Biliverdin reductase A is an enzyme, with serine/threonine/tyrosine kinase activation, converting biliverdin (BV) to bilirubin (BR) in heme degradation pathway. It has been reported to have anti-inflammatory and antioxidant effect in monocytes and human glioblastoma. However, the function of BVRA in polarized macrophage was unknown. This study aimed to investigate the effect of BVRA on macrophage activation and polarization in injured renal microenvironment. Classically activated macrophages (M1macrophages) and alternative activation of macrophages (M2 macrophages) polarization of murine bone marrow derived macrophage was induced by GM-CSF and M-CSF. M1 polarization was associated with a significant down-regulation of BVRA and Interleukin-10 (IL-10), and increased secretion of TNF-α. We also found IL-10 expression was increased in BVRA over-expressed macrophages, while it decreased in BVRA knockdown macrophages. In contrast, BVRA over-expressed or knockdown macrophages had no effect on TNF-α expression level, indicating BVRA mediated IL-10 expression in macrophages. Furthermore, we observed in macrophages infected with recombinant adenoviruses BVRA gene, which BVRA over-expressed enhanced both INOS and ARG-1 mRNA expression, resulting in a specific macrophage phenotype. Through in vivo study, we found BVRA positive macrophages largely existed in mice renal ischemia perfusion injury. With the treatment of the regular cytokines GM-CSF, M-CSF or LPS, excreted in the injured renal microenvironment, IL-10 secretion was significantly increased in BVRA over-expressed macrophages. In conclusion, the BVRA positive macrophage is a source of anti-inflammatory cytokine IL-10 in injured kidney, which may provide a potential target for treatment of kidney disease.

The traditional Chinese medicine bufalin, extracted from toad's skin, has been demonstrated to exert anticancer activities in various kinds of human cancers. The mechanisms of action lie in its capacity to induce apoptosis, or termed type I programmed cell death (PCD). However, type II PCD, or autophagy, participates in cancer proliferation, progression, and relapse, as well. Recent studies on autophagy seem to be controversial because of the dual roles of autophagy in cancer survival and death. In good agreement with previous studies, we found that 100 nM bufalin induced extensive HepG2 cell apoptosis. However, we also noticed bufalin triggered autophagy and enhanced Beclin-1 expression, LC3-I to LC3-II conversion, as well as decreased p62 expression and mTOR signaling activation in HepG2 cells. Blockage of autophagy by selective inhibitor 3-MA decreased apoptotic ratio in bufalin-treated HepG2 cells, suggesting a proapoptotic role of bufalin-induced autophagy. Furthermore, we investigated the underlying mechanisms of bufalin-induced autophagy. Bufalin treatment dose-dependently promoted AMPK phosphorylation while AMPK inhibition by compound C significantly attenuated bufalin-induced autophagy. Taken together, we report for the first time that bufalin induces HepG2 cells PCD, especially for autophagy, and the mechanism of action is, at least in part, AMPK-mTOR dependent.

TIR domain-containing proteins are essential for bacterial pathogens to subvert host defenses. This study describes a fish pathogen, Yersinia ruckeri SC09 strain, with a novel TIR domain-containing protein (STIR-2) that affects Toll-like receptor (TLR) function. STIR-2 was identified in Y. ruckeri by bioinformatics analysis. The toxic effects of this gene on fish were determined by in vivo challenge experiments in knockout mutants and complement mutants of the stir-2 gene. In vitro, STIR-2 downregulated the expression and secretion of IL-6, IL-1β, and TNF-α. Furthermore, the results of NF-κB-dependent luciferase reporter system, co-immunoprecipitation, GST pull-down assays, and yeast two-hybrid assay indicated that STIR-2 inhibited the TLR signaling pathway by interacting with myeloid differentiation factor 88 (MyD88). In addition, STIR-2 promoted the intracellular survival of pathogenic Yersinia ruckeri SC09 strain by binding to the TIR adaptor protein MyD88 and inhibiting the pre-inflammatory signal of immune cells. These results showed that STIR-2 increased virulence in Y. ruckeri and suppressed the innate immune response by inhibiting TLR and MyD88-mediated signaling, serving as a novel strategy for innate immune evasion.

