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Physical thickness of low-density polyethylene (LDPE) films might determine the release rate of phthalic acid esters (PAEs) & structural integrity and affect production efficiency. However, this critical issue is still unclear and little reported. Aging effects were evaluated in LDPE films with the thickness of 0.006, 0.008, 0.010 and 0.015 mm in a maize field of irrigation region. The Scanning electron microscope (SEM) results showed that the proportion of damaged area (Dam) to total area of LDPE films was massively lowered with increasing thickness after aging. The highest and lowest Dam was 32.2% and 3.5% in 0.006 and 0.015 mm films respectively. Also, the variations in peak intensity of asymmetric & symmetrical stretching vibrations (ASVI & SSVI) were detected using Fourier transform infrared spectrum (FTIR), indicating that the declines in peak intensity tended to be slower with thickness. Interestingly, the declines in physical integrity were tightly associated with increasing exhalation rate of PAEs. Average releasing rate of PAEs was 38.2%, 31.4%, 31.5% and 19.7% in LDPE films from 0.006 to 0.015 mm respectively. Critically, thicker film mulching can lead to greater soil water storage at plough layer (SWS-PL) and better thermal status, accordingly harvesting higher economic benefit. Therefore, LDPE film thickening may be a solution to reduce environmental risk but improve production efficiency in arid region. [Display omitted] • Structural damage of LDPE films increased with time but lowered with thickness. • The physical integrity was negatively associated with its releasing PAEs amount. • Aging characterization under SEM & FTIR showed the thickness-dependent degradation. • Thicker film mulching had better soil hydrothermal status for higher yield and WUE. • Film thickening was a critical solution to reduce pollution but improve productivity. [ABSTRACT FROM AUTHOR]

Complement C1q domain containing protein (C1qDC) is a vital recognition molecule and has an important effect on immunity. The C1qDCs exhibit opsonic activity in fish, while the mechanisms of C1qDCs in activation complement still remain unclear. This study explored immunological characteristics of a C1qDC from Japanese flounder (Paralichthys olivaceus) (PoC1qDC). PoC1qDC consists of 296 amino acid residues, possessing a collagen domain and a C1q domain. According to our results, PoC1qDC was expressed in 9 diverse tissue samples and showed up-regulation after bacterial challenge. Recombinant PoC1qDC (rPoC1qDC) activated normal serum bactericidal and hemolytic activities by interaction with Japanese flounder IgM, but not enhanced the complement activity of C3-depeleted serum. rPoC1qDC was significantly bound to various bacterial species and agglutination activity against Edwardsiella piscicida and Streptococcus iniae. Furthermore, rPoC1qDC showed direct interaction with peripheral blood leucocytes while enhancing phagocytic and chemotactic activity. When PoC1qDC was overexpressed in Japanese flounder before E. piscicida infection, bacterial replication was significantly inhibited in fish tissues. Consistently, when PoC1qDC expression in Japanese flounder was knocked down, bacterial replication was significantly enhanced. The above findings first suggested the role of PoC1qDC in teleost in mediating complement activation by interaction with IgM, which can positively influence bacterial infection. • PoC1qDC possesses a collagen domain and a C1q domain. • Bacterial infection enhanced PoC1qDC expression in spleen, kidney and blood. • Recombinant PoC1qDC mediated complement activation by interaction with IgM. • Recombinant PoC1qDC could bind to bacteria and exhibited agglutination activity. • PoC1qDC is required for optimal defense against bacterial infection. [ABSTRACT FROM AUTHOR]

CD59 is a complement regulatory protein that inhibits the formation of membrane attack complex of complement. In this study, we examined the expression and activity of tongue sole ( Cynoglossus semilaevis ) CD59 (CsCD59). CsCD59 possesses the conserved structural features of CD59 and shares 33%–46% sequence identities with other fish CD59. Expression of CsCD59 was high in liver, spleen, and muscle, and was stimulated by infection of bacterial pathogens. Recombinant CsCD59 (rCsCD59) exhibited an apparent inhibition effect on the activation of tongue sole serum complement. ELISA and microscopy detected binding of rCsCD59 to a number of Gram-negative and Gram-positive bacteria. Interaction with rCsCD59 did not affect bacterial viability but significantly enhanced bacterial resistance against the killing effect of fish serum. Together these results indicate that fish CD59 may to some degrees facilitate a general escape of bacteria from complement-mediated immunity. [ABSTRACT FROM AUTHOR]

