The adhesive strength of fiber post-to-canal dentin with Aniline green, Fotoenticine activated by PDT, green tea, and ozone as a final irrigant.

AIM: The effect of novel final disinfection protocols Malachite green (MG), Fotoenticine® (FTC), Green tea extract (GTE), and Ozonated water (OW) on the bond strength of prefabricated glass fiber posts (PGFP) adhered to canal dentin. MATERIAL AND METHOD: The canals of fifty premolars with closed apices were cleansed and obturated. The specimens were randomly assigned to one of five groups based on the final irrigant used, with the control group receiving NaOCl+EDTA and the experimental groups receiving MG, FTC, OW, and GTE. The GFP was cemented with a self-etching, dual-cure paste; the bond strength was estimated with a universal testing machine; and failure analysis was conducted with a stereomicroscope. RESULTS: The highest PBS was observed in the coronal third of Group 4 (using ozonated water as the final irrigant), whereas the lowest bond integrity was observed in the apical section of Group 2 (1.02-0.54 MPa) using Malachite green as the final irrigant. Group 1, Group 4, and Group 5 exhibited no significant difference in the bond integrity of GFP to dentin when compared to Group 2 (p>0.05). In addition, comparable bond score values were obtained for Groups 2 and 3 (p>0.05). CONCLUSION: The results of this study suggest that OW and GTE may be effective final disinfectants for root canals, as they increase the bond strength of resin-luting cement.

A Nickel-Containing Polyoxomolybdate as an Efficient Antibacterial Agent for Water Treatment.

In view of the water pollution issues caused by pathogenic microorganisms and harmful organic contaminants, nontoxic, environmentally friendly, and efficient antimicrobial agents are urgently required. Herein, a nickel-based Keggin polyoxomolybdate [Ni(L)(HL)]2H[PMo12O40] 4H2O (1, HL = 2-acetylpyrazine thiosemicarbazone) was prepared via a facile hydrothermal method and successfully characterized. Compound 1 exhibited high stability in a wide range of pH values from 4 to 10. 1 demonstrated significant antibacterial activity, with minimum inhibitory concentration (MIC) values in the range of 0.0019-0.2400 µg/mL against four types of bacteria, including Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), and Agrobacterium tumefaciens (A. tumefaciens). Further time-kill studies indicated that 1 killed almost all (99.9%) of E. coli and S. aureus. Meanwhile, the possible antibacterial mechanism was explored, and the results indicate that the antibacterial properties of 1 originate from the synergistic effect between [Ni(L)(HL)]+ and [PMo12O40]3-. In addition, 1 presented effective adsorption of basic fuchsin (BF) dyes. The kinetic data fitted a pseudo-second-order kinetic model well, and the maximum adsorption efficiency for the BF dyes (29.81 mg/g) was determined by the data fit of the Freundlich isotherm model. The results show that BF adsorption was dominated by both chemical adsorption and multilayer adsorption. This work provides evidence that 1 has potential to effectively remove dyes and pathogenic bacteria from wastewater.

Efficacy of malachite green mediated photodynamic therapy on treatment of Cutaneous Leishmaniasis: In vitro study.

BACKGROUND: Leishmaniasis is a common zoonotic disease that is transmitted by phlebotomus and causes several clinical conditions, from self healing lesion to deadly internal organ involvement. Photodynamic therapy (PDT) is a treatment method that leads to the generation of cytotoxic species and consequently to cell death and tissue destruction by visible light in the presence of a photosensitizer and oxygen. The aim of this study was to investigate effect of malachite green (MG)-mediated PDT in Leishmania tropica (L. tropica) promastigotes. MATERIAL AND METHODS: Parasites were incubated with 0.19, 0.39, 1.56, 3.25 and 6.25 µM of MG for one hour and subjected to 46.4 J/cm2 light irradiation. Trypan blue assay was used to evaluate the viability of the cells and mitochondirial activity alteration was determined by MTT. Morphological changes were analyzed by Giemsa staining and Scanning electron microscopy (SEM) analyses. Flow cytometry was used to quantify the fluorescence emitted by cell volume, JC-1, Cell Cycle and Annexin V/PI staining reagents. RESULTS: Malachite green mediated photodynamic therapy at 1.56 and 3.125 µM decreased the viability of the L. tropica promastigotes and induced changes in the mitochondrial membrane potential. L.tropica promastigotes was bloked in G0/G1 phase. The morphology of the parasite was affected at the 1.56 and 3.125 µM MG+PDT, resulting in rounded cells with loss of flagellum and irregular shape. CONCLUSIONS: This study demonstrated that antileishmanial effects through mitochondrial dysfunction, cell cycle arrest, and apoptosis-like cell death to parasites. This work showed PDT with MG effectedparasites. Therefore, MG-mediated PDT may provide a promising approach for L. tropica promastigotes.