qPCR as a powerful and attractive methodology has been widely applied to aquaculture researches for gene expression analyses. However, the suitable reference selection is critical for normalizing target genes expression in qPCR. In the present study, six commonly used endogenous controls were selected as candidate reference genes to evaluate and analyze their expression levels, stabilities and normalization to immune-related gene IgM expression during vaccination and infection in spleen of tilapia with RefFinder and GeNorm programs. The results showed that all of these candidate reference genes exhibited transcriptional variations to some extent at different periods. Among them, EF1A was the most stable reference with RefFinder, followed by 18S rRNA, ACTB, UBCE, TUBA and GAPDH respectively and the optimal number of reference genes for IgM normalization under different experiment sets was two with GeNorm. Meanwhile, combination the Cq (quantification cycle) value and the recommended comprehensive ranking of reference genes, EF1A and ACTB, the two optimal reference genes, were used together as reference genes for accurate analysis of immune-related gene expression during vaccination and infection in Nile tilapia with qPCR. Moreover, the highest IgM expression level was at two weeks post-vaccination when normalized to EF1A, 18S rRNA, ACTB, and EF1A together with ACTB compared to one week post-vaccination before normalizing, which was also consistent with the IgM antibody titers detection by ELISA.

Streptococcus iniae is a major fish pathogen that can also cause human bacteremia, cellulitis and meningitis. Screening for and identification of protective antigens plays an important role in developing therapies against S. iniae infections. In this study, we indicated that the α-enolase of S. iniae was not only distributed in the cytoplasm and associated to cell walls, but was also secreted to the bacterial cell surface. The functional identity of the purified recombinant α-enolase protein was verified by its ability to catalyze the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), and both the recombinant and native proteins interacted with human plasminogen. The rabbit anti-rENO serum blockade assay shows that α-enolase participates in S. iniae adhesion to and invasion of BHK-21 cells. In addition, the recombinant α-enolase can confer effective protection against S. iniae infection in mice, which suggests that α-enolase has potential as a vaccine candidate in mammals. We conclude that S. iniae α-enolase is a moonlighting protein that also associates with the bacterial outer surface and functions as a protective antigen in mice.

One of the leading causes of death in the world is cerebrovascular disease. Numerous Chinese traditional medicines, such as Cortex Moutan (root bark of Paeonia suffruticosa Andrew) and Radix Salviae miltiorrhizae (root and rhizome of Salvia miltiorrhiza Bunge), protect against cerebrovascular diseases and exhibit anti-atherosclerotic effects. Traditional medicines have been routinely used for a long time in China. In addition, these two herbs are prescribed together in clinical practice. Therefore, the pharmacodynamic interactions between the active constituents of these two herbs, which are paeonol (Pae) and danshensu (DSS), should be particularly studied. The study of Pae and DSS can provide substantial foundations in understanding their mechanisms and empirical evidence to support clinical practice. This study investigated the effects and possible mechanisms of the pharmacodynamic interaction between Pae and DSS on cerebrovascular malfunctioning in diabetes. Experimental diabetes was induced in rats, which was then treated with Pae, DSS, and Pae + DSS for eight weeks. Afterward, cerebral arteries from all groups were isolated and equilibrated in an organ bath with Krebs buffer and ring tension. Effects of Pae, DSS, and Pae + DSS were observed on vessel relaxation with or without endothelium as well as on the basal tonus of vessels from normal and diabetic rats. Indexes about oxidative stress were also determined. We report that the cerebral arteries from diabetic rats show decreased vascular reactivity to acetylcholine (ACh) which was corrected in Pae, DSS, and Pae + DSS treated groups. Furthermore, phenylephrine (PE)-induced contraction response decreased in the treated groups. Phenylephrine and CaCl(2)-induced vasoconstrictions are partially inhibited in the three treated groups under Ca2+-free medium. Pre-incubated with tetraethylammonium, a non-selective K+ channel blocker, the antagonized relaxation responses increased in DSS and Pae + DSS treated diabetic groups compared with those in diabetic and Pae-treated diabetic groups. In addition, superoxide dismutase activity and thiobarbituric acid reactive substances content significantly changed in the presence of Pae + DSS. We therefore conclude that both Pae and DSS treatments prevent diabetes-induced vascular damage. Furthermore, Pae + DSS prove to be the most efficient treatment regimen. The combination of Pae and DSS produce significant protective effects through the reduction of oxidative stress and through intracellular Ca2+ regulatory mechanisms.

