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Background: Asthma is a chronic inflammatory disease characterized by airway inflammation and airway hyperresponsiveness (AHR). IL-33 is considered as one of the most critical molecules in asthma pathogenesis. IL-33 is stored in nucleus and passively released during necrosis. But little is known about whether living cells can release IL-33 and how this process is regulated. Objective: We sought to investigate the role of polymerase I and transcript release factor (PTRF) in IL-33 release and asthma pathogenesis. Methods: Ovalbumin (OVA)-induced asthma model in PTRF+/− mice were employed to dissect the role of PTRF in vivo. Then, further in vitro experiments were carried out to unwind the potential mechanism involved. Results: In OVA asthma model with challenge phase, PTRF+/− mice showed a greater airway hyper-reaction, with an intense airway inflammation and more eosinophils in bronchoalveolar lavage fluid (BALF). Consistently, more acute type 2 immune response in lung and a higher IL-33 level in BALF were found in PTRF+/− mice. In OVA asthma model without challenge phase, airway inflammation and local type 2 immune responses were comparable between control mice and PTRF+/− mice. Knockdown of PTRF in 16HBE led to a significantly increased level of IL-33 in cell culture supernatants in response to LPS or HDM. Immunoprecipitation assay clarified Y158 as the major phosphorylation site of PTRF, which was also critical for the interaction of IL-33 and PTRF. Overexpression of dephosphorylated mutant Y158F of PTRF sequestered IL-33 in nucleus together with PTRF and limited IL-33 extracellular secretion. Conclusion: Partial loss of PTRF led to a greater AHR and potent type 2 immune responses during challenge phase of asthma model, without influencing the sensitization phase. PTRF phosphorylation status determined subcellular location of PTRF and, therefore, regulated IL-33 release.

FOXP3+ Regulatory T (Treg) cells play a key role in the maintenance of immune homeostasis and tolerance. Disruption of Foxp3 expression results in the generation of instable Treg cells and acquisition of effector T-cell-like function. Here we report that the E3 deubiquitinase USP21 prevents the depletion of FOXP3 at the protein level and restricts the generation of T-helper-1-like Treg cells. Mice depleted of Usp21 specifically in Treg cells display immune disorders characterized by spontaneous T-cell activation and excessive T-helper type 1 (Th1) skewing of Treg cells into Th1-like Treg cells. USP21 stabilizes FOXP3 protein by mediating its deubiquitination and maintains the expression of Treg signature genes. Our results demonstrate how USP21 prevents FOXP3 protein depletion and controls Treg lineage stability in vivo., The immunosuppressive role of regulatory T (Treg) cells largely depends on their virtue of expressing the transcription factor FOXP3. Here the authors show that the E3 deubiquitinase USP21 stabilizes FOXP3 by mediating its deubiquitination and helps to maintain the expression of Treg signature genes and Treg lineage stability in mice.

The heterogeneity and plasticity of T lymphocytes is critical for determining immune response outcomes. Functional regulatory T (Treg) cells are commonly characterized by stable FOXP3 expression and have reported to exhibit heterogeneous phenotypes under inflammatory conditions. However, the interplay between inflammation and Treg cell suppressive activity still remains elusive. Here, we utilized single-cell RNA sequencing to investigate how human Treg cells respond to the pro-inflammatory cytokine interleukin-6 (IL-6). We observed that Treg cells divided into two subpopulations after IL-6 stimulation. TIGIT− unstable Treg cells lost FOXP3 expression and gained an effector-like T cell phenotype, whereas TIGIT+ Treg cells retained robust suppressive function. Single cell transcriptome analysis revealed a spectrum of cellular states of IL-6-stimulated Treg cells and how cytochrome P450 family 1 subfamily A member 1 (CYP1A1) is a crucial regulator of Treg cell suppressive capability and stability. CYP1A1-deficient human Treg cells developed a Th17-like phenotype after IL-6 stimulation. Our findings implicate CYP1A1 as a previously unidentified regulator of Treg cells that may have target potential for clinical application for biotherapies.

