Protein Kinase CK2 Promotes Proliferation, Abnormal Differentiation, and Proinflammatory Cytokine Production of Keratinocytes via Regulation of STAT3 and Akt Pathways in Psoriasis.

Protein kinase CK2 is a constitutively active and ubiquitously expressed serine/threonine kinase that is closely associated with various types of cancers, autoimmune disorders, and inflammation. However, the role of CK2 in psoriasis remains unknown. Herein, the study indicated elevated expression of CK2 in skin lesions from patients with psoriasis and from psoriasis-like mice. In the psoriasis-like mouse model, the CK2-specific inhibitor CX-4945 ameliorated imiquimod-induced psoriasis symptoms with reduced proliferation, abnormal differentiation, inflammatory cytokine production (especially IL-17A) of keratinocytes, and infiltration of γδ T cells. In in vitro studies, exogenous CK2 promoted hyperproliferation and abnormal differentiation of human keratinocytes, which were reversed by the suppression of CK2 with CX-4945 or siRNA. Furthermore, knockdown of CK2 reduced IL-17A expression and abolished IL-17A-induced proliferation and inflammatory cytokine expression in keratinocytes. Interestingly, IL-17A increased the expression of CK2 in keratinocytes, thereby establishing a positive feedback loop. In addition, suppression of CK2 inhibited the activation of STAT3 and Akt signaling pathways in human keratinocytes and imiquimod-induced psoriatic lesions of mice. These findings indicate that a highly expressed CK2 level in the skin lesions is required in the development of psoriasis by promoting epidermal hyperplasia, abnormal differentiation, and inflammatory response via regulation of the STAT3 and Akt signaling pathways. CK2 may be a target for the treatment of psoriasis.

The preprogrammed anti-inflammatory phenotypes of CD11chigh macrophages by Streptococcus pneumoniae aminopeptidase N safeguard from allergic asthma.

BACKGROUND: Early microbial exposure is associate with protective allergic asthma. We have previously demonstrated that Streptococcus pneumoniae aminopeptidase N (PepN), one of the pneumococcal components, inhibits ovalbumin (OVA) -induced airway inflammation in murine models of allergic asthma, but the underlying mechanism was incompletely determined. METHODS: BALB/c mice were pretreated with the PepN protein and exposed intranasally to HDM allergen. The anti-inflammatory mechanisms were investigated using depletion and adoptive transfer experiments as well as transcriptome analysis and isolated lung CD11chigh macrophages. RESULTS: We found pretreatment of mice with PepN promoted the proliferation of lung-resident F4/80+CD11chigh macrophages in situ but also mobilized bone marrow monocytes to infiltrate lung tissue that were then transformed into CD11high macrophages. PepN pre-programmed the macrophages during maturation to an anti-inflammatory phenotype by shaping the metabolic preference for oxidative phosphorylation (OXPHOS) and also inhibited the inflammatory response of macrophages by activating AMP-activated protein kinase. Furthermore, PepN treated macrophages also exhibited high-level costimulatory signaling molecules which directed the differentiation into Treg. CONCLUSION: Our results demonstrated that the expansion of CD11chigh macrophages in lungs and the OXPHOS metabolic bias of macrophages are associated with reduced allergic airway inflammation after PepN exposure, which paves the way for its application in preventing allergic asthma.

CD5L attenuates allergic airway inflammation by expanding CD11chigh alveolar macrophages and inhibiting NLRP3 inflammasome activation via HDAC2.

Allergic asthma is an airway inflammatory disease dominated by type 2 immune responses and there is currently no curative therapy for asthma. CD5-like antigen (CD5L) has been known to be involved in a variety of inflammatory diseases. However, the role of CD5L in allergic asthma remains unclear. In the present study, mice were treated with recombinant CD5L (rCD5L) during house dust mite (HDM) and ovalbumin (OVA) challenge to determine the role of CD5L in allergic asthma, and the underlying mechanism was further explored. Compared with PBS group, serum CD5L levels of asthmatic mice were significantly decreased, and the levels of CD5L in lung tissues and bronchoalveolar lavage fluid (BALF) were significantly increased. CD5L reduced airway inflammation and Th2 immune responses in asthmatic mice. CD5L exerted its anti-inflammatory function by increasing CD11chigh alveolar macrophages (CD11chigh AMs), and the anti-inflammatory role of CD11chigh AMs in allergic asthma was confirmed by CD11chigh AMs depletion and transfer assays. In addition, CD5L increased the CD5L+ macrophages and inhibited NLRP3 inflammasome activation by increasing HDAC2 expression in lung tissues of asthmatic mice. Hence, our study implicates that CD5L has potential usefulness for asthma treatment.

