PPNet: Pyramid pooling based network for polyp segmentation.

Colonoscopy is the gold standard method for investigating the gastrointestinal tract. Localizing the polyps in colonoscopy images plays a vital role when doing a colonoscopy screening, and it is also quite important for the following treatment, e.g., polyp resection. Many deep learning-based methods have been applied for solving the polyp segmentation issue. However, precisely polyp segmentation is still an open issue. Considering the effectiveness of the Pyramid Pooling Transformer (P2T) in modeling long-range dependencies and capturing robust contextual features, as well as the power of pyramid pooling in extracting features, we propose a pyramid pooling based network for polyp segmentation, namely PPNet. We first adopt the P2T as the encoder for extracting more powerful features. Next, a pyramid feature fusion module (PFFM) combining the channel attention scheme is utilized for learning a global contextual feature, in order to guide the information transition in the decoder branch. Aiming to enhance the effectiveness of PPNet on feature extraction during the decoder stage layer by layer, we introduce the memory-keeping pyramid pooling module (MPPM) into each side branch of the encoder, and transmit the corresponding feature to each lower-level side branch. Experimental results conducted on five public colorectal polyp segmentation datasets are given and discussed. Our method performs better compared with several state-of-the-art polyp extraction networks, which demonstrate the effectiveness of the mechanism of pyramid pooling for colorectal polyp segmentation.

Improved Method to Characterize Leaf Surfaces, Guide Adjuvant Selection, and Improve Glyphosate Efficacy.

Glyphosate, one of the most widely used herbicides, plays an important role in controlling weeds and ensuring crop production. While using glyphosate, adjuvants are commonly added to improve its deposition on weeds and control efficacy. However, changes in weed leaf surface characteristics may reduce glyphosate penetration and contribute to evolved glyphosate resistance. Therefore, it is significant to introduce an improved method for regularizing leaf surface characterization and guide adjuvant selection to improve glyphosate efficacy. In this work, surface characteristics of typical weed leaves have been systematically investigated by 3D surface analysis and scanning electron microscopy, finally quantified by apparent surface free energy (ASFE) due to its comprehensive and quantitative evaluation of leaf surfaces. Moreover, the relationship between the weed leaf surface characteristics and the retention of glyphosate on weeds was established, further related to the control efficacy against weeds. To maximize the utilization rate of glyphosate, the types and concentrations of adjuvants should be regulated according to the ASFE of weeds. Our findings not only regularize the surface properties of weed leaves but also reveal their influencing mechanism on the deposition and biological activity of glyphosate, which provide effective guidance for the use of glyphosate.

Improvement of PD-1 Blockade Efficacy and Elimination of Immune-Related Gastrointestinal Adverse Effect by mTOR Inhibitor.

During the past decades, immunotherapy, especially the antibody-mediated immune checkpoint blockade (ICB) has shown durable tumor inhibition and changed the paradigm of cancer treatment. However, a growing body of evidence suggests that ICB treatment induces severe immune-related adverse events (irAEs), and the side effect even leads to the discontinuation of lifesaving treatment. Here, we found that ICB treatment induces colitis in melanoma patients and promotes the infiltration of CD8+ effector T cells into colitic lesions. Further transcriptomic dissection indicated the PI3K-AKT-mTOR pathway was highly activated in CD8+ effector T cells of colitic lesions. Moreover, we developed a mouse melanoma model to recapitulate the gastrointestinal toxicity of anti-PD-1 treatment in clinical settings. Anti-PD-1 treatment significantly contributed to the infiltration of CD8+ T cells, and correspondingly induced severe enteritis. Immunohistochemistry experiments showed that the PI3K-AKT-mTOR pathway of T cells was activated by anti-PD-1 treatment. Blockade of the pathway with mTOR inhibitor sirolimus not only inhibits tumor growth but also suppresses the T cell infiltration in colitic lesions. More importantly, combination with sirolimus and anti-PD-1 synergistically inhibits tumor growth via inducing the immunogenic cell death of tumor cells in vivo. In summary, our research demonstrated the principle of mTOR inhibitor and anti-PD-1 combinatorial therapeutic regimen, which provided a novel therapeutic strategy for irAEs in clinics.

The Yersinia Type III secretion effector YopM Is an E3 ubiquitin ligase that induced necrotic cell death by targeting NLRP3.

