Rational molecular design converting fascaplysin derivatives to potent broad-spectrum inhibitors against bacterial pathogens via targeting FtsZ.

The filamentous temperature-sensitive mutant Z protein (FtsZ), a key player in bacterial cell division machinery, emerges as an attractive target to tackle the plight posed by the ever growing antibiotic resistance over the world. Therefore in this regard, agents with scaffold diversities and broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens are highly needed. In this study, a new class of marine-derived fascaplysin derivatives has been designed and synthesized by Suzuki-Miyaura cross-coupling. Some compounds exhibited potent bactericidal activities against a panel of Gram-positive (MIC = 0.024-6.25 µg/mL) and Gram-negative (MIC = 1.56-12.5 µg/mL) bacteria including methicillin-resistant S. aureus (MRSA). They exerted their effects by dual action mechanism via disrupting the integrity of the bacterial cell membrane and targeting FtsZ protein. These compounds stimulated polymerization of FtsZ monomers and bundling of the polymers, and stabilized the resulting polymer network, thus leading to the dysfunction of FtsZ in cell division. In addition, these agents showed negligible hemolytic activity and low cytotoxicity to mammalian cells. The studies on docking and molecular dynamics simulations suggest that these inhibitors bind to the hydrophilic inter-domain cleft of FtsZ protein and the insights obtained in this study would facilitate the development of potential drugs with broad-spectrum bioactivities.

The study of the role of purified anti-mouse CD193 (CCR3) antibody in allergic rhinitis mouse animal models.

The pathogenesis of allergic asthma is similar to that of allergic rhinitis, with inflammation cells producing and releasing inflammatory mediators and cytokines closely related to CCR3.Based on the theory of "one airway, one disease", the use of CCR3 monoclonal antibody may have a similar effect on allergic rhinitis. However, there are few studies on CCR3 monoclonal antibody in allergic rhinitis. Therefore, the aim of this study was to investigate the effective concentration of CCR3 monoclonal antibody, to compare the effects of different methods of administration, and to examine the lung condition of allergic mice to investigate whether antibody treatment protects the lungs. In this study, we constructed a mouse model of allergic rhinitis and intraperitoneally injected different doses of CCR3 monoclonal antibody (5, 10, and 20 uL/mg) to observe its therapeutic effect: observing changes in tissue morphology of nasal mucosa, infiltration of inflammation, and using ELISA to detect changes in relevant inflammatory mediators and cytokines, studying the role of CCR3 mAb in inhibiting CCR3-related actions on the nasal mucosa of allergic rhinitis mice. Furthermore, In addition, the therapeutic effects of intraperitoneal injection (i.p.) and intranasal administration (i.n.) were studied on the basis of effective concentrations.

Design and synthesis of fascaplysin derivatives as inhibitors of FtsZ with potent antibacterial activity and mechanistic study.

The increase in antibiotic resistance has made it particularly urgent to develop new antibiotics with novel antibacterial mechanisms. Inhibition of bacterial cell division by disrupting filamentous temperature-sensitive mutant Z (FtsZ) function is an effective and promising approach. A series of novel fascaplysin derivatives with tunable hydrophobicity were designed and synthesized here. The in vitro bioactivity assessment revealed that these compounds could inhibit the tested Gram-positive bacteria including methicillin-resistant S. aureus (MRSA) (MIC = 0.049-25 µg/mL), B. subtilis (MIC = 0.024-12.5 µg/mL) and S. pneumoniae (MIC = 0.049-50 µg/mL). Among them, compounds B3 (MIC = 0.098 µg/mL), B6 (MIC = 0.098 µg/mL), B8 (MIC = 0.049 µg/mL) and B16 (MIC = 0.098 µg/mL) showed the best bactericidal activities against MRSA and no significant tendency to trigger bacterial resistance as well as rapid bactericidal properties. The cell surface integrity of bacteria was significantly disrupted by hydrophobic tails of fascaplysin derivatives. Further studies revealed that these highly active amphiphilic compounds showed low hemolytic activity and cytotoxicity to mammalian cells. Preliminary mechanistic exploration suggests that B3, B6, B8 and B16 are potent FtsZ inhibitors to promote FtsZ polymerization and inhibit GTPase activity of FtsZ, leading to the death of bacterial cells by inhibiting bacterial division. Molecular docking simulations and structure-activity relationship (SAR) study reveal that appropriate increase in the hydrophobicity of fascaplysin derivatives and the addition of additional hydrogen bonds facilitated their binding to FtsZ proteins. These amphiphilic fascaplysin derivatives could serve as a novel class of FtsZ inhibitors, which not only gives new prospects for the application of compounds containing this skeleton but also provides new ideas for the discovery of new antibiotics.