Chemokines are a large, diverse group of small cytokines that can be classified into several families, including the CC chemokine family, which plays a pivotal role in host defense by inducing leukocyte chemotaxis under physiological and inflammatory conditions. Here we studied 9 CC chemokines from half-smooth tongue sole ( Cynoglossus semilaevis ). Phylogenetic analysis divided these chemokines into four groups. The tissue specific expression patterns of the 9 chemokines under normal physiological conditions varied much, with most chemokines highly expressed in immune organs, while some other chemokines showing high expression levels in non-immune organs. In addition, the 9 chemokines exhibited similar or distinctly different expression profiles in response to the challenge of virus and intracellular and extracellular bacterial pathogens. These results indicate that in tongue sole, CC chemokines may be involved in different immune responses as homeostatic or inflammatory chemokines. [ABSTRACT FROM AUTHOR]

Megalocytivirus, a DNA virus belonging to the Iridoviridae family, is a severe pathogen to a wide range of marine and freshwater fish. In this study, using turbot ( Scophthalmus maximus ) as a host model, we examined the immunoprotective property of one megalocytivirus gene, ORF75, in the form of DNA vaccine (named pORF75). Immunofluorescence microscopy and RT-PCR analysis showed that P444, the protein encoded by ORF75, was naturally produced in the tissues of turbot during megalocytivirus infection, and that the vaccine gene in pORF75 was expressed in fish cells transfected with pORF75 and in the tissues of turbot immunized with pORF75. Following vaccination of turbot with pORF75, a high level of survival (73%) was observed against a lethal megalocytivirus challenge. Consistently, viral replication in the vaccinated fish was significantly inhibited. Immune response analysis showed that pORF75-vaccinated fish (i) exhibited upregulated expression of the genes involved in innate and adaptive immunity, (ii) possessed specific memory immune cells that showed significant response to secondary antigen stimulation, and (iii) produced specific serum antibodies which, when co-introduced into turbot with megalocytivirus, blocked viral replication. Furthermore, whole-genome transcriptome analysis revealed that ORF75 knockdown altered the transcription of 43 viral genes. Taken together, these results indicate that ORF75 encoded a highly protective immunogen that is also a global transcription regulator of megalocytivirus. [ABSTRACT FROM AUTHOR]

Edwardsiella tarda is a Gram-negative bacterium with a broad host range that includes a wide variety of farmed fish as well as humans. E. tarda has long been known to be able to survive in host serum, but the relevant mechanism is unclear. In this study, we investigated the fundamental question, i.e. whether E. tarda activated serum complement or not. We found that (i) when incubated with flounder serum, E. tarda exhibited a high survival rate (87.6%), which was slightly but significantly reduced in the presence of Mg 2+ ; (ii) E. tarda -incubated serum possessed strong hemolytic activity and bactericidal activity, (iii) compared to the serum incubated with a complement-sensitive laboratory Escherichia coli strain, E. tarda -incubated serum exhibited much less chemotactic activity, (iv) in contrast to the serum incubated with live E. tarda , the serum incubated with heat-inactivated E. tarda exhibited no apparent hemolytic capacity. Taken together, these results indicate for the first time that E. tarda circumvents serum attack by preventing, to a large extent, complement activation via the alternative pathway, and that heat-labile surface structures likely play an essential role in the complement evasion of E. tarda . [ABSTRACT FROM AUTHOR]

Abstract: Thioredoxin (Trx) is a redox protein that plays an important role in anti-oxidative stress and redox signaling. In a recent report, we observed that the Trx of tongue sole (Cynoglossus semilaevis), CsTrx1, is a reductase and an antioxidant with immunostimulatory property. In the current work, we studied the biological effect of a truncated CsTrx1, CsTrx80, which is composed of the N-terminal 80 residues of CsTrx1. We found that recombinant CsTrx80 (rCsTrx80) purified from Escherichia coli exhibited no insulin disulfide reductase activity; however, unlike rCsTrx1, which showed no apparent chemotactic activity, rCsTrx80 was able to bind to the head kidney monocytes of tongue sole and caused cellular migration in a dose-dependent manner. Mutation of the two cysteine residues in the highly conserved CXXC motif had no effect on the chemotactic activity of rCsTrx80. Like rCsTrx1, rCsTrx80 induced the expression of immune relevant genes in the target cells, but the induction patterns differed from that induced by rCsTrx1. In vivo study showed that when tongue sole were administered with rCsTrx80 before being infected with a bacterial pathogen, bacterial dissemination and colonization in fish tissues were significantly reduced compared to control fish. Taken together, these results indicate for the first time that a Trx80 derived from fish Trx possesses chemotactic and immunoregulatory properties which promote host resistance against bacterial infection. [Copyright &y& Elsevier]