Yersinia pestis uses type III effector proteins to target eukaryotic signaling systems. The Yersinia outer protein (Yop) M effector from the Y. pestis strain is a critical virulence determinant; however, its role in Y. pestis pathogenesis is just beginning to emerge. Here we first identify YopM as the structural mimic of the bacterial IpaH E3 ligase family in vitro, and establish that the conserved CLD motif in its N-terminal is responsible for the E3 ligase function. Furthermore, we show that NLRP3 is a novel target of the YopM protein. Specially, YopM associates with NLRP3, and its CLD ligase motif mediates the activating K63-linked ubiquitylation of NLRP3; as a result, YopM modulates NLRP3-mediated cell necrosis. Mutation of YopM E3 ligase motif dramatically reduces the ability of Y. pestis to induce HMGB1 release and cell necrosis, which ultimately contributes to bacterial virulence. In conclusion, this study has identified a previously unrecognized role for YopM E3 ligase activity in the regulation of host cell necrosis and plague pathogenesis.

Yersinia YopJ negatively regulates IRF3-mediated antibacterial response through disruption of STING-mediated cytosolic DNA signaling.

The Yersinia outer protein J (YopJ) plays a pivotal role in evading the host immune response and establishes a persistent infection in host cells after bacterial infection. YopJ is a cysteine protease and can act as a deubiquitinating enzyme that deubiquitinates several targets in multiple signaling pathways. Stimulator of interferon genes (STING) is a critical adapter for the induction of interferon regulatory factor 3 (IRF3) phosphorylation and subsequent production of the cytokines in response to nucleic acids in the cytoplasm. Our studies demonstrate that YopJ targets STING to inhibit IRF3 signaling. Specially, YopJ interacts with STING to block its ER-to-Golgi traffic and remove its K63-linked ubiquitination chains. Deubiquited STING perturbs the formation of STING-TBK1 complex and the activation of IRF3. The 172th cysteine of YopJ mediated STING deubiquitination and IRF3 signaling inhibition. Consequently, mice infected with WT and ΔYopJ/YopJ bacteria induced lower levels of IRF3 and IFN-ß, decreased inflammation and reduced staining of STING as compared to ΔYopJ and ΔYopJ/YopJ C172A strains infection. The data herein reveal a previously unrecognized mechanism by which YopJ modulates innate immune signaling.

Bacterial E3 Ubiquitin Ligase IpaH4.5 of Shigella flexneri Targets TBK1 To Dampen the Host Antibacterial Response.

IFN regulatory factors play a pivotal role in many cellular processes, including inflammatory and immune responses. Their activation is tightly regulated by TANK-binding kinase 1 (TBK1). In response to microbial components, TBK1 activates IFN regulatory factor 3 (IRF3) and cytokine expression. In this article, we show that TBK1 is a novel target of the IpaH4.5 protein, a Shigella type III effector possessing E3 ubiquitin ligase activity. Remarkably, IpaH4.5 interacts with TBK1 and promotes its K48-linked polyubiquitylation. Consequently, polyubiquitylated TBK1 undergoes proteasome-dependent degradation, which perturbs the phosphorylation, nuclear translocation, and activation of IRF3. Because IRF3 and TBK1 are required for restricting Shigella growth, we propose that the polyubiquitylation and degradation of TBK1 during Shigella infection are new bacterial strategies to modulate the host antibacterial responses.

miR-526a regulates apoptotic cell growth in human carcinoma cells.

MicroRNAs (miRNAs) play vital roles in the regulation of cell cycle, cell growth, apoptosis, and tumorigenesis. Our previous studies showed that miR-526a positively regulated innate immune response by suppressing CYLD expression, however, the functional relevance of miR-526a expression and cell growth remains to be evaluated. In this study, miR-526a overexpression was found to promote cancer cell proliferation, migration, and anchor-independent colony formation. The molecular mechanism(s) of miR-526a-mediated growth stimulation is associated with rapid cell cycle progression and inhibition of cell apoptosis by targeting CYLD. Taken together, these results provide evidence to show the stimulatory role of miR-526a in tumor migration and invasion through modulation of the canonical NF-κB signaling pathway.

MAVS Promotes Inflammasome Activation by Targeting ASC for K63-Linked Ubiquitination via the E3 Ligase TRAF3.