Renal fibrosis is an inevitable outcome of end-stage chronic kidney disease. During this process, epithelial cells lose E-cadherin expression. β-Catenin may act as a mediator by accumulation and translocation to the nucleus. Studies have suggested that CIP4, a Cdc42 effector protein, is associated with β-catenin. However, whether CIP4 contributes to E-cadherin loss in epithelial cells by regulating β-catenin translocation is unclear. In this study, we investigated the involvement of CIP4 in β-catenin translocation. Expression of CIP4 was upregulated in renal tissues of 5/6 nephrectomized rats and mainly distributed in renal tubular epithelia. In TGF-β1-treated NRK-52E cells, upregulation of CIP4 expression was accompanied by reduced expression of E-cadherin. CIP4 overexpression promoted the translocation of β-catenin to the nucleus, which was accompanied by reduced expression of E-cadherin even without TGF-β1 stimulation. In contrast, CIP4 depletion by using siRNA inhibited the translocation of β-catenin to the nucleus and reversed the decrease in expression of E-cadherin. The interaction between CIP4 and β-catenin was detected. We also show that β-catenin depletion could restore the expression of E-cadherin that was suppressed by CIP4 overexpression. In conclusion, these results suggest that CIP4 overexpression represses E-cadherin expression by promoting β-catenin translocation to the nucleus.

Models of benzalkonium chloride (BAC)-induced ocular disruption have been created and are widely used in various animals. This study aimed to compare the effects of BAC on the ocular surfaces of C57BL/6 and BALB/c mice. C57BL/6 and BALB/c mice were treated separately with BAC eye-drops at different concentrations. Eyes were evaluated by scoring epithelial disruption, corneal opacity and neovascularization in vivo, and by histological assays with hematoxylin/eosin (H/E) and periodic acid-Schiff stainings and by determining the expression of inflammatory factors in vitro on Days 7 and 14. The in vivo corneal epithelial disruption, corneal edema/opacity and neovascularization, which were in accordance with the results of the H/E staining and peaked at Day 7, were observed in a dose-dependent manner in the BAC-treated mice, with more severe signs in the C57BL/6 mice than the BALB/c mice. The loss of conjunctival goblet cells in the conjunctivas and the increasing expression of monocyte chemoattractant protein 1 (MCP-1), growth-regulated protein alpha (GROa) and macrophage inflammatory protein-1 alpha (MIP-1a) in the corneas were found in a dose-dependent manner in both strains of mice. Topical application of BAC can dramatically disrupt the ocular surfaces of C57BL/6 and BALB/c mice, and the disruptions were much more severe in the C57BL/6 mice that received high doses of BAC.