Abstract: Pentraxins are a family of evolutionarily conserved proteins that play an important part in innate immunity. C-reactive protein (CRP) is a member of the pentraxin family and in humans is known to be the major acute phase protein. In this work, we report the identification and analysis of a CRP, CsCRP, from half-smooth tongue sole (Cynoglossus semilaevis). CsCRP is composed of 228 amino acid residues and possesses a Pentraxin/CRP domain. Expression of CsCRP occurred in a wide range of tissues and was upregulated by pathogen infection in kidney, spleen, blood, and, in particular, liver. Following bacterial infection, CsCRP level in blood rose rapidly within 12 h and was approximately 3.8 fold of that of the basal level. Purified recombinant CsCRP (rCsCRP) was able to interact with Gram-negative and Gram-positive bacteria including those of pathogenic nature in a dose-dependent manner. When peripheral blood leukocytes (PBL) were infected with bacterial pathogen in the presence of rCsCRP, the respiratory burst and phagocytic capacity of the cells were increased to significant extents. Taken together, these results indicate that CsCRP is an acute phase protein that plays a role in innate immune defense against bacterial infection. [Copyright &y& Elsevier]

Abstract: Invasin is an outer membrane protein that is known to mediate entry of enteric bacteria into mammalian cells. In this study, we analyzed the function and immunoprotective potential of the invasin Inv1 from Edwardsiella tarda, a serious fish pathogen that can also infect humans. In silico analysis indicated that Inv1 possesses a conserved N-terminal DUF3442 domain and a C-terminal group 1 bacterial Ig-like domain. Subcellular localization analysis showed that Inv1 is exposed on cell surface and could be recognized by specific antibodies. Mutation of inv1 had no effect on bacterial growth but attenuates overall bacterial virulence and impaired the ability of E. tarda to attach and invade into host cells. Consistent with these observations, antibody blocking of Inv1 inhibited E. tarda infection of host cells. To examine the immunoprotective potential of Inv1, recombinant Inv1 (rInv1) corresponding to the DUF3442 domain was purified and used to vaccinate Japanese flounder (Paralichthys olivaceus). The results showed that rInv1 induced strong protection against lethal-dose challenge of E. tarda. ELISA analysis showed that rInv1-vaccinated fish produced specific serum antibodies that could enhance the serum bactericidal activity against E. tarda. Taken together, these results indicate that Inv1 is a surface-localized virulence factor that is involved in host infection and can induce effective immunoprotection when used as a subunit vaccine. [Copyright &y& Elsevier]

Abstract: Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46–94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn2+-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection. [Copyright &y& Elsevier]

In mammals, complement factor I (CFI) is a serine protease in serum and plays a pivotal role in the regulation of complement activation. In the presence of cofactor, CFI cleaves C3b to iC3b, and further degrades iC3b to C3c and C3d. In teleost, the function of CFI is poorly understood. In this study, we examined the immunological property of CFI from Japanese flounder (Paralichthys olivaceus) (PoCFI), a teleost species with important economic value. PoCFI is composed of 597 amino acid residues and possesses a trypsin-like serine protease (Tryp) domain. We found that PoCFI expressions occurred in nine different tissues and were upregulated by bacterial challenge. Recombinant PoCFI-Tryp (rPoCFI-Tryp) inhibited complement activation and degraded C3b in serum. rPoCFI-Tryp exhibited apparent binding capacities to a board-spectrum of bacteria and inhibited bacterial growth. These results provide the first evidence to indicate that CFI in teleost negatively regulates complement activation via degradation C3b, and probably plays a role in host immune defense against bacterial infection. • PoCFI possesses a trypsin serine protease (PoCFI-Tryp) domain. • PoCFI expression occurred in multiple tissues and was most abundant in liver. • Pathogen infection enhanced PoCFI expression in spleen, kidney and blood. • Recombinant PoCFI-Tryp (rPoCFI-Tryp) inhibited complement activation. • rPoCFI-Tryp was able to bind to bacteria and inhibited bacterial growth. [ABSTRACT FROM AUTHOR]

