VISTA deficiency attenuates antibody-induced arthritis and alters macrophage gene expression in response to simulated immune complexes

Background

In addition to activated T cells, the immune checkpoint inhibitor “V domain-containing Ig suppressor of T-cell activation” (VISTA) is expressed by myeloid cell types, including macrophages and neutrophils. The importance of VISTA expression by myeloid cells to antibody-induced arthritis and its potential for relevance in human disease was evaluated.

Methods

VISTA was immunolocalized in normal and arthritic human synovial tissue sections and synovial tissue lysates were subjected to western blot analysis. The collagen antibody-induced arthritis model (CAIA) was performed with DBA/1 J mice treated with antibodies against VISTA and with VISTA-deficient mice (V-KO). Total mRNA from arthritic joints, spleens, and cultured macrophages was analyzed with NanoString arrays. Cytokines secreted by splenic inflammatory macrophages were determined. In-vitro chemotaxis and signal transduction assays were performed with cultured macrophages.

Results

VISTA protein was localized to synovial membrane cells, neutrophils, and scattered cells in lymphocyte-rich foci and was detected by western blot analysis in normal synovium and synovium from rheumatoid arthritis patients. Deficiency of VISTA or treatment of mice with anti-VISTA monoclonal antibodies attenuated CAIA. Joint damage and MMP-3 expression were significantly reduced in V-KO mice. Surface expression of C5a receptor was reduced on monocytes, neutrophils, and cultured macrophages from V-KO. Upon Fc receptor engagement in vitro, gene expression by V-KO macrophages was altered profoundly compared to WT, including a significant induction of IL-1 receptor antagonist (IL1rn).

Conclusions

VISTA expression supports immune-complex inflammation in CAIA and VISTA is expressed in human synovium. VISTA supports optimal responses to C5a and modulates macrophage responses to immune complexes.

     

PureCLIP: capturing target-specific protein–RNA interaction footprints from single-nucleotide CLIP-seq data

The iCLIP and eCLIP techniques facilitate the detection of protein–RNA interaction sites at high resolution, based on diagnostic events at crosslink sites. However, previous methods do not explicitly model the specifics of iCLIP and eCLIP truncation patterns and possible biases. We developed PureCLIP (https://github.com/skrakau/PureCLIP), a hidden Markov model based approach, which simultaneously performs peak-calling and individual crosslink site detection. It explicitly incorporates a non-specific background signal and, for the first time, non-specific sequence biases. On both simulated and real data, PureCLIP is more accurate in calling crosslink sites than other state-of-the-art methods and has a higher agreement across replicates.     

A comparative evaluation of culture conditions for short-term maintenance (<24 hr) of human islets isolated using the Edmonton protocol

Once human islets are isolated, they are typically transplanted into type 1 diabetic recipients within 2 h of isolation. This time restriction makes it difficult for patients to travel from distant locations to receive an islet transplant and it also makes it difficult to complete pre-release quality control assessments (i.e., endotoxin and gram stain) before the expiration of the islet product. Therefore, there were two goals for this study. The first was to measure the stability of islets after a 24 h culture period using CMRL media 1066 (CMRL) supplemented with either fetal bovine serum (FBS); albumin or insulin transferrin and selenium (ITS). The second was to determine the impact of cell concentration and media depth on islet stability. The results of the study indicated that culture recoveries at 37 °C with CMRL + ITS (also known as Memphis media) were higher (64.1 ± 8.3%) than with CMRL supplemented with FBS (38.7 ± 9.7%) or albumin (47.6 ± 8.2%) and that post-culture islet viabilities, post-culture purities and stimulation indexes (SIs) were comparable. In the second series of experiments, the results showed that islets recoveries and SIs in cultures with low islet concentrations (300 IE/ml) were significantly better than cultures at high islet concentrations (1500 IE/ml). Additionally, at a shallow media depth (1.4 vs. 7 mm of media) the SI of the islets improved, and this effect was independent of the additive (i.e., FBS, albumin and ITS).     

