Mutation of the core pentamer, CCGAC, of two putative low temperature responsive elements (LTREs) in the 5'-proximal region for the winter Brassica napus cold-induced gene BN115 was carried out. Analyses of transient expression of the resultant mutated BN115 promoter-GUS fusions revealed the loss of low-temperature regulation by the promoter. This indicates that the CCGAC sequence is critical to the low-temperature response in the BN115 gene. In contrast, mutation of two G-boxes, CACGTG, staggered between the LTREs in the same region of the promoter did not alter cold-inducible gene expression. Replacement of a possible enhancer region of the BN115 promoter with the enhancer from the CaMV 35S promoter resulted in a several-fold increase in low temperature-induced GUS activity.

Cell-surface pattern recognition receptors (PRRs) are key components of the innate immune response in animals and plants. These receptors typically carry or associate with non-RD kinases to control early events of innate immunity signaling. Despite their importance, the mode of regulation of PRRs is largely unknown. Here we show that the rice PRR, XA21, interacts with XA21 binding protein 24 (XB24), a previously undescribed ATPase. XB24 promotes autophosphorylation of XA21 through its ATPase activity. Rice lines silenced for Xb24 display enhanced XA21-mediated immunity, whereas rice lines overexpressing XB24 are compromised for immunity. XB24 ATPase enzyme activity is required for XB24 function. XA21 is degraded in the presence of the pathogen-associated molecular pattern Ax21 when XB24 is overexpressed. These results demonstrate a function for this large class of broadly conserved ATPases in PRR-mediated immunity.non-RD kinase | pathogen-associated molecular pattern | pattern recognition receptor | ATPase | rice I nnate immunity is the first line of defense against pathogen attack and is activated rapidly following infection. In contrast to the adaptive immune system that depends on somatic gene rearrangements for the generation of antigen receptors with random specificities, the innate immune system uses a set of defined receptors for recognition of pathogen-associated molecular patterns (PAMPs) (1).Cell-surface pattern recognition receptors (PRRs) are key components of the innate immune response in animals and plants (2-8).In animals, recognition of PAMPs at the cell surface is largely carried out by the Toll-like receptor (TLR) family that contains leucine-rich repeats (LRRs) in the extracellular domain and a Toll-interleukin receptor (TIR) intracellular domain (9). Although TLRs recognize diverse molecules, they activate a common signaling pathway via association with non-RD (arginine-aspartic acid) kinases to induce a core set of defense responses (10, 11). For example, TLRs 1, 3, 5, 6, 7, 8, and 9 function through interleukin-1 receptor-associated kinase (IRAK1), whereas TLR3 and TLR4 function through receptor interacting-protein 1 (RIP1). Both IRAK1 and RIP1 are non-RD kinases (10). In plants, cell-surface recognition of PAMPs is carried out by PRRs that carry non-RD IRAK kinases integral to the receptor (ca. 35 in Arabidopsis and 328 in rice) (10). For example, rice XA21, XA26, Arabidopsis flagellin sensitive 2 (FLS2), elongation factor-Tu receptor (EFR), and barley Rpg1 (conferring stem rust-resistance) all contain an intracellular non-RD Ser/Thr kinase (8,(12)(13)(14)(15).Non-RD kinases typically carry a cysteine or glycine (in place of the arginine) before the catalytic aspartate residue and control early events of innate immunity signaling in animals and plants (16). Whereas RD kinases are regulated by autophosphorylation of the activation segment-a centrally located loop that sits close to the catalytic center-non-RD kinases are not autophosphorylated in the activation segment (17). These results s...