Interleukin-4 protects mice against lethal influenza and Streptococcus pneumoniae co-infected pneumonia.

Streptococcus pneumoniae co-infection post-influenza is a major cause of mortality characterized by uncontrolled bacteria burden and excessive immune response during influenza pandemics. Interleukin (IL)-4 is a canonical type II immune cytokine known for its wide range of biological activities on different cell types. It displays protective roles in numerous infectious diseases and immune-related diseases, but its role in influenza and S. pneumoniae (influenza/S. pneumoniae) co-infected pneumonia has not been reported. In our study, we used C57BL/6 wild-type (WT) and IL-4-deficient (IL-4-/- ) mice to establish co-infection model with S. pneumoniae after influenza virus infection. Co-infected IL-4-/- mice showed increased mortality and weight loss compared with WT mice. IL-4 deficiency led to increased bacterial loads in lungs without altering influenza virus replication, suggesting a role of IL-4 in decreasing post-influenza susceptibility to S. pneumoniae co-infection. Loss of IL-4 also resulted in aggravated lung damage together with massive proinflammatory cytokine production and immune cell infiltration during co-infection. Administration of recombinant IL-4 rescued the survival and weight loss of IL-4-/- mice in lethal co-infection. Additionally, IL-4 deficiency led to more immune cell death in co-infection. Gasdermin D (GSDMD) during co-infection was induced in IL-4-/- mice that subsequently activated cell pyroptosis. Treatment of recombinant IL-4 or inhibition of GSDMD activity by disulfiram decreased immune cell death and bacterial loads in lungs of IL-4-/- co-infected mice. These results suggest that IL-4 decreases post-influenza susceptibility to S. pneumoniae co-infection via suppressing GSDMD-induced pyroptosis. Collectively, this study demonstrates the protective role of IL-4 in influenza/S. pneumoniae co-infected pneumonia.

Cytosolic mtDNA released from pneumolysin-damaged mitochondria triggers IFN-ß production in epithelial cells.

Pneumolysin (Ply) is a major virulence factor of Streptococcus pneumoniae. Ply-induced interferon-ß (IFN-ß) expression in host macrophages has been shown to be due to the accumulation of mitochondrial deoxyribonucleic acid (mtDNA) in the cytoplasm during S. pneumoniae infection. Our findings extend this work to show human bronchial epithelial cells that reside at the interface of inflammatory injury, BEAS-2B, adapt to local cues by altering mitochondrial states and releasing excess mtDNA. The results in this research showed that purified Ply induced the expression of IFN-ß in human epithelial cells, which was accompanied by mitochondrial damage both in vivo and in vitro. The observations also were supported by the increased mtDNA concentrations in the bronchial lavage fluid of mice infected with S. pneumoniae. In summary, our study demonstrated that Ply triggered the production of IFN-ß in epithelial cells, and this response was mediated by mtDNA released from Ply-damaged mitochondria. It displayed an impressive modulation of IFN-ß response to S. pneumoniae in epithelial cells.

Nontypeable Haemophilus influenzae DNA stimulates type I interferon expression via STING signaling pathway.