Study of the correlation between the noncanonical pathway of pyroptosis and idiopathic inflammatory myopathy.

BACKGROUND: The pathogenesis of idiopathic inflammatory myopathy (IIM) is complex and unclear. The purpose of this study was to investigate whether the noncanonical pathway of pyroptosis is involved in the pathogenesis of IIM, and the intervention effect of drugs glyburide and bright blue G (BBG). METHODS: After the drug intervention, we detected the expression of the caspase-4, caspase-5, caspase-11, GSDMD, pannexin-1, NLRP3 and P2X7R proteins in skeletal muscle tissues from the six groups using Western blotting. We detected the expression of the caspase-11, GSDMD, pannexin-1, NLRP3 and P2X7R mRNAs in skeletal muscle tissues from the six groups using RT-qPCR and detected the serum IL-18 and IL-1ß levels in the six groups using ELISAs. RESULT: Lower expression levels of the P2X7R and NLRP3 proteins were observed in the EAM + BBG group than in the EAM1 group (P < 0.05). The expression of NLRP3 in the EAM + glyburide group was lower than in the EAM2 group (P < 0.05). Lower expression levels of the P2X7R and NLRP3 mRNAs were detected in the EAM + BBG group than in the EAM1 group (P < 0.05). NLRP3 was expressed at lower levels in the EAM + glyburide group than in the EAM2 group (P < 0.05). Lower serum IL-1ß levels were detected in the EAM + BBG group than in the EAM1 group (P < 0.05), and serum IL-1ß and IL-18 levels in the EAM + glyburide group were lower than those in the EAM2 group (P < 0.05). CONCLUSION: Our results suggest that the noncanonical pathway of pyroptosis may be involved in the pathogenesis of IIM, and glyburide and BBG exert certain intervention effects on its pathogenesis.

Role of TRPV4-P2X7 Pathway in Neuropathic Pain in Rats with Chronic Compression of the Dorsal Root Ganglion.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable non-selective cation channel that is involved in the development of neuropathic pain. P2X7 receptor (P2X7) belongs to a class of ATP-gated nonselective cation channels that plays an important role in neuropathic pain. Nevertheless, little is known about the interaction between them for neuropathic pain. In this paper, we investigated role of TRPV4-P2X7 pathway in neuropathic pain. We evaluated the effect of TRPV4-P2X7 pathway on neuropathic pain in a chronic compression of the dorsal root ganglion (DRG) (hereafter termed CCD) model. We analyzed the effect of P2X7 on mechanical and thermal hyperalgesia mediated by TRPV4 in CCD. Furthermore, we assessed the effect of TRPV4 on the expression of P2X7 and the release of IL-1ß and IL-6 in DRG after CCD. We found that intraperitoneal injection of TRPV4 agonist GSK-1016790A led to a significant increase of mechanical and thermal hyperalgesia in CCD, which was partially suppressed by P2X7 blockade with antagonist Brilliant Blue G (BBG). Then, we further noticed that GSK-1016790A injection increased the P2X7 expression of CCD, which was decreased by TRPV4 blockade with antagonist RN-1734 and HC-067047. Furthermore, we also discovered that the expressions of IL-1ß and IL-6 were upregulated by GSK-1016790A injection but reduced by RN-1734 and HC-067047. Our results provide evidence that P2X7 contributes to development of neuropathic pain mediated by TRPV4 in the CCD model, which may be the basis for treatment of neuropathic pain relief.

P2X7 receptor antagonist BBG inhibits endoplasmic reticulum stress and pyroptosis to alleviate postherpetic neuralgia.