Chemokines are a large family of chemotactic cytokines. Based on the arrangement of the first two cysteine residues, chemokines are divided into four groups, one of which is the CC chemokine group. In this study, we characterized a CC chemokine, CsCCL21, from half-smooth tongue sole ( Cynoglossus semilaevis ), and analyzed its activity. CsCCL21 contains two conserved N-terminal cysteine residues in a NCCL motif and is phylogenetically related to the CCL19/21/25 subgroup of CC chemokines. CsCCL21 was constitutively expressed in nine tissues and significantly upregulated by bacterial and viral infection. The recombinant CsCCL21 (rCsCCL21) induced migration of peripheral blood leukocytes. When the two conserved cysteine residues in the NCCL motif were mutated, the chemotactic activity of rCsCCL21 was abolished. rCsCCL21 enhanced the resistance of tongue sole against bacterial infection, but the mutant protein with NCCL mutation lacked this antibacterial effect. Taken together, these results suggest that CsCCL21 is a functional CC chemokine with the ability to recruit leukocytes and is involved in antibacterial immunity in a manner that requires the conserved NCCL motif. [ABSTRACT FROM AUTHOR]

Takifugu rubripes is a highly valued cultured fish in Asia, while pathogen infections can result in severe diseases and lead to substantial economic losses. Toll-like receptors (TLRs), as pattern recognition receptors, play a crucial role on recognition pathogens and initiation innate immune response. However, the immunological properties of teleost-specific TLR23 remain largely unknown. In this study, we investigated the biological functions of TLR23 (TrTLR23) from T. rubripes , found that TrTLR23 existed in various organs. Following bacterial pathogen challenge, the expression levels of TrTLR23 were significantly increased in immune related organs. TrTLR23 located on the cellular membrane and specifically recognized pathogenic microorganism. Co-immunoprecipitation and antibody blocking analysis revealed that TrTLR23 recruited myeloid differentiation primary response protein (MyD88), thereby mediating the activation of the ERK signaling pathway. Furthermore, in vivo showed that, when TrTLR23 is overexpressed in T. rubripes , bacterial replication in fish tissues is significantly inhibited. Consistently, when TrTLR23 expression in T. rubripes is knocked down, bacterial replication is significantly enhanced. In conclusion, these findings suggested that TrTLR23 played a critical role on mediation TLR23-MyD88-ERK axis against bacterial infection. This study revealed that TLR23 involved in the innate immune mechanism, and provided the foundation for development disease control strategies in teleost. • TrTLR23 is phylogenetically distantly related to teleost-specific TLR23. • Bacterial infection enhanced TrTLR23 expression in liver, spleen, and kidney. • TrTLR23 located on the cellular membrane and specifically recognized pathogen. • TrTLR23 recruited myeloid MyD88 and activated the ERK signaling pathway. • TrTLR23 is required for optimal defense against bacterial infection. [ABSTRACT FROM AUTHOR]

Abstract: Hepcidin is an antimicrobial peptide and a regulator of iron homeostasis. In turbot (Scophthalmus maximus), two types of hepcidins have been identified, which share approximately 50% sequence identity. In this study, we examined the antimicrobial potentials of the two hepcidins in the form of synthesized peptides, SmHep1P and SmHep2P. We found that SmHep1P and SmHep2P exhibited apparent bactericidal activities against both Gram-positive and Gram-negative bacteria in a dose-dependent manner. The bactericidal effect of SmHep1P was stronger against Gram-positive bacteria, while the bactericidal effect of SmHep2P was stronger against Gram-negative bacteria. Fluorescence and electron microscopy showed that both peptides were able to bind to the target bacterial cells and alter the surface structure of the cells. In vitro studies showed that SmHep1P and SmHep2P reduced bacterial invasion into cultured fish cells. In vivo studies showed that turbot administered with SmHep1P and SmHep2P exhibited significantly enhanced resistance against bacterial and viral infection. In both in vivo and in vitro studies, the antimicrobial activities of SmHep2P were in most cases significantly stronger than those of SmHep1P. Together these results indicate that the two hepcidins of turbot most likely possess antimicrobial properties and play a role in the innate immune defense against bacterial and viral pathogens. [Copyright &y& Elsevier]