Nontypeable Haemophilus influenzae (NTHI) is one of the leading causes of acute exacerbations of COPD (AECOPD). Although the immunoregulation function of NTHI outer member protein and endotoxin were confirmed, the role of NTHI DNA in activating immune responses remains to be elucidated. In this study, we found expression of IFN-ß and IFN stimulated gene CXCL10 in host cells was forcefully elevated after treating with NTHI and NTHI DNA. Interestingly, we tested increased level of STING in NTHI infected mice lung. Meanwhile, STING expression in lung of mimic COPD murine model was higher than healthy mice after NTHI infection. Importantly, knockout of STING or overexpression of STING, TBK1 and IRF3 respectively impaired or enhanced IFN-ß and CXCL10 expression during treating with NTHI and NTHI DNA. NTHI and NTHI DNA-induced I-IFN response appeared to be mediated by cGAS. Collectively, we suggested that NTHI DNA as a PAMP triggered I-IFN response, which was STING/TBK1/IRF3 dependent. SUMMARY: NTHI is the leading cause of acute exacerbations of COPD (AECOPD). Since AECOPD is an immune event, it is meaningful to elucidate the mechanism under NTHI induced immune response. It has been revealed that lipooligosaccharides and protein of NTHI could induce host immune response, but the function of NTHI nuclide acid during infection is unclear. In this research, we demonstrate NTHI DNA is a trigger for I-IFN expression, and the STING/TBK1/IRF3 pathway plays an integral role in sensing NTHI DNA to induce I-IFN expression. Moreover, by long-term intrabronchial infection of LPS, we constructed a mimic COPD murine model, in which the STING expression in lung tissues were higher than healthy mice after NTHI infection, which led us to surmise that NTHI cause AECOPD by inducing I-IFN production via STING signal pathway.

miR-29b Reverses T helper 1 cells/T helper 2 cells Imbalance and Alleviates Airway Eosinophils Recruitment in OVA-Induced Murine Asthma by Targeting Inducible Co-Stimulator.

Asthma is a complex chronic disease and the pathogenesis is still not entirely clear. In this study, we aimed to clarify the role and mechanism of miR-29b in the development of asthma. We observed that miR-29b levels were decreased in the lung and spleen of OVA-induced asthmatic mice. Reverse transcription-quantitative polymerase chain reaction and flow cytometry demonstrated that the inducible co-stimulator (ICOS) expression at mRNA and protein levels was elevated in the lung of asthmatic mice, and miR-29b expression in the lung of asthmatic mice was negatively associated with ICOS mRNA levels by Pearson Correlation analysis. Additional, flow cytometry showed that the percentage of CD4+ICOS+ T cells in the lung and spleen was regulated by miR-29b, and dual luciferase reporter assay confirmed ICOS was a target gene of miR-29b. Furthermore, miR-29b overexpression in asthmatic mice was induced with miR-29b agomir by intranasal administration; miR-29b alleviated total inflammatory cell infiltration and CCL24 levels, decreased IL-5 levels in bronchoalveolar lavage fluid and serum, and upregulated IFN-γ expression in serum. This study demonstrates that miR-29b targets ICOS, thereby reverses the imbalance of T helper 1 cells (Th1)/Th2 responses and decreases eosinophils recruitment in the airway, which are key features of allergic airway inflammation. Therefore, miR-29b might be an attractive candidate target for asthma treatment.

spd1672, a novel in vivo-induced gene, affects inflammatory response in a murine model of Streptococcus pneumoniae infection.

spd1672, a novel Streptococcus pneumoniae (hereinafter S. pn) gene induced in vivo, has been identified to contribute to the virulence of S. pn; however, the role of spd1672 during host innate immune reaction against S. pn infection is unknown. In the present study, mice were infected with wild-type D39 and mutant D39Δspd1672 strains. Compared with the D39-infected mice, reduced bacterial load and attenuated inflammatory response were observed in the D39Δspd1672-treated mice. The levels of proinflammatory cytokines, including IFN-γ, TNF-α, and IL-1ß, in the blood of D39Δspd1672-infected mice were lower than that in the D39-infected group. Additionally, attenuated activation of STAT3 and AKT was observed in the D39Δspd1672-infected mice. In conclusion, our data indicated that spd1672 expression modulates the release of proinflammatory cytokines, and AKT-STAT3 signaling appears to participate in the process. In conclusion, the present study extends our understanding of the role of an in vivo-induced gene in S. pn-host interaction.

Interleukin-17A Aggravates Middle Ear Injury Induced by Streptococcus pneumoniae through the p38 Mitogen-Activated Protein Kinase Signaling Pathway.