Experimental observation of the effect of immunotherapy on CD4+ T cells and Th1/Th2 cytokines in mice with allergic rhinitis.

The present study aims to investigate the effect of immunotherapy in a mouse model of allergic rhinitis (AR) and to explore the possible molecular mechanisms of action. An animal model of AR was established by sensitization and challenge of BALB/c mice with house dust mite (HDM) extract. The mice were injected subcutaneously with HDM for immunotherapy. AR nasal symptoms were evaluated according to the frequencies of nose rubbing and sneezing and the degree of rhinorrhea. The nasal mucosa and lung tissue architecture and inflammatory status by histological analysis; the infiltration of eosinophils in nasal lavage fluid (NALF) of mice was observed by Diff-Quik stain; ELISA-based quantification of serum HDM-specific IgE and TH1/TH2 cytokine concentration; and flow cytometry detected the number of serum CD4+/CD8+ cells to evaluate the mechanism of immunotherapy. It was found that after immunotherapy, the AR symptom score was reduced, the number of eosinophils in NALF was reduced, and the infiltration of inflammatory cells and tissue damage in the nasal mucosa and lung tissue were alleviated. Immunotherapy can increase the number of CD4+ T cells in the peripheral blood, increase the ratio of CD4+/CD8+ cells, increase the expression of Th1 cytokines such as IL-2 and IFN-γ, reduce the expression of Th2 cytokines such as IL-4 and IL-5. The results showed that repeated intraperitoneal injection of crude extract of HDM for sensitization, followed by nasal drops can effectively construct a mouse model of AR, and subcutaneous injection of immunotherapy in mice can reduce allergic inflammation in model mice and improve the inflammatory infiltration of the nasal cavity in allergic rhinitis. Immunotherapy can reduce the expression of inflammatory factors in AR, improve Th1/Th2 balance, and may play a role in the treatment of AR by improving the function of immune cells.

Knockdown of CCR3 gene inhibits Proliferation, migration and degranulation of eosinophils in mice by downregulating the PI3K/Akt pathway.

This study aimed to investigate the effects of CCR3 knockdown (CCR3-/-) on the proliferation, migration, and degranulation of the bone marrow eosinophils (EOS) in mice. Bone marrow cells from wild-type mice (WT) were harvested for primary culture and differentiated into mature EOS, which were then randomly divided into the control, 740Y-P, and LY294002 group. The effects of different concentrations of LY294002 (PI3K inhibitor) and 740Y-P (PI3K agonist) on the proliferation viability of EOS, expressions of EPO, Akt, and p-Akt proteins, and migration changes of EOS were detected. CCR3-/- mice were identified. Then, bone marrow cells of WT and CCR3-/- mice were differentiated into mature EOS and grouped into WT EOS, WT EOS + eotaxin (100 ng/mL), CCR3-/- EOS, and CCR3-/- EOS + eotaxin (100 ng/mL) group. The changes in EOS proliferation, migration, as well as expressions of EPO, Akt, and p-Akt proteins were detected. The number of migrated cells (P < 0.01) and expression of EPO (p < 0.05) in the 740Y-P group were higher than those in the control group, while opposite trends were observed for the LY294002 group. Expression levels of p-Akt and Akt in the LY294002 group were significantly lower than in the control group (all P < 0.01). Also, the expression of p-Akt in the 740Y-P group was significantly higher than that in the control group (p < 0.05). The proliferative activity of EOS, expression of EPO and p-Akt, and the number of migrated cells in the WT EOS group were higher than those in CCR3-/- EOS group (all P < 0.05). After adding eotaxin, the WT EOS group was higher than the other three groups (all P < 0.05). Mechanistically, CCR3-/- inhibited EOS's proliferation, migration, and degranulation by downregulating PI3K/Akt pathway. This data suggests that the knockout of the CCR3 gene in bone marrow cells may inhibit the function of EOS by downregulating the PI3K/Akt pathway, thereby affecting AR; thus, the CCR3 gene may be a target gene for AR therapy.

