SH2 domain-containing phosphatase 2 is a critical regulator of connective tissue mast cell survival and homeostasis in mice

Mast cells require KIT receptor tyrosine kinase signaling for development and survival. Here, we report that SH2 domain-containing phosphatase 2 (SHP2) signaling downstream of KIT is essential for mast cell survival and homeostasis in mice. Using a novel mouse model with shp2 deletion within mature mast cells (MC-shp2 knockout [KO]), we find that SHP2 is required for the homeostasis of connective tissue mast cells. Consistently with the loss of skin mast cells, MC-shp2 KO mice fail to mount a passive late-phase cutaneous anaphylaxis response. To better define the phenotype of shp2-deficient mast cells, we used an inducible shp2 knockout approach in bone marrow-derived mast cells (BMMCs) or cultured peritoneal mast cells and found that SHP2 promotes mast cell survival. We show that SHP2 promotes KIT signaling to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase and downregulation of the proapoptotic protein Bim in BMMCs. Also, SHP2-deficient BMMCs failed to repopulate mast cells in mast cell-deficient mice. Silencing of Bim partially rescued survival defects in shp2-deficient BMMCs, consistent with the importance of a KIT → SHP2 → Ras/ERK pathway in suppressing Bim and promoting mast cell survival. Thus, SHP2 is a key node in a mast cell survival pathway and a new potential therapeutic target in diseases involving mast cells.     

Social environment affects juvenile dispersal in great tits (Parus major)

1.?Habitat selection can affect individual fitness, and therefore, individuals are expected to assess habitat quality of potential breeding sites before settlement. 2.?We investigated the role of social environment on juvenile dispersal behaviour in the great tit (Parus major). Two main contradictory hypotheses can be formulated regarding social effects on juvenile dispersal as follows: (i) High fledgling density and sex ratio may enhance the intensity of local (kin) competition and, therefore, reduce individual survival chance, enhance emigration and reduce settlement ('repulsion' hypothesis) (ii) Alternatively, high fledgling density and sex ratio may signal high-quality habitat or lead to aggregation and thus increase individual survival chance, reduce emigration and enhance settlement ('attraction' hypothesis). 3.?To disentangle positive from negative effects of high density and male-biased sex ratio on dispersal, we manipulated the social composition of the fledgling population in 12 semi-isolated nest-box areas (plots) via a change of fledgling density (low/high) as well as fledgling sex ratio (female-biased/balanced/male-biased) across 3?years. We then tested whether experimental variation in male and female fledgling densities affected variation in local survival, emigration and settlement of juveniles, and whether social effects on survival and dispersal support the 'repulsion' or 'attraction' hypothesis. 4.?We found no experimental effects on local survival and emigration probabilities. However, consistent with the 'attraction' hypothesis, settlement was significantly and positively affected by local experimental sex ratio in each of the study years: both male and female juveniles avoided female-biased plots and settled more in plots that were balanced and male-biased the previous year. 5.?Our study provides unprecedented experimental evidence that local sex ratio plays a causal role in habitat selection. We suggest that settlers avoid female-biased plots because a high proportion of females may reflect the absence or the low quality of local resources in the habitat. Alternatively, male territory acquisition may be facilitated by a high local density of 'candidate' males, and therefore, juveniles were less successful in settling in female-biased plots.     

The γ3 chain of laminin is widely but differentially expressed in murine basement membranes: expression and functional studies

Laminins are heterotrimeric extracellular glycoproteins found in, but not confined to, basement membranes (BMs). They are important components in formation of the molecular networks of BMs as well as in cell polarity, cell differentiation and tissue morphogenesis. Each laminin is composed by an α, a β and a γ chain. Previous studies have shown that the γ3 chain is partnered with either the β1 chain (in placenta) or β2 chain (in the CNS) (Libby et al., 2000). Several studies, including our own, suggested that the γ3 chain is expressed in both apical and basal compartments (Koch et al., 1999; Gersdorff et al., 2005; Yan and Cheng, 2006). This study investigates the expression pattern of the γ3 chain in mouse. We developed three new γ3-reactive antibodies, and we show that the γ3 chain is present in BMs. The distribution pattern is considerably more restricted than that of the γ1 chain and within any tissue there is differential deposition into BM compartments. This is particularly true in the retina and brain, where γ3 is uniquely expressed in a subset of the vascular basement membranes and the pial surface. We used conventional genetic ablation techniques to remove the γ3 chain in mice; unlike other laminin null mice (α5, β2, γ1 nulls), these mice live a normal lifespan and have only minor abnormalities, the most striking of which are ectopic granule cells in the cerebellum and an apparent increase in capillary branching in the outer retina. These data support the suggestion that the γ3 chain is deposited in BMs and contributes some unique properties to their function, particularly in the nervous system.     

