A novel regulatory mechanism of the plant TIR immune signaling

Plant defense against microbial pathogens is mainly realized by pattern-triggered immunity (PTI) mediated by pattern recognition receptors (PRRs) at the cell surface, and effectortriggered immunity (ETI) mediated by nucleotide-binding leucine-rich repeat (NLR) immune receptors inside cells. Based on their N-terminal domains, plant NLRs can be divided into two categories: CC-NLRs (CNLs) with a coiled-coil (CC) domain and TIR-NLRs (TNLs) with a toll/interleukin 1 receptor (TIR) domain. Specific recognition of pathogen effectors induces oligomerization of NLRs, termed resistosomes, to transduce plant immune signaling. CNLs are able to form pentameric resistosomes upon activation and function as calcium (Ca2+)-permeable channels in the plasma membrane. Whether TNLs form resistosomes in response to pathogen infection remained an open question, although the TIR domain in TNLs has NADase activity that is required for TNL-mediated immunity. NADase activity, although essential, is not sufficient for TIR-triggered immune responses in plants, suggesting that other components may be required for TIR-mediated signaling. In my dissertation, I employed multiple approaches including biochemistry and structural biology to address these questions. The thesis contains three parts: In the first part, I present multiple lines of evidence showing that the Arabidopsis TNL RPP1 (for recognition of Peronospora parasitica 1) forms a tetrameric resistosome upon recognition of the cognate Hyaloperonospora arabidopsidis effector ATR1. Biochemical and structural data are summarized revealing the mechanism underlying the requirement of the RPP1 resistosome formation for NADase activity. The data from this study define the mechanism of direct effector recognition by a TNL, and demonstrate that the assembly of RPP1 resistosomes is required for TIR-encoded NADase activity and RPP1 function. In the second part, I describe biochemical evidence that TIR domain proteins also exhibit 2′,3′-cAMP/cGMP synthetase activity with RNA and probably DNA (RNA/DNA) as substrates. Then I present functional data supporting the physiological relevance of the synthetase activity in TIR-mediated immune responses. Structural data on a TIR domain protein bound by its dsDNA substrate are described, and the mechanisms of how TIR domain proteins encode both NADase and synthetase activities and how the two activities may act together to mediate TIR signaling are discussed. The data presented in this part reveal a novel enzymatic activity of plant TIR domain proteins and establish a role of 2′,3′-cAMP/cGMP in plant immunity. In the last section of my thesis, I describe experiments testing whether the RNase-like effector proteins associated with haustoria (RALPH effectors) have RNase activity

Sevoflurane ameliorates doxorubicin-induced myocardial injury by affecting the phosphorylation states of proteins in PI3K/Akt/mTOR signaling pathway

  Background: The effect of sevoflurane on the doxorubicin-induced myocardial injury was explored by investigating the phosphorylation states of proteins in phosphatidylinositol 3-kinase (PI3K)/Akt/mam­malian target of rapamycin (mTOR) signaling pathway. Methods: Myocardial injury rat models were induced by doxorubicin and evenly assigned into five groups according to different treatment: Doxorubicin group (DG, 200-μL saline solution), sevoflurane group (SevG, inhaled with 2.4% sevoflurane for 2 h), LY294002 group (LYG, Akt inhibitor, 0.3 mg/kg in 200-μL Dimethyl Sulfoxide [DMSO]), solvent DMSO control group (SG) and autophagy inhibitor 3-methyladenine (3-MA) group (MG, 30 mg/kg in 200-μL DMSO). The healthy rats were assigned to a contro1 group (CG, 200-μL saline solution). Myocardial apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The concentration of cardiac troponin I (cTnI) was detected by ELISA. The levels of total Akt (t-Akt), phosphorylated Akt (p-Akt), mammalian target of rapamycin (mTOR), phosphorylated-mTOR (p-mTOR) and autophagy marker LC3-II was detected by Western Blot. The experiments were also repeated at the cell level. Results: Terminal deoxynucleotidyl transferase dUTP nick end labeling analysis showed that the ap­optosis rates were high in DG and SG, reached the highest level in LYG, reduced in SevG and MG, and reached the lowest level in CG. The levels of p-Akt p-mTOR were low in groups DG and SG, reached the lowest level in LYG, increased in SevG and MG, and reached the highest level in CG. In contrast, LC3-II expression, apoptosis index and serum cTnI concentration were high in DG and SG, reached the highest level in LYG, reduced in SevG and MG, and reached the lowest level in CG (p < 0.05). Cell experiment showed similar results as with animal experiments. Conclusions: Sevoflurane ameliorates myocardial injury by affecting the phosphorylation states of the proteins in PI3K/Akt/mTOR signaling pathway and reducing the injury biomarker. (Cardiol J 2017; 24, 4: 409–418

Epigenetic regulation of CD271, a potential cancer stem cell marker associated with chemoresistance and metastatic capacity.