In this study, we examined the function of a Japanese flounder (Paralichthys olivaceus) microRNA (miRNA), pol-miR-363-3p. We found that pol-miR-363-3p targets an ubiquitin-specific protease (USP), USP32. USP is a family of deubiquitinating enzymes essential to the functioning of the ubiquitin proteasome system. In mammals, USP32 is known to be associated with cancer and immunity. In fish, the function of USP32 is unknown. We found that flounder USP32 (PoUSP32) expression was detected in the major tissues of flounder, particularly intestine. In vitro and in vivo studies showed that pol-miR-363-3p directly regulated PoUSP32 in a negative manner by interaction with the 3′UTR of PoUSP32. Overexpression of pol-miR-363-3p or interference with PoUSP32 expression in flounder cells significantly blocked Streptococcus iniae infection. Consistently, in vivo knockdown of pol-miR-363-3p or overexpression of PoUSP32 enhanced dissemination of S. iniae in flounder tissues, whereas in vivo knockdown of PoUSP32 inhibited S. iniae dissemination. In addition, pol-miR-363-3p knockdown also significantly promoted the tissue dissemination of the viral pathogen megalocytivirus, which, as well as S. iniae , regulated pol-miR-363-3p expression. Together these results revealed an important role of pol-miR-363-3p in flounder immune defense against bacterial and viral infection. • PoUSP32 was a target gene of pol-miR-363-3p and regulated negatively by the latter. • pol-miR-363-3p expression was regulated by Streptococcus iniae and megalocytivirus. • PoUSP32 knockdown/pol-miR-363-3p overexpression blocked S. iniae infection in vitro. • In vivo pol-miR-363-3p knockdown promoted S. iniae and megalocytivirus infection. • In vivo PoUSP32 overexpression/knockdown affected S. iniae invasion in flounder. [ABSTRACT FROM AUTHOR]

Interleukin 8 (IL8) is a CXC chemokine that plays a crucial role on promoting inflammatory response and immune regulation. In teleost, IL8 can induce the migration and activation of immune cells. However, the biological functions of IL8 are still unknown in Takifugu rubripes. In this study, we examined the biological characteristics of TrIL8 in T. rubripes. TrIL8 is composed of 98 residues and contained a chemokine CXC domain. We found that the TrIL8 expression was detected in diverse organs and significantly increased by Vibrio harveyi or Edwardsiella tarda challenge. The recombinant TrIL8 (rTrIL8) exhibited significantly the binding capacities to 8 tested bacteria. In addition, rTrIL8 could bind to peripheral blood leukocytes (PBL), and increased the expression of immune gene, resistance to bacterial infection, respiratory burst, acid phosphatase activity, chemotactic activity, and phagocytic activity of PBL. In the presence of rTrIL8, T. rubripes was enhanced the resistance to V. harveyi infection. These results indicated that TrIL8 is a chemokine and involved in the activation of immune cells against bacterial infection in teleost. • TrIL8 possesses a chemokine CXC domain. • Bacterial infection enhanced TrIL8 expression in liver, spleen, and kidney. • Recombinant TrIL8 (rTrIL8) induced chemotaxis of peripheral blood leukocytes (PBL). • rTrIL8 could bind to PBL and enhanced the immune activity of cells. • rTrIL8 enhanced the resistance to bacterial infection. [ABSTRACT FROM AUTHOR]