Stringent control of inflammasome signaling pathway is important for maintaining immunological balance, yet the molecular mechanisms responsible for its tight regulation are still poorly understood. In this study, we found that the signaling pathway dependent on mitochondrial antiviral signaling protein (MAVS) was required for the optimal activation of apoptosis-associated specklike protein (ASC)-dependent inflammasome. In particular, TNFR-associated factor 3 was found to be a direct E3 ligase for ASC. Ubiquitination of ASC at Lys(174) was critical for speck formation and inflammasome activation. Deficiency in MAVS or TNFR-associated factor 3 impaired ASC ubiquitination and cytosolic aggregates formation, resulting in reduced inflammasome response upon RNA virus infection. This study has identified a previously unrecognized role of MAVS in the regulation of inflammasome signaling and provided molecular insight into the mechanisms by which ubiquitination of ASC controls inflammasome activity through the formation of ASC specks.

Downregulation of microRNA miR-526a by enterovirus inhibits RIG-I-dependent innate immune response.

UNLABELLED: Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host-protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by inhibition of CYLD expression mediated by the microRNA miR-526a. We found that viral infection specifically upregulates miR-526a expression in macrophages via interferon regulatory factor (IRF)-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and suggest a novel mechanism for the evasion of the innate immune response controlled by EV71. IMPORTANCE: RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein; cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of the innate immune response controlled by EV71.

Overexpression of Mps1 in colon cancer cells attenuates the spindle assembly checkpoint and increases aneuploidy.

The spindle assembly checkpoint kinase Mps1 is highly expressed in several types of cancers, but its cellular involvement in tumorigenesis is less defined. Herein, we confirm that Mps1 is overexpressed in colon cancer tissues. Further, we find that forced expression of Mps1 in the colon cancer cell line SW480 enables cells to become resistant to both Mps1 inhibition-induced checkpoint depletion and cell death. Overexpression of Mps1 also increases genome instability in tumor cells owing to a weakened spindle assembly checkpoint. Collectively, our findings suggest that high levels of Mps1 contribute to tumorigenesis by attenuating the spindle assembly checkpoint.

Elevated expression of TANK-binding kinase 1 enhances tamoxifen resistance in breast cancer.

Resistance to antiestrogens is one of the major challenges in breast cancer treatment. Although phosphorylation of estrogen receptor α (ERα) is an important factor in endocrine resistance, the contributions of specific kinases in endocrine resistance are still not fully understood. Here, we report that an important innate immune response kinase, the IκB kinase-related TANK-binding kinase 1 (TBK1), is a crucial determinant of resistance to tamoxifen therapies. We show that TBK1 increases ERα transcriptional activity through phosphorylation modification of ERα at the Ser-305 site. Ectopic TBK1 expression impairs the responsiveness of breast cancer cells to tamoxifen. By studying the specimens from patients with breast cancer, we find a strong positive correlation of TBK1 with ERα, ERα Ser-305, and cyclin D1. Notably, patients with tumors highly expressing TBK1 respond poorly to tamoxifen treatment and show high potential for relapse. Therefore, our findings suggest that TBK1 contributes to tamoxifen resistance in breast cancer via phosphorylation modification of ERα.

MAVS regulates apoptotic cell death by decreasing K48-linked ubiquitination of voltage-dependent anion channel 1.

The mitochondrial antiviral signaling protein MAVS (IPS-1, VISA, or Cardif) plays an important role in the host defense against viral infection by inducing type I interferon. Recent reports have shown that MAVS is also critical for virus-induced apoptosis. However, the mechanism of MAVS-mediated apoptosis induction remains unclear. Here, we show that MAVS binds to voltage-dependent anion channel 1 (VDAC1) and induces apoptosis by caspase-3 activation, which is independent of its role in innate immunity. MAVS modulates VDAC1 protein stability by decreasing its degradative K48-linked ubiquitination. In addition, MAVS knockout mouse embryonic fibroblasts (MEFs) display reduced VDAC1 expression with a consequent reduction of the vesicular stomatitis virus (VSV)-induced apoptosis response. Notably, the upregulation of VDAC1 triggered by VSV infection is completely abolished in MAVS knockout MEFs. We thus identify VDAC1 as a target of MAVS and describe a novel mechanism of MAVS control of virus-induced apoptotic cell death.

Nalp3 inflammasome is activated and required for vascular smooth muscle cell calcification.