Cancer stem cells (CSCs) are considered to be the cause of tumor initiation, metastasis and recurrence. Additionally, CSCs are responsible for the failure of chemotherapy and radiotherapy. The isolation and identification of CSCs is crucial for facilitating the monitoring, therapy or prevention of cancer. We aimed to identify esophageal squamous cell carcinoma (ESCC) stem-like cells, the epigenetic mechanism and identify novel biomarkers for targeting ESCC CSCs. Sixty-three paired ESCC tissues and adjacent non-cancerous tissues were included in this study. CD271, which was identified as the CSC marker for melanoma, was assessed using quantitative PCR (qPCR). Using flow cytometry, we isolated CD271+ cells comprising 7.5% of cancer cells from the KYSE70 cell line. Sphere formation and anchorage-independent growth were analyzed in CD271+ and CD271- cancer cells, respectively. qPCR was used to detect stem-related genes and CCK-8 was performed to analyze the sensitivity to chemotherapy in the two groups. Bisulfite genomic sequencing was used to analyze the methylation status. CD271 expression was significantly higher in ESCC tissues than in adjacent non-cancerous tissues. Compared with CD271- cancer cells, CD271+ cancer cells showed a higher ability of sphere and colony formation, a high level expression of stem-related gene, and resistance to chemotherapy. The expression of CD271 was induced by a demethylation agent. In conclusion, CD271+ ESCC cells possess stem-like properties. CD271 can potentially act as a prognostic marker for ESCC, whose expression is regulated epigenetically

Hair cluster detection model based on dermoscopic images

Introduction: Hair loss has always bothered many people, with numerous individuals potentially facing the issue of sparse hair.Methods: Due to a scarcity of accurate research on detecting sparse hair, this paper proposes a sparse hair cluster detection model based on improved object detection neural network and medical images of sparse hair under dermatoscope to optimize the evaluation of treatment outcomes for hair loss patients. A new Multi-Level Feature Fusion Module is designed to extract and fuse features at different levels. Additionally, a new Channel-Space Dual Attention Module is proposed to consider both channel and spatial dimensions simultaneously, thereby further enhancing the model’s representational capacity and the precision of sparse hair cluster detection.Results: After testing on self-annotated data, the proposed method is proven capable of accurately identifying and counting sparse hair clusters, surpassing existing methods in terms of accuracy and efficiency.Discussion: Therefore, it can work as an effective tool for early detection and treatment of sparse hair, and offer greater convenience for medical professionals in diagnosis and treatment

Structural polymorphisms within a common powdery mildew effector scaffold as a driver of coevolution with cereal immune receptors

In plants, host-pathogen coevolution often manifests in reciprocal, adaptive genetic changes through variations in host nucleotide-binding leucine-rich repeat immune receptors (NLRs) and virulence-promoting pathogen effectors. In grass powdery mildew (PM) fungi, an extreme expansion of a RNase-like effector family, termed RALPH, dominates the effector repertoire, with some members recognized as avirulence (AVR) effectors by cereal NLR receptors. We report the structures of the sequence-unrelated barley PM effectors AVR$_{A6}$, AVR$_{A7}$, and allelic AVR$_{A10}$/AVR$_{A22}$ variants, which are detected by highly sequence-related barley NLRs MLA6, MLA7, MLA10, and MLA22 and of wheat PM AVR$_{PM2}$ detected by the unrelated wheat NLR PM2. The AVR effectors adopt a common scaffold, which is shared with the RNase T1/F1 family. We found striking variations in the number, position, and length of individual structural elements between RALPH AVRs, which is associated with a differentiation of RALPH effector subfamilies. We show that all RALPH AVRs tested have lost nuclease and synthetase activities of the RNase T1/F1 family and lack significant binding to RNA, implying that their virulence activities are associated with neo-functionalization events. Structure-guided mutagenesis identified six AVR$_{A6}$ residues that are sufficient to turn a sequence-diverged member of the same RALPH subfamily into an effector specifically detected by MLA6. Similar structure-guided information for AVR$_{A10}$ and AVR$_{A22}$ indicates that MLA receptors detect largely distinct effector surface patches. Thus, coupling of sequence and structural polymorphisms within the RALPH scaffold of PMs facilitated escape from NLR recognition and potential acquisition of diverse virulence functions