Postherpetic neuralgia (PHN) is the most common complication of acute herpes zoster. The treatment of PHN remains a challenge for clinical pain management. The present study investigated the P2X7 receptor antagonist brilliant blue G (BBG) whether inhibits endoplasmic reticulum stress and pyroptosis (a necrotic form of cell death) and alleviates PHN. Varicella zoster virus (VZV)-infected CV-1 cells were used to induce PHN model. Mechanical paw withdrawal thresholds were measured using an ascending series of von Frey filaments. Immunohistochemistry was used to detect the expression of P2X7R in nerve tissues. Western blot was used to determine the expression of endoplasmic reticulum (ER) stress and pyroptosis-related molecules. The expression of IL-1ß and IL-18 in tissue homogenate was detected by ELISA. The PHN rat has the lower paw withdrawal threshold, but higher expression of P2X7 in nerve tissues. And, endoplasmic reticulum stress was activated and pyroptosis was increased in PHN rats. BBG can decrease pain thresholds and reduce ER stress and pyroptosis in PHN rats. In addition, ER stress activator tunicamycin (TM) can reverse the effect of BBG on the paw withdrawal thresholds, endoplasmic reticulum stress, and pyroptosis. Therefore, P2X7 receptor antagonist BBG alleviates PHN by activating ER stress and reducing pyroptosis.

Properties of the Extracellular Polymeric Substance Layer from Minimally Grown Planktonic Cells of Listeria monocytogenes.

The bacterium Listeria monocytogenes is a serious concern to food processing facilities because of its persistence. When liquid cultures of L. monocytogenes were prepared in defined media, it was noted that planktonic cells rapidly dropped out of suspension. Zeta potential and hydrophobicity assays found that the cells were more negatively charged (-22, -18, -10 mV in defined media D10, MCDB 202 and brain heart infusion [BHI] media, respectively) and were also more hydrophobic. A SEM analysis detected a capsular-like structure on the surface of cells grown in D10 media. A crude extract of the extracellular polymeric substance (EPS) was found to contain cell-associated proteins. The proteins were removed with pronase treatment. The remaining non-proteinaceous component was not stained by Coomassie blue dye and a further chemical analysis of the EPS did not detect significant amounts of sugars, DNA, polyglutamic acid or any other specific amino acid. When the purified EPS was subjected to attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, the spectra obtained did not match the profile of any of the 12 reference compounds used. An x-ray diffraction (XRD) analysis showed that the EPS was amorphous and a nuclear magnetic resonance (NMR) analysis detected the presence of glycerol. An elemental energy dispersive x-ray (EDX) analysis showed traces of phosphorous as a major component. In conclusion, it is proposed that the non-proteinaceous component may be phospholipid in nature, possibly derived from the cell wall lipoteichoic acid.

Comparative proteomic analysis provides insight into the key proteins as potential targets underlying the effect of malachite green against Ichthyophthirius multifiliis.

Target identification is important for drug discovery. Unfortunately, no drug targets have been found in Ichthyophthirius multifiliis until now and further limited development of the novel drug for Ichthyophthiriasis. In this study, an iTRAQ-based quantitative proteomic analysis was used to find the target of malachite green (MG), exhibiting greater efficacy than the existing drugs, against I. multifiliis trophonts in situ. We also verified the proteomic results by RT-qPCR, TEM and cell apoptosis assay. Our results showed that major variations in protein abundance were found among many of the ribosome proteins, indicating ribosome might be a candidate target. Furthermore, GO and KEGG pathway analyses of differentially expressed proteins (DEPs) revealed that ribosome and PI3K-Akt signalling pathway were remarkably enriched. Taken together, the above DEPs were also verified by RT-qPCR and morphological observations. This study provides insights into the key proteins enriched in PI3K-Akt signal pathway and ribosome pathway as potential targets of MG killing I. multifiliis, which could be served as targets for other less toxic drugs and be tested as potential treatments for I. multifiliis.

BBG enhances OLT1177-induced NLRP3 inflammasome inactivation by targeting P2X7R/NLRP3 and MyD88/NF-κB signaling in DSS-induced colitis in rats.

