Mechanisms of Tim-3 Signal Transduction in the Modulation of Downstream TCR Signaling

T-cell immunoglobulin and mucin 3 protein (Tim-3) is a type-I transmembrane protein known to negatively regulate Th1 and Th17 CD4 T cells, and CD8 T cell mediated immune responses. Tim-3 expression level correlates with the severity of T cell exhaustion under conditions of chronic viral infection and tumor burden and Tim-3 antibodies can reverse this dysfunctional phenotype. Exploring the signal transduction mechanism of Tim-3, I demonstrated that ectopic expression of Tim-3 enhanced NFAT/AP-1, NFAT and NF-κB reporter activity induced by TCR stimulation, and enhanced AP-1 reporter activity independent of TCR stimulation. Two tyrosines in the cytosolic tail of Tim-3 are responsible for the potentiation of signal transduction by TCR in a redundant manner. I demonstrated that the Src family kinase (SFK) Fyn can phosphorylate Y256 and Y263 residues of Tim-3. An SH2 domain screen and co-immunoprecipitation identified p85 PI3K interaction with Tim-3 in a tyrosine phosphorylation-dependent manner. I also found that Tim-3 expression upregulates phosphorylation of PLC-γ1 and MAP kinases ERK1/2, p38, and JNK. Finally, I demonstrated that Tim-3 upregulates phosphorylation of ribosomal protein S6, a downstream target of PI3K and ERK, and IL-2 secretion induced by TCR stimulation. I conclude that potentiation of NFAT/AP1 and NF-κB activity by Tim-3 is mediated by enhancement of the PI3K, PLC-γ1 and ERK pathways. Thus, we have demonstrated a paradoxical activating function of Tim-3 in TCR signaling, while Tim-3 is known as a negative regulator of Th1 and Tc1 T cell mediated immune responses. This augmentation of TCR signaling by Tim-3 may contribute to driving and/or maintaining T cell exhaustion. Alternatively, Tim-3 may have a dual activation and inhibition role depending on the ligands available at a particular stage of T cell activation

A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes

BACKGROUND: Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF) animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines). RESULTS: We screened ~15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. CONCLUSIONS: We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future investigations of the complicated relationships between apoptosis and autophagy

Crystal Structure of the TLR4-MD-2 Complex with Bound Endotoxin Antagonist Eritoran

SummaryTLR4 and MD-2 form a heterodimer that recognizes LPS (lipopolysaccharide) from Gram-negative bacteria. Eritoran is an analog of LPS that antagonizes its activity by binding to the TLR4-MD-2 complex. We determined the structure of the full-length ectodomain of the mouse TLR4 and MD-2 complex. We also produced a series of hybrids of human TLR4 and hagfish VLR and determined their structures with and without bound MD-2 and Eritoran. TLR4 is an atypical member of the LRR family and is composed of N-terminal, central, and C-terminal domains. The β sheet of the central domain shows unusually small radii and large twist angles. MD-2 binds to the concave surface of the N-terminal and central domains. The interaction with Eritoran is mediated by a hydrophobic internal pocket in MD-2. Based on structural analysis and mutagenesis experiments on MD-2 and TLR4, we propose a model of TLR4-MD-2 dimerization induced by LPS

Rare variant testing across methods and thresholds using the multi-kernel sequence kernel association test (MK-SKAT)

Analysis of rare genetic variants has focused on region-based analysis wherein a subset of the variants within a genomic region is tested for association with a complex trait. Two important practical challenges have emerged. First, it is difficult to choose which test to use. Second, it is unclear which group of variants within a region should be tested. Both depend on the unknown true state of nature. Therefore, we develop the Multi-Kernel SKAT (MK-SKAT) which tests across a range of rare variant tests and groupings. Specifically, we demonstrate that several popular rare variant tests are special cases of the sequence kernel association test which compares pair-wise similarity in trait value to similarity in the rare variant genotypes between subjects as measured through a kernel function. Choosing a particular test is equivalent to choosing a kernel. Similarly, choosing which group of variants to test also reduces to choosing a kernel. Thus, MK-SKAT uses perturbation to test across a range of kernels. Simulations and real data analyses show that our framework controls type I error while maintaining high power across settings: MK-SKAT loses power when compared to the kernel for a particular scenario but has much greater power than poor choices

Development of a Truss-Arch Model Unified for Flexure and Shear-Critical Interaction of Structural Concrete Members

Based on force-deformation behavior computed using the Compatibility Strut and Tie Model (C STM), a theoretical limit analysis model is developed to predict the ultimate shear-carrying capacity of shear-critical structural concrete beams with and without transverse reinforcement. The Truss-Arch Model Unified (TAMU) is validated based on experimental observations and from previous studies reported in the literature. The limit analysis approach assumes the failure mechanism occurs when the principal diagonal arch reaches its softened peak strength. Two truss models, with a vertical tie or an inclined tie, are used depending on whether the members are reinforced with or without shear steel, respectively. Explicit solutions for the principal strain ratio and ultimate shear strength are derived based on truss and arch contribution to flexibility and strength. A large database of test data consisting of 839 beams is assembled for the substantiation of the proposed method and also to conduct a comparative assessment of existing code-based shear analysis methods. The proposed TAMU approach predicts well the limit load capacities against the database. When compared with existing code-based shear analysis approaches, the TAMU based approach demonstrates superior accuracy with less dispersion due to modeling and aleatory uncertainty in both D-and B-regions. The TAMU approach is further extended to account for axial strain effects which may be present due to the presence of either prestress, axial load, or both. A full-scale experimental program is conducted on reinforced and prestressed concrete bent caps to investigate the effect of prestressing force, shear reinforcement spacing, interior voids, and axial loads on the bent cap. The specimens are analyzed by the TAMU method and other existing code-based analysis methods. The C-STM and TAMU analysis methods are able to accurately predict the experimental test outcomes somewhat better than existing code analysis approaches

LRRK2 kinase plays a critical role in manganese-induced inflammation and apoptosis in microglia.