[Study on expression and mechanism of serum differential proteins after rush immunotherapy of allergic rhinitis].

Objective:To detect the expression of differentially expressed proteins in serum of patients with allergic rhinitis who were allergic to dust mites before and after 6-day rush immunotherapy. The three differentially expressed proteins, CRP, CTHRC1 and WDR89, were detected and identified. The immunoregulatory effects and significance of these three differentially expressed proteins in rush immunotherapy of allergic rhinitis were analyzed and discussed. Method:The serum samples of 15 patients with allergic rhinitis, 15 patients with rush immunotherapy and 10 patients with healthy control group were collected. The samples were studied by isobaric tags for relative and absolute quantitation（iTRAQ） technique. The related differential proteins were determined by two-dimensional gel electrophoresis and mass spectrometry, and the rationality of the screened differential proteins was tested and verified by Cluster3.0 software and Java TreeView software. Finally, the selected CRP, CTHRC1 and WDR89 proteins were identified by enzyme-linked immunosorbent assay（ELISA）. Result:In this study, 893 proteins were detected and 53 differential proteins were identified. Compared with healthy control group, 24 proteins which was statistically significant were found in allergic rhinitis group, which were closely related to the occurrence of allergic rhinitis, including 10 up-regulated proteins and 14 down-regulated proteins. Compared with the allergic rhinitis group, patients with allergic rhinitis underwent 6 days of rush immunotherapy. There were 29 proteins whose expression of proteins with a difference of P value of less than 0.05 and 1.2 times higher, which were related to the effect after the incremental phase of rush immunotherapy was completed, of which 12 were up-regulated and 17 were down-regulated. Compared with healthy control group, the expression of up-regulated of allergic rhinitis group and the expression of down-regulated protein after 6 days of rush immunotherapy were CTHRC1, WDR89; Compared with healthy control group, AR group was down-regulated and the expression of up-regulated protein after 6 days of rush immunotherapy was CRP. CRP, CTHRC1 and WDR89 proteins were identified by enzyme linked immunosorbent assay（ELISA）, and it was found that the differential expression of CTHRC1 and WDR89 in AR and RIT was statistically significant（P<0.05）, but the differential expression of serum CRP in AR and RIT was not statistically significant（P>0.05）. Conclusion:Serum protein CTHRC1 and WDR89 are closely related to the pathogenesis of allergic rhinitis, and played a role in the regulation of rush immunotherapy, while serum protein CRP has no significant effect on AR and RIT.

[Safety comparison of omalizumab and glucocorticoid in rush allergen immunotherapy].