Chronic ulceration of the colon is associated with the activation of TLR4/NF-κB and P2X7R/NLRP3 signaling pathways. We investigated the effect of individual or combined administration of BBG, a P2X7R blocker, and OLT1177, a selective NLRP3 inhibitor, in the dextran sodium sulfate-induced ulcerative colitis (UC) rat model. The ulcerative rats were treated orally with brilliant blue G (BBG) (50 mg/kg/day) or OLT1177 (200 mg/kg/day) or a combination of both. Myd88 and NF-κB levels were measured by ELISA, qRT-PCR, and immunohistochemical staining. Cytokines known to be associated with TLR4/NF-κB or P2X7R/NLRP3 signaling were measured by ELISA. P2X7R and NLRP3 expression were measured by ELISA and qRT-PCR. The administration of BBG or OLT1177 ameliorated the toxic effects of DSS on the colon as they restored normal colonic macroscopic and microscopic morphology. BBG administration, but not OLT1177, reduced the expression of Myd88, NF-κB, IL-6, and TNF-α in addition to lowering P2X7R and oxidative stress levels. Individual BBG or OLT1177 administration decreased NLRP3 inflammasome recruitment and subsequent activation of caspase-1, IL-1ß, and IL-18. However, the combined administration of OLT1177 with BBG potentiated its inhibitory effect on the NLRP3, which was reflected by the additional suppressive effect on caspase-1, IL-1ß, IL-18 levels. In conclusion, BBG/OLT1177 exhibited complementary effects and effectively ameliorated UC. This novel approach provides a basis for the clinical application of this combination for the treatment of IBDs and might also be promising for the pharmacological intervention of other NLRP3 inflammasome-dependent inflammatory conditions.

P2X7 receptor antagonism increases regulatory T cells and reduces clinical and histological graft-versus-host disease in a humanised mouse model.

Graft-versus-host disease (GVHD) is a severe inflammatory response arising from allogeneic haematopoietic stem cell transplantation. Previous studies revealed that antagonism of the P2X7 receptor with Brilliant Blue G (BBG) reduced liver GVHD but did not alter clinical GVHD in a humanised mouse model. Therefore, the present study aimed to trial a modified injection regime using more frequent dosing of BBG to improve outcomes in this model of GVHD. NOD-scid IL2Rγnull (NSG) mice were injected intraperitoneally (i.p.) with 10 × 106 human peripheral blood mononuclear cells (hPBMCs) (day 0), then daily with BBG (50 mg/kg) or saline (days 0-10). BBG significantly reduced clinical score, mortality and histological GVHD compared with saline treatment (endpoint). BBG significantly increased proportions of human regulatory T cells (Tregs) and human B cells and reduced serum human interferon-γ compared with saline treatment prior to development of clinical GVHD (day 21). To confirm the therapeutic benefit of P2X7 antagonism, NSG mice were injected i.p. with 10 × 106 hPBMCs (day 0), then daily with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (300 mg/kg) or saline (days 0-10). PPADS increased human Treg proportions compared with saline treatment (day 21), but potential clinical benefits were confounded by increased weight loss with this antagonist. To investigate the role of P2X7 antagonism on Treg survival, hPBMCs were cultured in reduced serum conditions to promote cell death. BBG increased proportions of Tregs (and B cells) compared with saline under these conditions. In conclusion, P2X7 antagonism reduces clinical and histological GVHD in a humanised mouse model corresponding to an increase in human Tregs.

Suppression of Malachite Green-Induced Toxicity to Human Liver Cells Utilizing Host-Guest Chemistry of Cucurbit[7]uril.

We investigated a host-guest complex between cucurbit[7]uril and malachite green, and its effect on the toxicity to human liver cells. The host-guest complexation was evaluated by a UV/vis titration and electrospray-ionization mass spectrometry. Interestingly, the host-guest complex resulted in remarkable suppression of the toxicity of malachite green in its practical concentration range (ca. ∼6 µM). This study is one step forward to the active control of the biological effects of potent toxicants utilizing host-guest chemistry.

pH-triggered solubility and cytotoxicity changes of malachite green derivatives incorporated in liposomes for killing cancer cells.