Acute otitis media (AOM) is one of the most common bacterial infectious diseases in children aged 2 to 7 years worldwide. We previously demonstrated that interleukin-17A (IL-17A) promotes an acute inflammatory response characterized by the influx of neutrophils into the middle ear cavity during Streptococcus pneumoniae-induced AOM. In general, the inflammatory response is viewed as an effector that frequently causes local tissue damage. However, little is known about the pathogenic effects of IL-17A in AOM. Here, we investigated the pathogenic effects of IL-17A by using wild-type (WT) and IL-17A knockout (KO) mouse models. The results showed that the pathogenic effects of AOM, including weight loss, histopathological changes, and proinflammatory cytokine production, were more severe in WT mice than in IL-17A KO mice, suggesting that IL-17A aggravates tissue damage in AOM. Furthermore, these pathogenic effects were found to be dependent on p38 mitogen-activated protein kinase (MAPK) and could be reversed in the presence of a p38 MAPK-specific inhibitor. It was also demonstrated that IL-17A promoted the production of neutrophil myeloperoxidase (MPO) through the p38 MAPK signaling pathway, which was responsible for the middle ear tissue injury. These data support the conclusion that IL-17A contributes to middle ear injury through the p38 MAPK signaling pathway.

A purification method for tag-free human cystatin C recombinant protein expressed in Escherichia coli.

To obtain recombinant cystatin C (CysC) protein, which can be used in immunological diagnostic kits, we focused on the preparation of tag-free CysC. The 6 × His-TF-CysC fusion protein was found to overexpress in soluble form in cells of BL21-Gold (DE3)/pCold TF-CysC, which had been induced with isopropyl-D-1-thiogalactopyranoside. Subsequently, we established a protein purification method for tag-free CysC using immobilized metal-affinity chromatography and size-exclusion chromatography. In this method, glutathione-S-transferase-human rhinovirus 3C proteases were used to remove the protein tags. High homogeneity of the purified CysC was determined by SDS-PAGE, while the purity of the tag-free CysC was ascertained to be above 95%. With a yield of 25 mg/L from bacterial culture, the biological activity of the tag-free CysC was evaluated as inhibitors like natural CysC. The performance of this purification method was successfully evaluated in the preparation of other low molecular weight heterologous proteins in Escherichia coli.

TLR2 promotes macrophage recruitment and Streptococcus pneumoniae clearance during mouse otitis media.

BACKGROUND: The natural course of otitis media (OM) in most children is acute and self-limiting; however, approximately 10-20% of children can experience persistent or recurrent OM. Determining the host factors that influence outcome of OM will help us design better therapies. This study focused on the role of Toll-like receptor 2 (TLR2) in a pneumococcal OM mouse model. METHODS: The middle ears (MEs) of wild-type (WT) and TLR2-/- mice were inoculated with Streptococcus pneumoniae (Spn) serotype 19F via transbullar injection. ME TLR2 expression in WT mice was determined by qRT-PCR and immunofluorescence. ME pathological manifestations, inflammatory response, and pneumococcal clearance between WT and TLR2-/- mice were compared after Spn inoculation. RESULTS: TLR2 expression in ME mucosa was markedly enhanced following infection with Spn in WT mice. In contrast to WT mice, TLR2-/- mice exhibited unaffected early ME inflammatory response. During late stage of ME infection, however, the absence of TLR2 can lead to reduced macrophage recruitment, impaired Spn clearance, and prolonged ME inflammation. CONCLUSION: Our results demonstrate that TLR2 signaling is critical for bacterial clearance and timely resolution of inflammation in OM induced by Spn.

Interleukin 4 Deficiency Reverses Development of Secondary Pseudomonas aeruginosa Pneumonia During Sepsis-Associated Immunosuppression.

