrum of EC membrane proteins regulated by inflammation.     

Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing

Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:974–982, 2015     

Pro‐haemostatic effect of DDAVP is partially derived through non‐classical (CD14dim /CD16 ++) monocytes residing the spleen

The splenic endothelial Weibel‐palade bodies are one of the most important candidate organelles to release von Willebrand factor upon stimulation with desmopressin. However, the presence of functional desmopressin‐specific receptor has not yet been demonstrated on endothelial cells. Experimental evidences are in favour of an indirect pro‐haemostatic effect of desmopressin, but the exact mediator and its cellular origin are largely elusive. Here, we report partially hampered desmopressin response in a splenectomised severe haemophilia A/Beta Thalassemia patient without any genetic variant relevant to his incomplete desmopressin response. To further investigate the role of the spleen in this phenomenon, the release of VWF from desmopressin‐treated human splenic endothelial cells was assessed in vitro. As a result, desmopressin induced the release of VWF from endothelial cells when the cells were co‐cultured with non‐classical (CD14^dim/CD16⁺⁺), but not other subtypes of monocytes or PBMCs. This in vitro study which resembles close proximity of endothelial cells of sinusoids to monocyte reservoir reside in parenchyma of subcapsular red pulp of the spleen sheds a light upon the role of this highly vascularized VWF‐producing organ in driving indirect effect of desmopressin.     

Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1β secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1β production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.     

Queuosine Biosynthesis Is Required for Sinorhizobium meliloti-Induced Cytoskeletal Modifications on HeLa Cells and Symbiosis with Medicago truncatula

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.