Objective The role of tumor-associated macrophages (TAMs) in tumor microenvironment remains controversial due to the two different polarized subsets of TAMs. Here, we performed a meta-analysis to evaluate the correlation between subpopulations of TAMs and clinical outcomes in patients with ovarian cancer. Methods A comprehensive search in PUBMED/Medline and EMBASE databases was performed. The association between TAMs and patient prognosis of ovarian cancer was estimated with hazard ratios (HRs) and their corresponding 95% confidence intervals (95% CIs) using a random-effect model. Additionally, sensitivity analysis and Begg's test were conducted. Results Nine studies including 794 patients were enrolled in the meta-analysis. The results showed that higher M1/M2 ratio in tumor tissues was associated with a favorable overall survival (OS) (HR = 0.449, 95% CI = 0.283–0.712, P = 0.001). Elevated intra-islet M1/M2 TAMs ratio showed a positive correlation for OS (HR = 0.510, 95% CI = 0.264–0.986, P = 0.045). No significant relation was observed between OS and CD68 + TAMs (HR = 0.99, 95% CI = 0.88–1.11, P = 0.859), CD163 + TAMs (HR = 1.04, 95% CI = 0.92–1.16, P = 0.544) or CD163 +/CD68 + TAMs ratio (HR = 1.628, 95% CI = 0.529–5.008, P = 0.395). Worse progression-free survival (PFS) was associated with high density of CD163 + TAMs (HR = 2.157, 95% CI = 1.406–3.312, P = 0.000) and higher ratio of CD163 +/CD68 + TAMs (HR = 3.223, 95% CI = 1.805–5.755, P = 0.000). Elevated M1/M2 TAMs ratio predicted better PFS of ovarian cancer (HR = 0.490, 95% CI = 0.270–0.890, P = 0.019). Furthermore, high density of CD163 + and CD68 + TAMs was observed in ovarian cancer with advanced TNM stage. Conclusion In our study, it was revealed that CD163 + TAMs infiltration was associated with poor prognosis of ovarian cancer and high M1/M2 macrophages ratio in tumor tissues predicted better prognosis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Polygonum capitatum (PC) has both antibacterial and anti-inflammatory effects. • PC has advantages in the treatment of Helicobacter pylori -associated gastritis(HAG). • Network pharmacology lets us quickly predict the action mechanism of PC. • We found that PC affects the phosphorylation of IκBα, NF-κB p65, p38MAPK, and ERK1/2. • PC can significantly inhibite the nuclear translocation of NF-κB p65 and p-p38MAPK. Helicobacter pylori (H. pylori) infection is a common disease that can cause H. pylori -associated gastritis (HAG), peptic ulcers, and gastric cancer. As a traditional Chinese medicine, Polygonum capitatum (PC) manifests its unique advantages in the prevention and treatment of complex diseases and chronic diseases, due to its ability to clear heat, detoxify and relieve pain, promote blood circulation, and remove blood stasis. In order to explore the molecular mechanism of PC for HAG, the study collected the predicted targets of active compounds, conducted functional analysis by the STRING database, collected HAG differential expression genes, and conducted KEGG enrichment analysis on the intersection of predicted targets and differential expression genes of gastritis by Cluego. The results show that PC works mainly by affecting phosphorylation of IκBα, NF-κB p65, p38MAPK, and ERK1/2 and nuclear transposition of NF-κB p65 and p-p38MAPK, which has been proved by in vivo and in vitro experiments. These results suggest that PC may act on HAG with multiple targets and pathways, and play a key role in the process of HAG treatment. [ABSTRACT FROM AUTHOR]

• Phosphorylation of RssB happens on the interface thus weakens the interaction σS. • IraD interacts with the RssB at higher affinity regardless of phosphorylation. • A new two-tier mechanism for controlled proteolysis of σS is proposed. RssB is fully activated during logarithmic growth, promoting σS degradation. Phosphorylation on D58 partially switches the degradation off as the phosphorylation reduces RssB affinity for σS. The binding of anti-adaptor protein IraD to RssB prevents any RssB- σS interaction and shuts off σS degradation completely. Regulation of bacterial stress responding σS is a sophisticated process and mediated by multiple interacting partners. Controlled proteolysis of σS is regulated by RssB which maintains minimal level of σS during exponential growth but then elevates σS level while facing stresses. Bacteria developed different strategies to regulate activity of RssB, including phosphorylation of itself and production of anti-adaptors. However, the function of phosphorylation is controversial and the mechanism of anti-adaptors preventing RssB-σS interaction remains elusive. Here, we demonstrated the impact of phosphorylation on the activity of RssB and built the RssB-σS complex model. Importantly, we showed that the phosphorylation site - D58 is at the interface of RssB-σS complex. Hence, mutation or phosphorylation of D58 would weaken the interaction of RssB with σS. We found that the anti-adaptor protein IraD has higher affinity than σS to RssB and its binding interface on RssB overlaps with that for σS. And IraD-RssB complex is preferred over RssB-σS in solution, regardless of the phosphorylation state of RssB. Our study suggests that RssB possesses a two-tier mechanism for regulating σS. First, phosphorylation of RssB provides a moderate and reversible tempering of its activity, followed by a specific and robust inhibition via the anti-adaptor interaction. [ABSTRACT FROM AUTHOR]