BACKGROUND: Interleukin 4 (IL-4) is an important cytokine that may modulate development of secondary bacterial pneumonia during sepsis-induced immunosuppression. METHODS: We established an experimental model of cecal ligation and puncture (CLP)-induced sublethal polymicrobial sepsis followed by secondary Pseudomonas aeruginosa pulmonary infection, RESULTS: IL-4-deficient mice that underwent CLP were resistant to secondary pulmonary P. aeruginosa infection. As compared to wild-type mice, IL-4 knockout (KO) mice displayed improved survival and better bacterial clearance. Furthermore, IL-4 KO mice exhibited enhanced lung inflammation, neutrophil recruitment to airspaces, and elevated pulmonary cytokine production, with significantly increased tumor necrosis factor α (TNF-α) production. Neutralization of TNF-α could reverse the enhanced protection against secondary P. aeruginosa pneumonia in septic IL-4 KO mice, indicating that the resistance of septic IL-4 KO mice to secondary bacterial pneumonia was partially mediated by TNF-α. In addition, IL-4 priming displayed marked impairment of the ability of alveolar macrophages to phagocytose and kill P. aeruginosa in vitro, and this defect was associated with decreased activation of Akt, JNK, p38MAPK, and ERK intracellular signaling pathways by IL-4. Finally, neutralization of IL-4 in septic mice could improve survival and clearance of bacteria from the lungs of septic mice infected with P. aeruginosa. CONCLUSIONS: Our findings provide new insight for immunopathologic mechanisms of sepsis-induced secondary bacterial pneumonia.

Mucosal immunization with the live attenuated vaccine SPY1 induces humoral and Th2-Th17-regulatory T cell cellular immunity and protects against pneumococcal infection.

Mucosal immunization with attenuated vaccine can protect against pneumococcal invasion infection, but the mechanism was unknown. Our study found that mucosal delivery with the live attenuated SPY1 vaccine strain can confer T cell- and B cell-dependent protection against pneumococcal colonization and invasive infection; yet it is still unclear which cell subsets contribute to the protection, and their roles in pneumococcal colonization and invasion remain elusive. Adoptive transfer of anti-SPY1 antibody conferred protection to naive µMT mice, and immune T cells were indispensable to protection examined in nude mice. A critical role of interleukin 17A (IL-17A) in colonization was demonstrated in mice lacking IL-17A, and a vaccine-specific Th2 immune subset was necessary for systemic protection. Of note, we found that SPY1 could stimulate an immunoregulatory response and that SPY1-elicited regulatory T cells participated in protection against colonization and lethal infection. The data presented here aid our understanding of how live attenuated strains are able to function as effective vaccines and may contribute to a more comprehensive evaluation of live vaccines and other mucosal vaccines.

c-Jun N-terminal kinase and Akt signalling pathways regulating tumour necrosis factor-α-induced interleukin-32 expression in human lung fibroblasts: implications in airway inflammation.

Airway inflammatory diseases such as chronic obstructive pulmonary disease (COPD) and asthma are associated with elevated expression of interleukin-32 (IL-32), a recently described cytokine that appears to play a critical role in inflammation. However, so far, the regulation of pulmonary IL-32 production has not been fully established. We examined the expression of IL-32 by tumour necrosis factor-α (TNF-α) in primary human lung fibroblasts. Human lung fibroblasts were cultured in the presence or absence of TNF-α and/or other cytokines/Toll-like receptor (TLR) ligands or various signalling molecule inhibitors to analyse the expression of IL-32 by quantitative RT-PCR and ELISA. Next, activation of Akt and c-Jun N-terminal kinase (JNK) signalling pathways was investigated by Western blot. Interleukin-32 mRNA of four spliced isoforms (α, ß, γ and δ) was up-regulated upon TNF-α stimulation, which was associated with a significant IL-32 protein release from TNF-α-activated human lung fibroblasts. The combination of interferon-γ and TNF-α induced enhanced IL-32 release in human lung fibroblasts, whereas IL-4, IL-17A, IL-27 and TLR ligands did not alter IL-32 release in human lung fibroblasts either alone, or in combination with TNF-α. Furthermore, the activation of Akt and JNK pathways regulated TNF-α-induced IL-32 expression in human lung fibroblasts, and inhibition of the Akt and JNK pathways was able to suppress the increased release of IL-32 to nearly the basal level. These data suggest that TNF-α may be involved in airway inflammation via the induction of IL-32 by activating Akt and JNK signalling pathways. Therefore, the TNF-α/IL-32 axis may be a potential therapeutic target for airway inflammatory diseases.