Objective:To observe the safety of omalizumab and glucocorticoid in the dose-increasing phase of rush allergen immunotherapy（RIT）. Method:The clinical data of 88 patients with allergic rhinitis treated with RIT were retrospectively studied, including gender, age, pre-treatment total VAS score, blood EOS%, serum total IgE, local and systemic adverse reactions. Of all patients, fifty-seven were treated with omalizumab combined with RIT（experimental group） and thirty-one were treated with hormone/antiallergic drugs combined with RIT（control group）. The safety of the two groups was compared in the dose-increasing phase. Result:There was no grade Ⅰ systemic adverse reactions during the whole process in the experimental group, while Grade Ⅱ systemic adverse reactions were 4 cases（7.1%） during the period of hospitalization, 2 cases（3.6%） after the first injection after discharge, zero（0） after the second injection after discharge. No local pruritus and induration were observed. During the period of hospitalization, the first and second injection after discharge, control group had grade Ⅰ level systemic adverse reactions were 1 case（3.4%）, 2 cases（6.9%）, 1 case（3.4%） at different time point, respectively. Grade Ⅱ systemic adverse reactions were 5 cases（17.2%）, 1 case（3.4%）, zero（0） at different time point, respectively. Local injection site itching was observed in 8 patients（5 cases were mild and 3 cases were moderate） and 4 cases（13.8%） had induration during hospitalization. Conclusion:Omalizumab combined with RIT not only shortens the duration of dose-increasing phase of specific immunotherapy, but also increases the safety of the dose-increasing phase during hospitalization, the first and second injection after discharge and improves patient compliance.

CCR3-shRNA promotes apoptosis and inhibits chemotaxis and degranulation of mouse mast cells.

Mast cells (MCs) are the major effector cells of allergic rhinitis (AR). The present study aimed to investigate the effects of C-C chemokine receptor type 3 (CCR3) on the proliferation, apoptosis, chemotaxis and activated degranulation of mouse MCs. Mouse bone marrow-derived MCs were cultured in vitro, purified and identified using toluidine blue staining and flow cytometry. Three different CCR3-short hairpin (shRNA) lentiviral vectors were constructed and transfected into MCs, and the mRNA and protein expression levels of CCR3 were assessed by reverse transcription-quantitative PCR and western blotting. Proliferation and apoptosis of the MCs were measured using Cell Counting kit-8 (CCK-8) assays and flow cytometry, respectively. MC chemotaxis was assessed by Transwell assay and quantified using flow cytometry. The activation of MC degranulation was examined using ELISAs. The results demonstrated that MCs were appropriately isolated, and identified that CCR3-shRNA2 presented the higher knockdown effect among the three shRNAs tested. Following 96 h of transfection, the results of CCK-8 and flow cytometry assays demonstrated that CCR3-shRNA2 inhibited MC proliferation and promoted MC apoptosis. The results from the Transwell assay indicated that CCR3-shRNA2 restrained MC chemotaxis, whereas ELISA results demonstrated that CCR3-shRNA2 suppressed MC degranulation. In conclusion, CCR3-shRNA2 effectively downregulated CCR3 mRNA and protein expression levels in mouse MCs. In addition, CCR3-shRNA2 promoted MC apoptosis and suppressed the proliferation, chemotaxis and degranulation of mouse MCs, suggesting that CCR3-shRNA2 may serve as a therapeutic tool for the treatment of allergic rhinitis.

Effects of lentivirus-mediated CCR3 RNA interference on the function of mast cells of allergic rhinitis in mice.

The CC chemokine receptor 3 (CCR3) expressed by eosinophils, mast cells and Th2 cells is closely related to allergic diseases. The objective of this study was to explore whether silencing of CCR3 with short hairpin RNAs (shRNAs) delivered by a lentiviral vector could impact the function of mast cells in a murine model of allergic rhinitis (AR) in vivo. The murine model of allergic rhinitis (AR) inducing by ovalbumin (OVA) was constructed, and the BALB/c mice were divided into normal control group, AR group, controlshRNA treated group and lentiviral CCR3-shRNA treated group. The recombinant lentivirus vectors which express a short hairpin RNA (shRNA) targeting the CCR3 were dropped into the nasal cavity of OVA-sensitized mice before the challenges. Real-time fluorescence quantitative PCR and western blotting were performed to observe inhibitory effect of CCR3 gene. Nasal symptoms of mice and OVA-specific IgE in each group were assessed. Concentrations of histamine, tryptase and Prostaglandin D2 (PGD2) in bone marrow, peripheral blood and nasal mucosa were analyzed. Furthermore, histological analysis and electron microscopy analysis were applied to detect the histology changes of nasal mucosa and the infiltration of mast cells in nasal mucosa. The results showed that administration of CCR3shRNA could effectively inhibit the expression of the CCR3 gene in bone marrow, peripheral blood and nasal mucosa, which reduced the nasal symptoms, the level of OVA-specific IgE, the inflammatory cells and mast cells infiltration into nasal cavity, and relieved the histopathological changes of nasal mucosa. In addition, intervention of CCR3shRNA could reduce the levels of the histamine, tryptase and PGD2 in bone marrow, peripheral blood and nasal mucosa. These results suggest that inhibition of CCR3 gene expression by shRNAs lentiviral vectors can effectively attenuate migration, infiltration and degranulation of mast cells in local tissues and alleviate the inflammation of allergic rhinitis mice.