Novel UNC‐44 AO13 ankyrin is required for axonal guidance in C. elegans,contains six highly repetitive STEP blocks separated by seven potential transmembrane domains,and is localized to neuronal processes and the periphery of neural cell bodies

Conventional ankyrins are cortical cytoskeletal proteins that form an ankyrin‐spectrin meshwork underlying the plasma membrane. We report here the unusual structure of a novel ankyrin (AO13 ankyrin, 775,369 Da, 6994 aa, pI = 4.45) that is required for proper axonal guidance in Caenorhabditis elegans. AO13 ankyrin contains the ANK repeat and spectrin‐binding domains found in other ankyrins, but differs from all others in that the acidic carboxyl region contains six blocks of serine/threonine/glutamic acid/proline rich (STEP) repeats separated by seven hydrophobic domains. The STEP repeat blocks are composed primarily of sequences related to ETTTTTTVTREHFEPED(E/D)X_nVVESEEYSASGSPVPSE (E/K)DVE(H/R)VI, and the hydrophobic domains contain sequences related to PESGEESDGEGFGSKVLGFAKK[AGMVAGGVVAAPVALAAVGA]KAAYDALKKDDDEE, which includes a potential transmembrane domain (in brackets). Recombinant protein fragments of AO13 ankyrin were used to prepare polyclonal antisera against the spectrin‐binding domain (AO271 Ab), the conventional ankyrin regulatory domain (AO280 Ab), the AO13 ankyrin STEP domain (AO346 Ab), the AO13 ankyrin STEP + hydrophobic domain (AO289 Ab), and against two carboxyl terminal domain fragments (AO263 Ab and AO327 Ab). Western blot analysis with these Ab probes demonstrated multiple protein isoforms. By immunofluorescence microscopy, the antispectrin‐binding and regulatory domain (AO271 and AO280) antibodies recognized many cell types, including neurons, and stained the junctions between cells. The AO13 ankyrin‐specific (AO289 and AO346) antibodies showed a neurally restricted pattern, staining nerve processes and the periphery of neural cell bodies. These results are consistent with a role for AO13 ankyrin in neural development. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 333–349, 2002; DOI 10.1002/neu.10036     

Using GaBi 3 to perform life cycle assessment and life cycle engineering

The growing availability of software tools has increased the speed of generating LCA studies. Databases and visual tools for constructing material balance modules greatly facilitate the process of analyzing the environmental aspects of product systems over their life cycle. A robust software tool, containing a large LCI dataset and functions for performing LCIA and sensitivity analysis will allow companies and LCA practitioners to conduct systems analyses efficiently and reliably. This paper discusses how the GaBi 3 software tool can be used to perform LCA and Life Cycle Engineering (LCE), a methodology that combines life cycle economic, environmental, and technology assessment. The paper highlights important attributes of LCA software tools, including high quality, well-documented data, transparency in modeling, and data analysis functionality. An example of a regional power grid mix model is used to illustrate the versatility of GaBi 3.     

Developmental constraints revealed by co-variation within and among molar rows in two murine rodents

SUMMARY Morphological integration corresponds to interdependency between characters that can arise from several causes. Proximal causes of integration include that different phenotypic features may share common genetic sets and/or interact during their development. Ultimate causes may be the prolonged effect of selection favoring integration of functionally interacting characters, achieved by the molding of these proximal causes. Strong and direct interactions among successive teeth of a molar row are predicted by genetic and developmental evidences. Functional constraints related to occlusion, however, should have selected more strongly for a morphological integration of occluding teeth and a corresponding evolution of the underlying developmental and genetic pathways. To investigate how these predictions match the patterns of phenotypic integration, we studied the co‐variation among the six molars of the murine molar row, focusing on two populations of house mice (Mus musculus domesticus) and wood mice (Apodemus sylvaticus). The size and shape of the three upper and lower molars were quantified and compared. Our results evidenced similar patterns in both species, size being more integrated than shape among all the teeth, and both size and shape co‐varying strongly between adjacent teeth, but also between occluding teeth. Strong co‐variation within each molar row is in agreement with developmental models showing a cascade influence of the first molar on the subsequent molars. In contrast, the strong co‐variation between molars of the occluding tooth rows confirms that functional constraints molded patterns of integration and probably the underlying developmental pathways despite the low level of direct developmental interactions occurring among molar rows. These patterns of co‐variation are furthermore conserved between the house mouse and the wood mouse that diverged >10 Ma, suggesting that they may constitute long‐running constraints to the diversification of the murine rodent dentition.     