Receptor-like kinases (RLKs)3 play important roles in many biological processes, including development and immunity responses, in both animals and plants. Most RLKs possess intrinsic protein kinase activity and regulate downstream signaling through phosphorylation and dephosphorylation events (1-5). Kinases are classified as arginine-aspartate (RD) or non-RD kinases. RD kinases carry a conserved arginine (Arg) immediately preceding the catalytic aspartate (Asp) (6). RD kinases are regulated by autophosphorylation of the activation segment, a centrally located loop positioned close to the catalytic center.In contrast to RD kinases, non-RD kinases typically carry a cysteine or glycine in place of the arginine. We have previously reported that non-RD kinases are associated with the control of early signaling events in both plant and animal innate immunity (7). For example, in humans, non-RD kinases associate with proteins belonging to the Toll-like receptor (TLR) family, which contain leucine-rich repeats in the extracellular domain and Toll-interleukin receptor intracellular domains (8). TLRs recognize pathogen-associated molecular patterns at the cell surface and then activate a common signaling pathway through an association with non-RD kinases to induce a core set of defense responses (9). TLR1, TLR3, TLR5, TLR6, TLR7, TLR8, and TLR9 function through the non-RD interleukin-1 receptor-associated kinase (IRAK1), whereas TLR3 and TLR4 function through the non-RD receptor interacting-protein 1 (10). In plants, RLKs demonstrated to function in mediating innate immunity fall into the non-RD class (10) or are associated with non-RD RLKs (11-13). These include the Arabidopsis pattern recognition receptors (PRRs), flagellin sensitive 2 (FLS2) (14) and elongation factor-Tu receptor (15), the rice PRRs, such as XA21 (16), XA26 (17), and Pid2 (or called Pi-d2) (18), the barley PRG1 (resistance to Puccinia graminis f. sp. tritici) (19), and the Arabidposis RD RLK BAK1 that associates with the non-RD RLK FLS2 (11). Unlike RD kinases, non-RD kinases do not autophosphorylate their activation segments (6,10,20). Given the demonstrated importance of the non-RD class of kinases in innate immunity, there is great interest in understanding their mode of action.RLKs contain a juxtamembrane (JM) domain located between the transmembrane and kinase domains. It is now clear that the JM domain can play an important role in regulating the function of kinase. For example, deletion of the JM domain of the epidermal growth factor (ErbB-1) kinase (an RD RLK) results in a severe loss of tyrosine phosphorylation (1). Two conserved tyrosine phosphorylation sites Tyr 605 and Tyr 611 of EphB2 are essential for EphB2 kinase autophosphorylation and biological responses (21,22). Phosphorylation of the JM domain of the type I transforming growth factor-␤ (T␤R-I, an RD RLK) eliminates the binding site for the FKBP12 (12-kDa FK506-binding protein) inhibitor protein, leading to activation of the T␤R-I kinase (23, 24). To date, the role of the

Purpose: Given that heterogeneous expression and variants of antigens on solid tumors are responsible for relapse after chimeric antigen receptor (CAR)-T cell therapy, we hypothesized that combinatorial targeting two tumor-associated antigens would lessen this problem and enhance the antitumor activity of T cells. Methods: The co-expression level of CD70 and B7-H3 was analyzed in multiple tumor tissue samples. Further, two putative antigens were identified in The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis database. Two CD70 targeted CARs with different antigen binding domain, truncated CD27 and CD70 specific single-chain antibody fragment (scFv), were designed to screen a more suitable target-antigen binding moiety. Accordingly, we designed a bivalent tandem CAR (TanCAR) and further assessed the anti-tumor efficacy of TanCAR-T cells in vitro and in vivo . Results: Our results indicated that co-expression of CD70 and B7-H3 was observed on multiple tumor types including kidney, breast, esophageal, liver, colon cancer, glioma as well as melanoma. The CD70 targeted CAR-T cells with binding moiety of CD70 specific scFv exhibit a higher affinity and antitumor effect against CD70 + tumor cells. TanCAR-T cells induced enhanced ability of cytolysis and cytokine release over unispecific CAR-T cells when encountering tumor cells expressing two target-antigens. Further, low doses of TanCAR-T cells could also effectively control the lung cancer and melanoma xenografts and improved overall survival of the treated animals. Conclusion: TanCAR-T cells targeting CD70 and B7-H3 exhibit enhanced antitumor functionality and improve the problem of antigenic heterogeneity and variant in the treatment against solid tumor and melanoma.