A novel protein, RafX, is important for common cell wall polysaccharide biosynthesis in Streptococcus pneumoniae: implications for bacterial virulence.

Teichoic acid (TA), together with peptidoglycan (PG), represents a highly complex glycopolymer that ensures cell wall integrity and has several crucial physiological activities. Through an insertion-deletion mutation strategy, we show that ΔrafX mutants are impaired in cell wall covalently attached TA (WTA)-PG biosynthesis, as evidenced by their abnormal banding patterns and reduced amounts of WTA in comparison with wild-type strains. Site-directed mutagenesis revealed an essential role for external loop 4 and some highly conserved amino acid residues in the function of RafX protein. The rafX gene was highly conserved in closely related streptococcal species, suggesting an important physiological function in the lifestyle of streptococci. Moreover, a strain D39 ΔrafX mutant was impaired in bacterial growth, autolysis, bacterial division, and morphology. We observed that a strain R6 ΔrafX mutant was reduced in adhesion relative to the wild-type R6 strain, which was supported by an inhibition assay and a reduced amount of CbpA protein on the ΔrafX mutant bacterial cell surface, as shown by flow cytometric analysis. Finally, ΔrafX mutants were significantly attenuated in virulence in a murine sepsis model. Together, these findings suggest that RafX contributes to the biosynthesis of WTA, which is essential for full pneumococcal virulence.

An efficient alternative marker for specific identification of Mycobacterium tuberculosis.

Rapid and accurate identification of mycobacteria to the species level is important to provide epidemiological information and to guide the appropriate treatment, especially identification of the Mycobacterium tuberculosis (MTB) which is the leading pathogen causing tuberculosis. The genetic marker named as Mycobacterium tuberculosis specific sequence 90 (mtss90) was screened by a bioinformatics software and verified by a series of experiments. To test its specificity, 266 strains of microorganisms and human cells were used for the mtss90 conventional PCR method. Moreover, the efficiency of mtss90 was evaluated by comparing 16S rDNA (Mycobacterium genus-specific), IS6110 (specific identification of MTB complex), mtp40 (MTB-specific) and PNB/TCH method (traditional bacteriology testing) in Mycobacterium strains. All MTB isolates were mtss90 positive. No amplification was observed from any other tested strains with M. microti as an exception. Compared with the traditional PNB/TCH method, the coincidence rate was 99.1 % (233/235). All of the mtss90 positive strains were IS6110 and 16S rDNA positive, indicating a 100 % coincidence rate (216/216) between mtss90 and these two genetic markers. Additionally, mtss90 had a better specificity than mtp40 in the identification of MTB. Lastly, a real-time PCR diagnostic assay was developed for the rapid identification of MTB. In conclusion, mtss90 may be an efficient alternative marker for species-specific identification of MTB and could be used for the diagnosis of tuberculosis combined with other genetic markers.

Synergy of IL-27 and TNF-α in regulating CXCL10 expression in lung fibroblasts.

IL-27 is involved in inflammatory reactions. CXCL10 is an important chemokine contributing to airway inflammatory disease. In this study, we investigated whether IL-27 modulated the synthesis of CXCL10 in primary human lung fibroblasts (HLFs). HLFs were activated by IL-27 alone, or in combination with other cytokines. CXCL10 synthesis was measured by real-time PCR and ELISA. An examination of transcriptional regulation was performed via the transient transfection of promoter constructs, whereas mRNA stability was assessed by actinomycin D chase and real-time PCR. The underlying signaling pathways were studied by Western blotting and intracellular staining, using flow cytometry. Our results demonstrated that IL-27 induced and synergized with TNF-α to up-regulate CXCL10 mRNA and protein concentrations in a steroid-insensitive manner. This synergistic CXCL10 production was dependent on the transcriptional regulation of CXCL10 gene promoter activity and the enhanced stability of CXCL10 mRNA because of IL-27 and TNF-α, and this synergism was regulated by the activation of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-OH kinase (PI3K)-Akt dominantly, and in small part via NF-κB. Interestingly, IL-27 promoted the basal and enhanced TNF-α-induced phosphorylation of p38 MAPK and Akt, but not IκBα. Moreover, enhanced CXCL10 mRNA stability occurred via a p38 MAPK-dependent pathway. Finally, clinical analysis showed that IL-27 was detected in the bronchoalveolar lavage of patients with asthma, chronic obstructive pulmonary disease (COPD), and pulmonary tuberculosis (PTB), and increased IL-27 concentrations were correlated with increased CXCL10 concentrations in patients with COPD and PTB. Our findings suggest that IL-27 has the potential to amplify airway inflammation via the induction of CXCL10 from HLFs, in combination with TNF-α.