BACKGROUND: The calcification of blood vessels correlates with increased morbidity and mortality in patients with atherosclerosis, diabetes, and end-stage kidney disease. Increased inflammasome activation has been shown to play an important role in the pathogenesis of atherosclerosis. However, the contribution of inflammasome activation on the development of vascular calcification has not been investigated. METHODS: ß-Glycerophosphate (ß-GP) was used as a procedure to induce extensive artery calcification in primary vascular smooth muscle cells (VSMCs). Analysis of the levels of Nalp3 inflammasome complex was performed by quantitative real-time PCR and western blotting. The effect of Nalp3 deficiency on VSMC calcification was examined after transfecting Nalp3 siRNA into cultured VSMCs. RESULTS: We demonstrated for the first time that the mRNA levels of Nalp3 inflammasome complex including Nalp3, ASC and caspase1 were upregulated in calcifying VSMCs, resulting in increased IL-1ß secretion. Inhibition of inflammasome activation by Nalp3 RNA interference reduced IL-1ß secretion and inhibited VSMC calcification. Further analysis of clinical popliteal artery specimens showed an upregulation of inflammasome complex mRNA levels (4/5) and caspase1 activity (5/5) compared with their non-calcified adjacent tissues, indicating that Nalp3 inflammasome was tightly correlated with arterial calcification disease. CONCLUSION: Our findings indicate that activation of the Nalp3-mediated inflammatory response pathway is an important venue associated with host response and pathogenesis of VSMC calcification.

Identification of tyrosine-9 of MAVS as critical target for inducible phosphorylation that determines activation.

BACKGROUND: Innate immunity to viruses involves receptors such as RIG-I, which senses viral RNA and triggers an IFN-ß signaling pathway involving the outer mitochondrial membrane protein MAVS. However, the functional status of MAVS phosphorylation remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate for the first time that MAVS undergoes extensive tyrosine phosphorylation upon viral infection, indicating that MAVS phosphorylation might play an important role in MAVS function. A tyrosine-scanning mutational analysis revealed that MAVS tyrosine-9 (Y9) is a phosphorylation site that is required for IFN-ß signaling. Indeed, MAVS Y9F mutation severely impaired TRAF3/TRAF6 recruitment and displayed decreased tyrosine phosphorylation in response to VSV infection compared to wild type MAVS. Functionally, MAVS Y9 phosphorylation contributed to MAVS antiviral function without interfering with its apoptosis property. CONCLUSIONS/SIGNIFICANCE: These experiments identify a novel residue of MAVS that is crucially involved in the recruitment of TRAF3/TRAF6 and in downstream propagation of MAVS signaling.

Inhibition of HBV replication by theophylline.

We have suggested recently that ATM-Rad3-Related (ATR) DNA damage signaling pathway, which responds to single-strand breaks in DNA, was activated in response to HBV infection. ATR knockdown cells showed decreased HBV DNA yields, implying HBV infection and replication activate and exploit the activated DNA damage response. Host cell proteins may constitute an attractive target for anti-HBV-1 therapeutics, since development of drug resistance against compounds targeting these cellular cofactor proteins is unlikely. In this study, we show that one of the clinically used compounds of ATR and ataxia telangiectasia-mutated (ATM) kinases inhibitor, theophylline (Tp), significantly reduced the yield of HBV DNA, HBsAg and HBeAg in HepG2215 cell culture system, furthermore, Tp could also suppress serum HBV DNA and HBsAg levels in the HBV-transgenic mice. Consistent with this result, immunohistology also showed reduced intensity of HBsAg staining on livers from Tp-treatment group. Taken together, these data indicated the feasibility of therapeutic approaches that target host cell proteins by inhibiting a cellular gene that was required for HBV replication and provided a potential approach for the prevention and treatment of HBV infection.

The hepatitis B virus X protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein.

Previous studies have shown that both hepatitis A virus and hepatitis C virus inhibit innate immunity by cleaving the mitochondrial antiviral signaling (MAVS) protein, an essential component of the virus-activated signaling pathway that activates NF-kappaB and IFN regulatory factor-3 to induce the production of type I IFN. For human hepatitis B virus (HBV), hepatitis B s-Ag, hepatitis B e-Ag, or HBV virions have been shown to suppress TLR-induced antiviral activity with reduced IFN-beta production and subsequent induction of IFN-stimulated genes. However, HBV-mediated suppression of the RIG-I-MDA5 pathway is unknown. In this study, we found that HBV suppressed poly(deoxyadenylate-thymidylate)-activated IFN-beta production in hepatocytes. Specifically, hepatitis B virus X (HBX) interacted with MAVS and promoted the degradation of MAVS through Lys(136) ubiquitin in MAVS protein, thus preventing the induction of IFN-beta. Further analysis of clinical samples revealed that MAVS protein was downregulated in hepatocellular carcinomas of HBV origin, which correlated with increased sensitivities of primary murine hepatocytes isolated from HBX knock-in transgenic mice upon vesicular stomatitis virus infections. By establishing a link between MAVS and HBX, this study suggests that HBV can target the RIG-I signaling by HBX-mediated MAVS downregulation, thereby attenuating the antiviral response of the innate immune system.