The CD4(+)CD25(+)FOXP3(+) regulatory T (Treg) cells are critical for maintaining immune tolerance in healthy individuals and are reported to restrict anti-inflammatory responses and thereby promote tumor progression, suggesting them as a target in the development of antitumor immunotherapy. Forkhead box P3 (FOXP3) is a key transcription factor governing Treg lineage differentiation and their immune-suppressive function. Here, using Treg cells, as well as HEK-293T and Jurkat T cells, we report that the stability of FOXP3 is directly and positively regulated by the E3 ubiquitin ligase ring finger protein 31 (RNF31), which catalyzes the conjugation of atypical ubiquitin chains to the FOXP3 protein. We observed that shRNA-mediated RNF31 knockdown in human Treg cells decreases FOXP3 protein levels and increases levels of interferon-γ, resulting in a Th1 helper cell–like phenotype. Human Treg cells that ectopically expressed RNF31 displayed stronger immune-suppressive capacity, suggesting that RNF31 positively regulates both FOXP3 stability and Treg cell function. Moreover, we found that RNF31 is up-regulated in Treg cells that infiltrate human gastric tumor tissues compared with their counterparts residing in peripheral and normal tissue. We also found that elevated RNF31 expression in intratumoral Treg cells is associated with poor survival of gastric cancer patients, suggesting that RNF31 supports the immune-suppressive functions of Treg cells. Our results suggest that RNF31 could be a potential therapeutic target in immunity-based interventions against human gastric cancer.

Regulatory T cells (Treg cells) are essential for maintaining immune tolerance, and the dysfunction of Treg cells may cause autoimmune diseases and tumors. Forkhead box P3 (FOXP3) is the key transcription factor controlling Treg cell development and suppressive function. Mouse double minute 2 homolog (MDM2), an E3 ubiquitin ligase, has been identified as an oncoprotein that mediates the ubiquitination and degradation of tumor suppressor p53; however, whether it has functions in Treg cells remains unknown. Here, we demonstrate that MDM2 positively regulates human Treg cell suppressive function via its mediated ubiquitination and stabilization of FOXP3. Knockdown of MDM2 with shRNA in human primary Treg cells leads to the impaired ability of FOXP3 to regulate the expression levels of downstream genes and the attenuated suppressive capacity of Treg cells, due to FOXP3 instability. Consistently, MDM2 overexpression in human Treg cells enhances FOXP3 stability and Treg cell suppressive capacity. Mechanistically, MDM2 interacts with FOXP3, and mainly mediates monoubiquitination and polyubiquitination of FOXP3, thus stabilizing the protein level of FOXP3. We have also found lysine residues in FOXP3 required for MDM2-mediated ubiquitination. In addition, TCR/CD28 signaling upregulates the expression level of MDM2 and its mediated FOXP3 ubiquitination in human Treg cells. Therefore, our findings reveal that MDM2 in Treg cells could be a potential therapeutic target for treating autoimmune diseases and tumors.

Regulatory T cells (Tregs) are a subpopulation of T cells that are specialized in suppressing immune responses. Here we show that the arginine methyl transferase protein PRMT5 can complex with FOXP3 transcription factors in Tregs. Mice with conditional knock out (cKO) of PRMT5 expression in Tregs develop severe scurfy-like autoimmunity. In these PRMT5 cKO mice, the spleen has reduced numbers of Tregs, but normal numbers of Tregs are found in the peripheral lymph nodes. These peripheral Tregs that lack PRMT5, however, display a limited suppressive function. Mass spectrometric analysis showed that FOXP3 can be di-methylated at positions R27, R51, and R146. A point mutation of Arginine (R) 51 to Lysine (K) led to defective suppressive functions in human CD4 T cells. Pharmacological inhibition of PRMT5 by DS-437 also reduced human Treg functions and inhibited the methylation of FOXP3. In addition, DS-437 significantly enhanced the anti-tumor effects of anti-erbB2/neu monoclonal antibody targeted therapy in Balb/c mice bearing CT26Her2 tumors by inhibiting Treg function and induction of tumor immunity. Controlling PRMT5 activity is a promising strategy for cancer therapy in situations where host immunity against tumors is attenuated in a FOXP3 dependent manner.