C­C chemokine receptor type 3 gene knockout alleviates inflammatory responses in allergic rhinitis model mice by regulating the expression of eosinophil granule proteins and immune factors.

The present study aimed to investigate the effects of C­C chemokine receptor type 3 (CCR3) gene knockout on allergic rhinitis (AR) in mice, as well as the underlying molecular mechanisms. Ovalbumin was administrated to CCR3+/+ and CCR3­/­ BALB/c mice to establish an AR model. The mice were divided into four groups: i) Normal control (CG), ii) AR model (AR), iii) CCR3 knockout CG (CCR3­/­CG) and iv) AR model with CCR3 knockout (CCR3­/­AR). Histological sections of nasal mucosae were examined by hematoxylin and eosin staining, which revealed that CCR3 knockout suppressed the invasion of inflammatory cells and relieved the damage of nasal mucosae. Peripheral blood smear and nasal­washing smears were evaluated by Wright's staining. Eosinophil (EOS) numbers in nasal mucosae, peripheral blood, and nasal washings of the various groups were ranked in the order: AR>CCR3­/­AR>CG>CCR3­/­. mRNA expression levels of CCR3, EOS peroxidase (EPO), EOS cationic protein (ECP), and major basic protein (MBP) in the peripheral serum and nasal washings were detected by reverse transcription­polymerase chain reaction. Interferon­Î³ (IFN­Î³), interleukin (IL)­4, IL­10, and immunoglobulin E (IgE) protein levels in the peripheral serum and nasal washings were investigated by ELISA. CCR3 mRNA expression was not detected in the CCR3­/­ and CCR3­/­AR groups, whereas expression levels in the AR group were markedly higher compared with expression in the CG group. Compared with the CG­associated groups (i.e., the CG and CCR3­/­CG groups), the levels of EPO, ECP, MBP, IL­4, and IgE were significantly increased in the AR­associated groups (that is, R and CCR3­/­AR). In addition, the CCR3­/­AR group mice produced significantly lower levels of EPO, ECP, MBP, IL­4 and IgE compared with the AR group, whereas the expression levels of IFN­Î³ and IL­10 were increased. CCR3 gene knockout may alleviate EOS invasion and the inflammatory response in AR model mice by reducing the expression levels of EPO, ECP, MBP, IL­4, and IgE, and increasing the expression of IL­10 and IFN­Î³.

Utilization of simulated flue gas for cultivation of Scenedesmus dimorphus.

Effects of flue gas components on growth of Scenedesmus dimorphus were investigated and two methods were carried out to eliminate the inhibitory effects of flue gas on microalgae. S. dimorphus could tolerate CO(2) concentrations of 10-20% and NO concentrations of 100-500 ppm, while the maximum SO(2) concentration tolerated by S. dimorphus was 100 ppm. Addition of CaCO(3) during sparging with simulated flue gas (15% CO(2), 400 ppm SO(2), 300 ppm NO, balance N(2)) maintained the pH at about 7.0 and the algal cells grew well (3.20 g L(-1)). By intermittent sparging with flue gas controlled by pH feedback, the maximum biomass concentration and highest CO(2) utilization efficiency were 3.63 g L(-1) and 75.61%, respectively. These results indicated that S. dimorphus could tolerate high concentrations of CO(2) and NO, and the methods of CaCO(3) addition and intermittent sparging have great potential to overcome the inhibition of flue gas on microalgae.