c-Abl tyrosine kinase interacts with MAVS and regulates innate immune response.

The tyrosine kinase, c-Abl, plays important roles in many aspects of cellular function. Previous reports showed that c-Abl is involved in NF-kappaB signaling. However, the functions of c-Abl in innate immunity are still unknown. Here we demonstrate that the mitochondrial antiviral signaling (MAVS) protein can be physically associated with c-Abl in vivo and in vitro. MAVS interacted with c-Abl through its Card and TM domain. A phosphotyrosine-specific antibody indicated that MAVS was phosphorylated by c-Abl. Functional impairment of c-Abl attenuated MAVS or VSV induced type-I IFN production. Importantly, c-Abl knockdown in MCF7 cells displayed impaired MAVS-mediated NF-kappaB and IRF3 activation. Taken together, our results suggest that c-Abl modulates innate immune response through MAVS.

Negative regulation of MAVS-mediated innate immune response by PSMA7.

Innate immunity to viruses involves receptors such as Retinoic Acid Induced Gene-1 (RIG-I), which senses viral RNA and triggers a signaling pathway involving the outer mitochondrial membrane protein mitochondrial antiviral signaling (MAVS). Recent work has identified that NLRX1, a member of another class of innate immune receptors, sequesters MAVS away from RIG-I and thereby prevents mitochondrial antiviral immunity. In this study, we demonstrate that the proteasome PSMA7 (alpha4) subunit associates with MAVS in vivo and in vitro. Expression of PSMA7 results in a potent inhibition of RIG-1 and MAVS-mediated IFN-beta promoter activity; conversely, depletion of PSMA7 with small interference RNA enhances virus-induced type I IFN production, with consequent reduction of virus replication. Furthermore, a striking reduction in the abundance of endogenous MAVS with overexpressed PSMA7 was found and virus infection leads to transient increase in the endogenous PSMA7 protein level. Cumulatively, these results suggest that PSMA7 is a negative regulator of the MAVS-mediated innate immunity that probably serves to attenuate the establishment of an antiviral state during viral infection, highlighting the biological significance of PSMA7-MAVS association as an important cellular regulatory control.

Cellular DNA repair cofactors affecting hepatitis B virus infection and replication.

AIM: To investigate whether hepatitis B virus (HBV) infection activates DNA damage response and DNA repair cofactors inhibit HBV infection and replication. METHODS: Human hepatocyte cell line HL7702 was studied. Immunoblotting was performed to test the expression of ataxia telangiectasia-mutated (ATM)-Rad3-related protein (ATR), p21 and the level of phosphorylation of Chk1, p53, H2AX, ATM in HBV-infected or non-infected-cells. Special short RNAi oligos was transfected to induce transient ATR knockdown in HL7702. ATR-ATM chemical inhibitors caffeine (CF) and theophylline (TP), or Chk1 inhibitor 7-hydroxystaurosporine (UCN01) was studied to determine whether they suppress cellular DNA damage response and MG132 inhibits proteasome. RESULTS: The ATR checkpoint pathway, responding to single-strand breaks in DNA, was activated in response to HBV infection. ATR knockdown cells decreased the HBV DNA yields, implying that HBV infection and replication could activate and exploit the activated DNA damage response. CF/TP or UCN01 reduced the HBV DNA yield by 70% and 80%, respectively. HBV abrogated the ATR-dependent DNA damage signaling pathway by degrading p21, and introduction of the p21 protein before HBV infection reduced the HBV DNA yield. Consistent with this result, p21 accumulation after MG132 treatment also sharply decreased the HBV DNA yield. CONCLUSION: HBV infection can be treated with therapeutic approaches targeting host cell proteins by inhibiting a cellular gene required for HBV replication or by restoring a response abrogated by HBV, thus providing a potential approach to the prevention and treatment of HBV infection.