BackgroundOccupational exposure to chromium compounds may result in adverse health effects. This study aims to investigate whether low-level hexavalent chromium (Cr(VI)) exposure can cause DNA damage in electroplating workers.Methods157 electroplating workers and 93 control subjects with no history of occupational exposure to chromium were recruited in Hangzhou, China. Chromium levels in erythrocytes were determined by graphite furnace atomic absorption spectrophotometer. DNA damage in peripheral lymphocytes was evaluated with the alkaline comet assay by three parameters: Olive tail moment, tail length and percent of DNA in the comet tail (tail DNA%). Urinary 8-OHdG levels were measured by ELISA.ResultsChromium concentration in erythrocytes was about two times higher in electroplating workers (median: 4.41 μg/L) than that in control subjects (1.54 μg/L, P < 0.001). The medians (range) of Olive tail moment, tail length and tail DNA% in exposed workers were 1.13 (0.14-6.77), 11.17 (3.46-52.19) and 3.69 (0.65-16.20), and were significantly higher than those in control subjects (0.14 (0.01-0.39), 3.26 (3.00-4.00) and 0.69 (0.04-2.74), P < 0.001). Urinary 8-OHdG concentration was 13.65 (3.08-66.30) μg/g creatinine in exposed workers and 8.31 (2.94-30.83) μg/g creatinine in control subjects (P < 0.001). The differences of urinary 8-OHdG levels, Olive tail moment, tail length and tail DNA% between these two groups remained significant (P < 0.001) even after stratification by potential confounding factors such as age, gender, and smoking status. Chromium exposure was found to be positively associated with chromium levels in erythrocytes, urinary 8-OHdG levels, Olive tail moment, tail length and tail DNA%. Positive dose-response associations were also found between chromium levels in erythrocytes and Olive tail moment, tail length and tail DNA%.ConclusionThe findings in this study indicated that there was detectable chromium exposure in electroplating workers. Low-level occupational chromium exposure induced DNA damage.

Lysine acetylation in proteins is a dynamic and reversible PTM and plays an important role in diverse cellular processes. In this study, using lysine-acetylation (Kac) peptide enrichment coupled with nano HPLC/MS/MS, we initially identified the acetylome in the silkworms. Overall, a total of 342 acetylated proteins with 667 Kac sites were identified in silkworm. Sequence motifs analysis around Kac sites revealed an enrichment of Y, F, and H in the +1 position, and F was also enriched in the +2 and -2 positions, indicating the presences of preferred amino acids around Kac sites in the silkworm. Functional analysis showed the acetylated proteins were primarily involved in some specific biological processes. Furthermore, lots of nutrient-storage proteins, such as apolipophorin, vitellogenin, storage proteins, and 30 K proteins, were highly acetylated, indicating lysine acetylation may represent a common regulatory mechanism of nutrient utilization in the silkworm. Interestingly, Ser2 proteins, the coating proteins of larval silk, were found to contain many Kac sites, suggesting lysine acetylation may be involved in the regulation of larval silk synthesis. This study is the first to identify the acetylome in a lepidoptera insect, and expands greatly the catalog of lysine acetylation substrates and sites in insects.

Recently, B7-H3 was frequently reported to be overexpressed in various cancer types and has been suggested to be a promising target for cancer immunotherapy. In the present study, we analyzed the mRNA expression of B7-H3 in The Cancer Genome Atlas (TCGA) database and validated its expression across multiple cancer types. We then generated a novel B7-H3-targeted chimeric antigen receptor (CAR) and tested its antitumor activity both in vitro and in vivo. The B7-H3 expression heterogeneity and variation were frequent. Moderate or even high expression levels of B7-H3 were also observed in some tumor-adjacent tissues, but the staining intensity was weaker than that in tumor tissues. B7-H3 expression was absent or very low in normal tissues and organs. Flow cytometry indicated that the mean expression level of B7-H3 in eight bone marrow specimens from patients with acute myeloid leukemia (AML) was 57.2% (range 38.8-80.4). Furthermore, we showed that the B7-H3-targeted CAR-T cells exhibited significant antitumor activity against AML and melanoma in vitro and in xenograft mouse models. In conclusion, although B7-H3 represents a promising pan-cancer target, and B7-H3-redirected CAR-T cells can effectively control tumor growth, the expression heterogeneity and variation have to be carefully considered in translating B7-H3-targeted CAR-T cell therapy into clinical practice.

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