Neutralization of IL- 10 produced by B cells promotes protective immunity during persistent HCV infection in humanized mice

Chronic HCV infection can lead to cirrhosis and is associated with increased mortality. Interleukin (IL)- 10- producing B cells (B10 cells) are regulatory cells that suppress cellular immune responses. Here, we aimed to determine whether HCV induces B10 cells and assess the roles of the B10 cells during HCV infection. HCV- induced B10 cells were enriched in CD19hi and CD1dhiCD5+ cell populations. HCV predominantly triggered the TLR2- MyD88- NF- κB and AP- 1 signaling pathways to drive IL- 10 production by B cells. In a humanized murine model of persistent HCV infection, to neutralize IL- 10 produced by B10 cells, mice were treated with pcCD19scFv- IL- 10R, which contains the genes coding the anti- CD19 single- chain variable fragment (CD19scFv) and the extracellular domain of IL- 10 receptor alpha chain (sIL- 10Ra). This treatment resulted in significant reduction of B10 cells in spleen and liver, increase of cytotoxic CD8+ T- cell responses against HCV, and low viral loads in infected humanized mice. Our results indicate that targeting B10 cells via neutralization of IL- 10 may offer a novel strategy to enhance anti- HCV immunotherapy.HCV predominantly triggers the TLR2- MyD88- NF- κB and AP- 1 signaling pathways to drive IL- 10 production by B cells. Neutralization of IL- 10 produced by B10 cells promotes anti- HCV immunity in a humanized murine model of persistent HCV infection. These results provide insight into a novel immunotherapy strategy for HCV treatment.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162732/2/eji4736.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162732/1/eji4736_am.pd

Perioperative dynamic alterations in peripheral regulatory T and B cells in patients with hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>Intratumoral and circulating regulatory T cells (Tregs) have been shown to be critical in the pathogenesis of hepatocellular carcinoma (HCC). However there is limited knowledge on the alterations of regulatory B cells (Bregs). We here investigated perioperative dynamic alterations of peripheral circulating Tregs and Bregs in HCC patients to reveal the relationship between regulatory lymphocytes and its clinical implications.</p> <p>Methods</p> <p>36 patients with HCC, 6 with chronic hepatitis B infection and 10 healthy donors were enrolled for this study. Frequencies of peripheral Tregs and Bregs were measured by flow cytometry with antibodies against CD4, CD25, CD127, CD19 and IL-10 before, and after radical surgery. Then, clinical informatics of HCC patients was achieved through Digital Evaluation Score System (DESS) for the assessment of disease severity. Finally, we analysed correlations between digitalized clinical features and kinetics of circulating regulatory lymphocytes.</p> <p>Results</p> <p>Level of circulating CD4⁺CD25⁺CD127^-Tregs in HCC patients was significantly lower than that in healthy donors and patients with chronic hepatitis B infection before surgery, but was increased after surgery. Preoperative level of CD19⁺IL-10⁺Bregs in HCC patients was also significantly lower than the other groups. However it dramatically was elevated right after surgery and remained elevated compared to controls (about 7 days after surgery, <it>P </it>= 0.04). Frequency of circulating Tregs was correlated with circulating leukocytes, ferritin, and clinical features suggesting tumor aggressiveness including portal vein thrombosis, hepatic vein involvement and advanced clinical stages. Frequency of circulating Bregs was associated with Hepatitis B e Antigen (HBeAg) and Hepatitis B virus (HBV) DNA copy number. In addition, DESS was significantly and positively correlated with other staging systems.</p> <p>Conclusion</p> <p>Frequencies of peripheral Tregs and Bregs in HCC patients increased after surgery. These results suggest that a postoperative combination of therapies against Tregs and Bregs may be beneficial for better outcome of HCC patients after resection.</p

Compressed glassy carbon: An ultrastrong and elastic interpenetrating graphene network

Carbon’s unique ability to have both sp2 and sp3 bonding states gives rise to a range of physical attributes, including excellent mechanical and electrical properties. We show that a series of lightweight, ultrastrong, hard, elastic, and conductive carbons are recovered after compressing sp2-hybridized glassy carbon at various temperatures. Compression induces the local buckling of graphene sheets through sp3 nodes to form interpenetrating graphene networks with long-range disorder and short-range order on the nanometer scale. The compressed glassy carbons have extraordinary specific compressive strengths—more than two times that of commonly used ceramics—and simultaneously exhibit robust elastic recovery in response to local deformations. This type of carbon is an optimal ultralight, ultrastrong material for a wide range of multifunctional applications, and the synthesis methodology demonstrates potential to access entirely new metastable materials with exceptional properties