Aiming at the problem of low accuracy, high computational complexity and incomplete edge information of most image splicing localization algorithm, this paper proposes a new image splicing localization algorithm. First, the SLIC image segmentation algorithm is used to segment the image. Secondly, the noise estimation value of each super-pixel block is calculated by the FAST noise estimation algorithm. Then, weight of each image block is calculated through noise and image features. Finally, the noise value sequence is processed by clustering and statistical processing to determine the pixels of the background area and the splicing area, thus the splicing area is located. In this paper, the algorithm is tested on the color image database of Columbia, and compared with the existing image splicing localization algorithms based on block-segmentation and based on pixel. The experiment shows that the proposed algorithm can preserve the connection between image features, hold the edge of the splicing area, and effectively improve the efficiency of localization detection under the premise of ensuring the accuracy of image splicing localization.

Tissue-resident immune cells play critical roles in regulating tissue function and homeostasis. Obesity-associated visceral adipose tissue inflammation is attributed to the accumulation of M1 macrophages which produce inflammatory cytokines like TNF-α, IL-6, and expansion of effector T cells like Th1 cells, CD8

T cells play a critical role to defend against tumor and maintain immune homeostasis. The diverse functions of T cells require precise regulation of metabolic pathways. Recent studies reveal that metabolic changes are tightly linked to the activation and function of T cells. Given the importance of these cells in tumor progression, it is important to understand how the tumor microenvironment regulates metabolism of T cells and how the metabolic reprogramming of T cells affects tumor growth. Here, we review new findings and discuss how metabolic reprogramming of different types of T cells affects the immune response in tumors.

Tissue-resident immune cells play critical roles in regulating tissue function and homeostasis. Obesity-associated visceral adipose tissue inflammation is attributed to the accumulation of M1 macrophages which produce inflammatory cytokines like TNF-α, IL-6, and expansion of effector T cells like Th1 cells, CD8+ cytotoxic T cells which produce interferon-γ to further add to the severity of inflammation in the visceral adipose tissue. Regulatory T cells have been reported to exert key roles in suppressing inflammation, thus maintaining the homeostasis of immune responses, and visceral adipose Tregs exert critical roles in defending against obesity-associated metabolic disorders. They inhibit the infiltration of effector T cells and facilitate the reconstitution of adipose tissue macrophages from M1 to M2 phenotype. What is more, they can take up lipids from the adipocytes through CD36 which is driven by PPARγ. Here we review the recent progress in adipose tissue-resident regulatory T cells (Tregs), a subpopulation of CD4+ T cells which suppress adipose tissue inflammation.

Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10–100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery.

Thrombolytic agents play an important role in the treatment of thrombus diseases. In clinic, traditional thrombolytic agents always resulted in the side effect of systemic thrombolysis because of the shortcomings, such as short half-life, poor specificity of thrombus. Recently, with the development of knowledge of the relationship between their structure and function, a series of new thrombolytic agents were constructed by means of molecular biological techniques. Developing new types of thrombolytic agents is of great significance for more effective treatment of cardiovascular diseases. The latest knowledge of the structure-function relationship of t-PA and u-PA and the progress in development of new thrombolytic agents are reviewed.

This paper presents a novel algorithm named Double-layer Multi-expression Programming (DMEP). Then DMEP model is applied to the prediction of 28-day Portland cement compressive strength. We compare DMEP model with other four soft computing models, namely Multi-Expression Programming model (MEP), Gene Expression Programming model (GEP), Neural Networks model (NN) and Fuzzy logic model (FL) on 28-day cement strength prediction. The experiment results show that DMEP model has a lower rate in RMSE and MAE, and the prediction average percentage error of the cement strength data using DMEP model is reduced to 2.18%, which is lower than other four models. The results obtained from the computational tests demonstrate that DMEP is a promising technique for the prediction of cement strength.

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.