Regional Variations in Biomass Distribution in Brazilian Savanna Woodland

The Cerrado, the savanna biome in central Brazil, mostly comprised of woodland savanna, is experiencing intense and fast land use changes. To understand the changes in Cerrado carbon stocks, we present an overview of biomass distribution in different Cerrado vegetation types (i.e., grasslands, shrublands and forestlands). We surveyed 26 studies including 170 Cerrado sites. The grasslands presented mean total biomass of 24 Mg/ha, with 70 percent allocated in the belowground portion. In shrublands, the mean total biomass was 58 Mg/ha being 58 percent in the belowground portion. Finally, in forestlands the mean total biomass was 98 Mg/ha with 18 percent as belowground biomass. The surveyed studies presented 12 allometric equations for biomass estimate, most involving both diameter and height. Data on wood density for Cerrado shrubs and trees are not abundant and the average value was 0.66 g/cm³, similar to that found in the central portion of the Amazon Forest. We also examined the relationship between total precipitation and dry‐season intensity with biomass variation in the Cerrado shrubland using data from tropical rainfall measurement mission (TRMM) for the period 2000–2010. Dry‐season precipitation amount in cerrado areas in severe drought regions explained 29 percent of the variation in aboveground woody biomass. This finding is important in the face of the predictions of longer and more severe dry seasons in the region due to climate change.     

Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG

Clathrin depletion by ribonucleic acid interference (RNAi) impairs mitotic spindle stability and cytokinesis. Depletion of several clathrin-associated proteins affects centrosome integrity, suggesting a further cell cycle function for clathrin. In this paper, we report that RNAi depletion of CHC17 (clathrin heavy chain 17) clathrin, but not the CHC22 clathrin isoform, induced centrosome amplification and multipolar spindles. To stage clathrin function within the cell cycle, a cell line expressing SNAP-tagged clathrin light chains was generated. Acute clathrin inactivation by chemical dimerization of the SNAP-tag during S phase caused reduction of both clathrin and ch-TOG (colonic, hepatic tumor overexpressed gene) at metaphase centrosomes, which became fragmented. This was phenocopied by treatment with Aurora A kinase inhibitor, suggesting a centrosomal role for the Aurora A-dependent complex of clathrin, ch-TOG, and TACC3 (transforming acidic coiled-coil protein 3). Clathrin inactivation in S phase also reduced total cellular levels of ch-TOG by metaphase. Live-cell imaging showed dynamic clathrin recruitment during centrosome maturation. Therefore, we propose that clathrin promotes centrosome maturation by stabilizing the microtubule-binding protein ch-TOG, defining a novel role for the clathrin-ch-TOG-TACC3 complex.     

Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer <Emphasis Type="Italic">Streptomyces collinus</Emphasis> Tü 365

Streptomycetes are prolific sources of novel biologically active secondary metabolites with pharmaceutical potential. S. collinus Tü 365 is a Streptomyces strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein biosynthesis interacting with elongation factor EF-Tu. Genome Mining revealed 32 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of Streptomyces collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS-NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC–MS analyses, most of the uncharacterized gene clusters are not expressed under standard laboratory conditions. With this study we aimed to characterize the genome information of S. collinus Tü 365 to make use of gene clusters, which previously have not been described for this strain. We were able to connect the gene clusters of a lanthipeptide, a carotenoid, five terpenoid compounds, an ectoine, a siderophore and a spore pigment-associated gene cluster to their respective biosynthesis products.