Three different malachite green leuco derivatives (MG-Xs) are incorporated in liposomes. In all three cases, a substituent (X) is covalently linked to the central carbon atom, abbreviated as MG-OH, MG-OCH3, and MG-CN. The three MG-X compounds are solubilized separately in liposome membranes and become cationic (MG+) and water soluble under acidic conditions. MG+ is consequently released from the liposome to the aqueous exterior. Their release behavior corresponds to their ionization ability: MG-OH > MG-OCH3 > MG-CN. The cellular uptake of the liposomes, the cytotoxic effect, and the location of MG+ in cancer cells are investigated using murine cells derived from colon cancer (Colon 26 cells) and human embryonic kidney cells (HEK 293 cells). The toxic effect on cancer cells is correlated to the ionization ability of MG-Xs. The liposomes effectively deliver MG+via the endocytic pathway, resulting in the cytotoxicity of liposomes containing MG-OH which is higher than that of free MG-OH and MG+. The difference in the phospholipids constituting the liposome membranes barely had an effect on the ionization ratio and the cytotoxicity of MG-OH. Confocal fluorescence microscopic observations revealed that MG+ is ultimately transported into the nuclei after being released in acidic cellular compartments.

P2X7 receptor antagonism inhibits tumour growth in human high-grade gliomas.

Gliomas, the most common primary brain cancer, are highly infiltrative and extremely difficult to treat. Despite advancements, current treatment is limited, with patients surviving for a median of 14-15 months post-diagnosis. Previous research has demonstrated the upregulation of a purinergic receptor, P2X7R, in human gliomas. P2X7R is expressed on both glioma cells and microglia within the glioma microenvironment. It is hypothesized that P2X7R contributes to tumour growth and proliferation via immune-mediated mechanisms involving tumour cells and surrounding microglia. We sought to elucidate the role of P2X7R in a human glioblastoma cell line (U251) and on surgically resected human glioma samples. We treated U251 and human glioma cultures for 72 h with P2X7R antagonists, Brilliant Blue G (BBG), oxidized ATP (oATP) and AZ10606120. Cell counting via fluorescence confocal microscopy was conducted to assess tumour proliferation. We observed no significant reductions in tumour cell numbers following P2X7R antagonism with BBG (20 µM) and oATP (250 µM) in both U251 cells and human glioma samples. Interestingly, there was a significant reduction in tumour cell number in both U251 cells (p = 0.0156) and human glioma samples (p = 0.0476) treated with varying concentrations of AZ10606120. When compared with the conventional chemotherapeutic agent, temozolomide, AZ10606120 was also found to more effectively inhibit tumour proliferation in U251 cells (p < 0.0001). Our pilot results demonstrate a potential trophic role of P2X7R where its inhibition by AZ10606120, a potent antagonist, hinders glioma growth directly or through the inactivation of microglia. This sheds new light on P2X7R as a therapeutic target for human gliomas.

Brilliant blue G, a P2X7 receptor antagonist, attenuates early phase of renal inflammation, interstitial fibrosis and is associated with renal cell proliferation in ureteral obstruction in rats.

BACKGROUND: Previous study showed that purinergic P2X7 receptors (P2X7R) reach the highest expression in the first week after unilateral ureteral obstruction (UUO) in mice, and are involved in the process of inflammation, apoptosis and fibrosis of renal tissue. We, herein, document the role of purinergic P2X7 receptors activation on the third day of UUO, as assessed by means of BBG as its selective inhibitor. METHODS: We investigated the effects of brilliant blue G (BBG), a P2X7R antagonist, in the third day of kidney tissue response to UUO in rats. For this purpose, male Wistar rats submitted to UUO or sham operated, received BBG or vehicle (V), comprising four groups: UUO-BBG, UUO-V, sham-BBG and sham-V. The kidneys were harvested on day 3 UUO and prepared for histology, immunohistochemistry (P2X7R, PCNA, CD-68, α-sma, TGF-ß1, Heat-shock protein-47, TUNEL assay), quantitative real-time PCR (IL-1ß, procollagens type I, III, and IV) for mRNA quantification. RESULTS: The group UUO-V presented an enhancement in tubular cell P2X7-R expression, increase influx of macrophages and myofibroblasts, HSP-47 and TGF- ß1 expression. Also, upregulation of procollagen types I, III, and IV, and IL-1ß mRNAs were seen. On the other hand, group UUO-BBG showed lower expression of procollagens and IL-1ß mRNAs, as well as less immunoreactivity of HSP-47, TGF-ß, macrophages, myofibroblasts, and tubular apoptosis. This group also presented increased epithelial cell proliferation. CONCLUSION: BBG, a known highly selective inhibitor of P2X7R, attenuated renal inflammation, collagen synthesis, renal cell apoptosis, and enhanced renal cell proliferation in the early phase of rat model of UUO.