Screening and identification of DnaJ interaction proteins in Streptococcus pneumoniae.

Streptococcus pneumoniae DnaJ is recognized as a virulence factor whose role in pneumococcal virulence remains unclear. Here, we attempted to reveal the contribution of DnaJ in pneumococcal virulence from the identification of its interacting proteins using co-immunoprecipitation method. dnaJ was cloned into plasmid pAE03 generating pAE03-dnaJ-gfp which was used to transform S. pneumoniae D39 strain. Then anti-GFP coated beads were used to capture GFP-coupled proteins from the bacterial lysate. The resulting protein mixtures were subjected to SDS-PAGE and those differential bands were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. We finally obtained nine proteins such as DnaK, Gap, Eno, SpxB using this method. Furthermore, to confirm the interaction between DnaJ and these candidates, bacterial two-hybrid system was employed to reveal, for example, the interaction between DnaJ and DnaK, Eno, SpxB. Further protein expression experiments suggested that DnaJ prevented denaturation of Eno and SpxB at high temperature. These results help to understand the role of DnaJ in the pathogenesis of S. pneumoniae.

PdPtB Electrochemiluminescence Nanoenhancer and SiC@Au-PEDOT Nanowires-Based Detection of ß-Amyloid Oligomers in Alzheimer's Disease.

ß-Amyloid oligomers (AßOs) are promising biomarkers for the diagnosis of Alzheimer's disease (AD). The present research introduces a novel electrochemiluminescence (ECL) immunosensor based on PdPtB nanoenhancer and SiC@Au-PEDOT nanowires (NWs) for the specific and ultrasensitive detection of AßOs. The PdPtB nanoenhancer exhibited excellent oxidase-like catalytic activity with in situ generation of reactive oxygen species (ROS) to enhance luminol ECL in neutral media. In addition, SiC@Au-PEDOT NWs were utilized as a biocompatible and conductive substrate for the modification of the glassy carbon electrode (GCE). With this design, the ECL immunosensor showed outstanding AßOs analytical performance without exogenous coreactant. The ECL immunosensor demonstrated a favorable linear range of 20 pM to 20 nM and a detection limit of 10 pM under optimized conditions with potential straightforward clinical application. In general, the developed ECL immunosensor provides a promising strategy for the early diagnosis of AD.

Detoxified pneumolysin derivative ΔA146Ply inhibits triple- negative breast cancer metastasis mainly via mannose receptor-mediated autophagy inhibition.

The detoxified pneumolysin derivative ΔA146Ply has been proven to have a direct anti-triple negative breast cancer effect by our group, but its work model remains unclear. In this study, we focused on its ability to inhibit triple-negative breast cancer metastasis. We found that ΔA146Ply suppressed the migration and invasion of triple-negative breast cancer cells by activating mannose receptor and toll-like receptor 4. Their activation triggers the activation of the mammalian target of rapamycin signaling, sequentially leading to autophagy, transforming growth factor-ß1, and epithelial-mesenchymal transition inhibition. Furthermore, the combination of doxorubicin and ΔA146Ply significantly inhibited triple-negative breast cancer progression and prolonged survival in tumor-bearing mice. Taken together, our study provides an alternative microbiome-based mannose receptor-targeted therapy for triple-negative breast cancer and a novel theoretical and experimental basis for the downstream signaling pathway of the mannose receptor.