ATP-binding cassette (ABC) transporters are a superfamily of proteins that transport nutrient substances and secondary metabolites through cell membranes. They also act as an uptake system for N,N'-diacetylchitobiose (GlcNAc)2 in Streptomyces coelicolor. (GlcNAc)2 is an important inducer of chitinase. However, whether the ABC transporter in Trichoderma spp. is also responsible for (GlcNAc)2 uptake and chitinase induction has not yet been confirmed. In this study, we applied RNA interference and overexpression technologies to alter the expression level of the ABC-B transporter in order to detect changes in its transportation ability and the expression level of inducible endo-chitinase ECH42-an important biocontrol enzyme in Trichoderma asperellum. The results revealed that, after interference with the expression of the ABC-B transporter, T. asperellum T4 was only able to grow normally when glucose was the only carbon source. Compared with the wild-type, the efficiency of (GlcNAc)2 by the overexpression strain evidently increased, along with the activity level of ECH42. In conclusion, one of the functions of the ABC-B transporter in T. asperellum is the uptake and transport of (GlcNAc)2 into cells, and chitobiose is a strong inducer of ECH42 in T. asperellum T4.

Aerobic denitrification is considered as a promising biological method to eliminate the nitrate contaminants in waterbodies. However, the molecular mechanism of this process varies in different functional bacteria. In this study, the nitrogen removal characteristics for a newly isolated aerobic denitrifier Bacillus subtilis JD-014 were investigated, and the potential functional genes involved in the aerobic denitrification process were further screened through transcriptome analysis. JD-014 exhibited efficient denitrification performance when having sodium succinate as the carbon source with the range of nitrate concentration between 50 and 300 mg/L. Following the transcriptome data, most of the up-regulated differentially expressed genes (DEGs) were associated with cell motility, carbohydrate metabolism, and energy metabolism. Moreover, gene nirsir annotated as sulfite reductase was screened out and further identified as a regulator participating in the nitrogen removal process within JD-014. The findings in present study provide meaningful information in terms of a comprehensive understanding of genetic regulation of nitrogen metabolism, especially for Bacillus strains.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen causing severe diseases, such as hemorrhagic colitis and lethal hemolytic uremic syndrome. The signal-sensing capability of EHEC O157:H7 at specific host colonization sites via different two-component systems (TCSs) is closely related to its pathogenicity during infection. However, the types of systems involved and the regulatory mechanisms are not fully understood. Here, we investigated the function of the TCS BarA/UvrY regulator UvrY in the pathogenicity regulation of EHEC O157:H7. Our results showed that UvrY acts as a positive regulator of EHEC O157:H7 for cellular adherence and mouse colonization through the transcriptional activation of the locus for enterocyte effacement (LEE) pathogenic genes. Furthermore, this regulation is mediated by the LEE island master regulator, Ler. Our results highlight the significance of UvrY in EHEC O157:H7 pathogenicity and underline the unknown importance of BarA/UvrY in colonization establishment and intestinal adaptability during infection.

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are two key factors for natural rubber biosynthesis. To further understand the roles of these proteins in rubber formation, six different genes for latex abundant REF or SRPP proteins, including REF138,175,258 and SRPP117,204,243, were characterized from Hevea brasiliensis Reyan (RY) 7-33-97. Sequence analysis showed that REFs have a variable and long N-terminal, whereas SRPPs have a variable and long C-terminal beyond the REF domain, and REF258 has a β subunit of ATPase in its N-terminal. Through two-dimensional electrophoresis (2-DE), each REF/SRPP protein was separated into multiple protein spots on 2-DE gels, indicating they have multiple protein species. The abundance of REF/SRPP proteins was compared between ethylene and control treatments or among rubber tree clones with different levels of latex productivity by analyzing 2-DE gels. The total abundance of each REF/SRPP protein decreased or changed a little upon ethylene stimulation, whereas the abundance of multiple protein species of the same REF/SRPP changed diversely. Among the three rubber tree clones, the abundance of the protein species also differed significantly. Especially, two protein species of REF175 or REF258 were ethylene-responsive only in the high latex productivity clone RY 8-79 instead of in RY 7-33-97 and PR 107. Some individual protein species were positively related to ethylene stimulation and latex productivity. These results suggested that the specific protein species could be more important than others for rubber production and post-translational modifications might play important roles in rubber biosynthesis.