Long-term exposure to elevated levels of manganese (Mn) causes manganism, a neurodegenerative disorder with Parkinson's disease (PD)-like symptoms. Increasing evidence suggests that leucine-rich repeat kinase 2 (LRRK2), which is highly expressed in microglia and macrophages, contributes to the inflammation and neurotoxicity seen in autosomal dominant and sporadic PD. As gene-environment interactions have emerged as important modulators of PD-associated toxicity, LRRK2 may also mediate Mn-induced inflammation and pathogenesis. In this study, we investigated the role of LRRK2 in Mn-induced toxicity using human microglial cells (HMC3), LRRK2-wild-type (WT) and LRRK2-knockout (KO) RAW264.7 macrophage cells. Results showed that Mn activated LRRK2 kinase by phosphorylation of its serine residue at the 1292 position (S1292) as a marker of its kinase activity in macrophage and microglia, while inhibition with GSK2578215A (GSK) and MLi-2 abolished Mn-induced LRRK2 activation. LRRK2 deletion and its pharmacological inhibition attenuated Mn-induced apoptosis in macrophages and microglia, along with concomitant decreases in the pro-apoptotic Bcl-2-associated X (Bax) protein. LRRK2 deletion also attenuated Mn-induced production of reactive oxygen species (ROS) and the pro-inflammatory cytokine TNF-α. Mn-induced phosphorylation of mitogen-activated protein kinase (MAPK) p38 and ERK signaling proteins was significantly attenuated in LRRK2 KO cells and GSK-treated cells. Moreover, inhibition of MAPK p38 and ERK as well as LRRK2 attenuated Mn-induced oxidative stress and cytotoxicity. These findings suggest that LRRK2 kinase activity plays a critical role in Mn-induced toxicity via downstream activation of MAPK signaling in macrophage and microglia. Collectively, these results suggest that LRRK2 could be a potential molecular target for developing therapeutics to treat Mn-related neurodegenerative disorders

Synergy Effect Study of Poly(N-isoacrylamide) with Tetra Butyl Phosphonium Bromide on Methane Hydrate Formation

Natural gas hydrate plugging is one of the costly and challenging problems for the oil and gas industry, especially for subsea fields. One way to prevent gas hydrate formation at low cost is the use of low-dosage hydrate inhibitors (LDHI). Poly(N-isopropylmethacrylamide) (PNIPAM) with amide group have previously been shown to be an outstanding low-dosage hydrate inhibitor (LDHI). PNIPAMs are usually polymerized via radical polymerization, which can allow control over the molecular weight. We have synthesized PNIPAMs using control radical polymerization giving a fairly high degree of polymerization control. Additionally various Kinetic Hydrate Inhibitors (KHIs) were tested as CH4 hydrate inhibitors with polymeric hydrate inhibitor, PNIPAM. Furthermore, to check the synergetic effects of tetra butyl phosphonium bromide (TBPB), PNIPAM and their mixture TBPB-PNIPAM were tested as KHIs for methane gas hydrate formation. In this paper we present results on the performance of various polymeric inhibitors in KHI tests on methane gas in stirred autoclaves and on structure I hydrate formation. When PNIPAM alone was used as a single KHI, PNIPAM exhibited worst performance among the KHIs tested in this study. However the mixing of TBPB with PNIPAM further extended the induction time and reduced the CH4 hydrate growth rate. TBPB was an excellent synergist in blends with PNIPAM for kinetic hydrate inhibition of structure I forming CH4 hydrate. In summary, the mixture TBPB-PNIPAM was proven that great synergist for prevent the CH4 hydrate and good potential in achieving the industrial application of Oil & Gas production technology and therefore was a significantly meaningful discovery in the field of energy production.publishedVersio

A genetic screen for modifiers of <it>Drosophila </it>caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes

Abstract Background Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF) animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines). Results We screened ~15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. Conclusions We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future investigations of the complicated relationships between apoptosis and autophagy.</p

Rare variant testing across methods and thresholds using the multi-kernel sequence kernel association test (MK-SKAT)

Analysis of rare genetic variants has focused on region-based analysis wherein a subset of the variants within a genomic region is tested for association with a complex trait. Two important practical challenges have emerged. First, it is difficult to choose which test to use. Second, it is unclear which group of variants within a region should be tested. Both depend on the unknown true state of nature. Therefore, we develop the Multi-Kernel SKAT (MK-SKAT) which tests across a range of rare variant tests and groupings. Specifically, we demonstrate that several popular rare variant tests are special cases of the sequence kernel association test which compares pair-wise similarity in trait value to similarity in the rare variant genotypes between subjects as measured through a kernel function. Choosing a particular test is equivalent to choosing a kernel. Similarly, choosing which group of variants to test also reduces to choosing a kernel. Thus, MK-SKAT uses perturbation to test across a range of kernels. Simulations and real data analyses show that our framework controls type I error while maintaining high power across settings: MK-SKAT loses power when compared to the kernel for a particular scenario but has much greater power than poor choices