Computational modeling-based discovery of novel classes of anti-inflammatory drugs that target lanthionine synthetase C-like protein 2

Background

Lanthionine synthetase component C-like protein 2 (LANCL2) is a member of the eukaryotic lanthionine synthetase component C-Like protein family involved in signal transduction and insulin sensitization. Recently, LANCL2 is a target for the binding and signaling of abscisic acid (ABA), a plant hormone with anti-diabetic and anti-inflammatory effects.

Methodology/Principal Findings

The goal of this study was to determine the role of LANCL2 as a potential therapeutic target for developing novel drugs and nutraceuticals against inflammatory diseases. Previously, we performed homology modeling to construct a three-dimensional structure of LANCL2 using the crystal structure of lanthionine synthetase component C-like protein 1 (LANCL1) as a template. Using this model, structure-based virtual screening was performed using compounds from NCI (National Cancer Institute) Diversity Set II, ChemBridge, ZINC natural products, and FDA-approved drugs databases. Several potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked compound (NSC61610) in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a mouse model of dextran sodium sulfate (DSS)-induced colitis. Our findings showed that the lead compound, NSC61610, activated peroxisome proliferator-activated receptor gamma in a LANCL2- and adenylate cyclase/cAMP dependent manner in vitro and ameliorated experimental colitis by down-modulating colonic inflammatory gene expression and favoring regulatory T cell responses.

Conclusions/Significance

LANCL2 is a novel therapeutic target for inflammatory diseases. High-throughput, structure-based virtual screening is an effective computational-based drug design method for discovering anti-inflammatory LANCL2-based drug candidates.     

Isolation of immune cells from primary tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors. Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4(+) T helper cells via adoptive transfer, promotes CD8(+) T cells to maintain pro-inflammatory effector functions. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.     

Enzymatic modification of 2,6-dimethoxyphenol for the synthesis of dimers with high antioxidant capacity

2,6-Dimethoxyphenol is a phenolic compound that is extensively used for the measurement of laccase activity, but is often not exploited for its potential as an antioxidant compound. Since laccase can be used to modify phenolic antioxidants as a way of improving their activities, the present study investigated the laccase-mediated oxidation of 2,6-dimethoxyphenol in biphasic or homogenous aqueous-organic media for the production of compounds with higher antioxidant capacity than the starting substrate. The main product was a dimer (m/z 305.0672), which was further characterized as a symmetrical CC linked 3,3′,5,5′-tetramethoxy biphenyl-4,4′-diol. In the monophasic system, the dimer was preferentially formed when acetone was used as co-solvent, while in the biphasic system, formation of the dimer increased as the concentration of ethyl acetate was increased from 50 to 90%. The dimer showed higher antioxidant capacity than the substrate (≈2×) as demonstrated by standard antioxidant assays (DPPH and FRAP). These results demonstrate that a product of the laccase-catalysed oxidation of 2,6-dimethoxyphenol can find useful application as a bioactive compound.     

Subterranean Sympatry: An Investigation into Diet Using Stable Isotope Analysis

In the Western Cape three species of mole-rat occur in sympatry, however, little is known about differences in their dietary preferences. Dietary composition of the three species; the common mole-rat (Cryptomys hottentotus hottentotus), the Cape mole-rat (Georychus capensis) and the Cape dune mole-rat (Bathyergus suillus) were examined using stable isotope analysis. Blood, fur and claw samples were collected from 70 mole-rats, in addition to several potential food items, to assess food selection of the three species under natural conditions. Overall there was a significant difference in the isotopic composition (δ¹³C and δ¹⁵N) between all three species and significant differences in their diet composition. There were also significant differences between tissues in all three species suggesting temporal variation in diet. The small size and colonial lifestyle of C. h. hottentotus allows it to feed almost 100% on bulbs, while the solitary and larger species G. capensis and B. suillus fed to a greater extent on other resources such as grasses and clover. B. suillus, the largest of the species, had the most generalized diet. However, overall all species relied most heavily upon geophytes and consumed the same species suggesting competition for resources could exist. We also showed a high level of individual variation in diet choices. This was most pronounced in B. suillus and G. capensis and less so in C. h. hottentotus. We demonstrate that stable isotope analysis can successfully be applied to examine dietary patterns in subterranean mammals and provide insights into foraging patterns and dietary variation at both the inter and intra population level.