By exploring Ssp DnaE intein-catalyzed protein trans-splicing we investigated the ligation of expression product of ATP-binding cassette transporter A1 (ABCA1) gene in E. coli.The ABCA1 cDNA was broken into two halves of N-part and C-part before Cys978 codon which meets the splicing required conserved residue, and then fused to 123 and 36 amino acid-containing N terminal and C terminal coding sequences of Ssp DnaE intein naturally occurring trans-splicing intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pET-28a (+). After transformation into E. coli BL21 (DE3) cells followed by induction the expression of recombinant proteins and the ligation of ABCA1 were observed.Through IPTG induction for expression of recombinant protein it displayed an obvious protein band as predicted size of ABCA1 on SDS-PAGE gel. Western blotting using His-Tag specific antibody confirmed that this protein band is trans-spliced ABCA1.The data demonstrated that Ssp DnaE intein can efficiently catalyze the ligation of ABCA1 providing an evidence for our ongoing study on ABCA1 gene transfer by a dual AAV vector system to circumvent AAV volume limitation in gene therapy of Tangier disease which resulted from ABCA1 gene mutations.

Most studies regarding the role of epidermal growth factor (EGF) receptor (EGFR) C-terminal domain in EGFR internalization are done in the context of EGFR kinase activation. We recently showed that EGF-induced EGFR internalization is directly controlled by receptor dimerization, rather than kinase activation. Here we studied the role of EGFR C-terminus in EGF-induced EGFR internalization with or without EGFR kinase activation. We showed that graduate truncation of EGFR from C-terminus to 1044 did not affect EGF-induced EGFR endocytosis with or without kinase activation. However, truncation to 991 or further completely inhibited EGFR endocytosis. Graduate truncation within 991-1044 progressively lower EGF-induced EGFR endocytosis with most significant effects observed for residues 1005-1017. The endocytosis patterns of mutant EGFRs are independent of EGFR kinase activation. The residues 1005-1017 were also required for EGFR internalization triggered by non-ligand-induced receptor dimerization. This indicates that residues 1005-1017 function as an internalization motif, rather than a dimerization motif, to mediate EGFR internalization. Furthermore, we showed that the di-leucine motif 1010LL1011 within this region is essential in mediating EGF-induced rapid EGFR internalization independent of kinase activation. We conclude that EGFR C-terminal sequences 1005-1017 and the 1010LL1011 motif are essential for EGF-induced EGFR endoytosis independent of EGFR kinase activation and autophosphorylation.

Focusing of urokinase-type plasminogen activator (uPA) to the cell surface via binding to its specific receptor (uPAR, CD87) is critical for tumor invasion and metastasis. Consequently, the inhibition of uPA-uPAR interaction on the cell surface might be a promising anti-invasion and anti-metastasis strategy. We examined the effects of cDNA transfection of the human uPA amino-terminal fragment (ATF) on invasion and metastasis of cancer cells. First, a highly metastatic human lung giant-cell carcinoma cell line (PG), used as the target cell for evaluation of this effect, was demonstrated to express both uPA and uPAR. Then, ATF, which contains an intact uPAR binding site but is catalytically inactive, was designed as an antagonist of uPA-uPAR interaction and was transfected into PG cells. [(3)H]-Thymidine incorporation and cell growth curves indicated that expressed ATF did not affect the proliferation of transfected cells. However, analysis by scanning electron microscopy revealed that ATF changed the host cells from the typical invasive phenotype to a noninvasive one. Correspondingly, the modified Boyden chamber test in vitro showed that ATF expression significantly decreased the invasive capacity of transfected cells. Furthermore, in the spontaneous metastasis model, it was confirmed in vivo that expressed ATF remarkably inhibited lung metastasis of implanted ATF-transfected PG cells. In summary, autocrine ATF could act as an antagonist of uPA-uPAR interaction, and ATF cDNA transfection could efficiently inhibit the invasion and metastasis of the cancer cells. Inhibition of uPA-uPAR interaction on the cell surface might be a promising anti-invasion and anti-metastasis strategy.