P2X7 receptor inhibition ameliorates dendritic spine pathology and social behavioral deficits in Rett syndrome mice.

Dysregulated immunity has been implicated in the pathogenesis of neurodevelopmental disorders but its contribution to synaptic and behavioral deficits in Rett syndrome (RTT) remains unknown. P2X7 receptors (P2X7Rs) are unique purinergic receptors with pro-inflammatory functions. Here, we report in a MECP2-deficient mouse model of RTT that the border of the cerebral cortex exhibits increased number of inflammatory myeloid cells expressing cell-surface P2X7Rs. Total knockout of P2X7Rs in MECP2 deficient mice decreases the number of inflammatory myeloid cells, restores cortical dendritic spine dynamics, and improves the animals' neurological function and social behavior. Furthermore, either genetic depletion of P2X7Rs in bone-marrow derived leukocytes or pharmacological block of P2X7Rs primarily outside of the central nervous system parenchyma, recapitulates the beneficial effects of total P2X7R depletion on the social behavior. Together, our results highlight the pathophysiological roles of P2X7Rs in a mouse model of RTT.

The triphenylmethane dye brilliant blue G is only moderately effective at inhibiting amyloid formation by human amylin or at disaggregating amylin amyloid fibrils, but interferes with amyloid assays; Implications for inhibitor design.

The development of inhibitors of islet amyloid formation is important as pancreatic amyloid deposition contributes to type-2 diabetes and islet transplant failure. The Alzheimer's Aß peptide and human amylin (h-amylin), the polypeptide responsible for amyloid formation in type-2 diabetes, share common physio-chemical features and some inhibitors of Aß also inhibit amyloid formation by h-amylin and vice versa. Thus, a popular and potentially useful strategy to find lead compounds for anti-amylin amyloid agents is to examine compounds that have effects on Aß amyloid formation. The triphenylmethane dye, brilliant blue G (BBG, Sodium;3-[[4-[(E)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-N-ethyl-3-methylanilino]methyl]benzenesulfonate) has been shown to modulate Aß amyloid formation and inhibit Aß induced toxicity. However, the effects of BBG on h-amylin have not been examined, although other triphenylmethane derivatives inhibit h-amylin amyloid formation. The compound has only a modest impact on h-amylin amyloid formation unless it is added in significant excess. BBG also remodels preformed h-amylin amyloid fibrils if added in excess, however BBG has no significant effect on h-amylin induced toxicity towards cultured ß-cells or cultured CHO-T cells except at high concentrations. BBG is shown to interfere with standard thioflavin-T assays of h-amylin amyloid formation and disaggregation, highlighting the difficulty of interpreting such experiments in the absence of other measurements. BBG also interferes with ANS based assays of h-amylin amyloid formation. The work highlights the differences between inhibition of Aß and h-amylin amyloid formation, illustrates the limitation of using Aß inhibitors as leads for h-amylin amyloid inhibitors, and reinforces the difficulties in interpreting dye binding assays of amyloid formation.

Brilliant Blue G Inhibits Inflammasome Activation and Reduces Disruption of Blood-Spinal Cord Barrier Induced by Spinal Cord Injury in Rats.

BACKGROUND Brilliant blue G (BBG) is a P2X7 receptor inhibitor that has been reported to improve spinal cord injury (SCI) in previous studies, but the specific mechanism has been unclear. In this study, we investigated the effects of BBG on inflammasomes and blood-spinal cord barrier (BSCB) permeability after SCI. MATERIAL AND METHODS The experimental rats were randomly divided into 3 groups: sham, SCI, and SCI+BBG. The expression of P2X7 and inflammasome-related proteins was measured by Western blot and immunohistochemistry, while IL-1ß and IL-18 levels were measured by using an enzyme-linked immunosorbent assay (ELISA) kit. The permeability of the BSCB was evaluated by Evans Blue (EB) exosmosis, and histological alterations were observed by hematoxylin-eosin staining. Motor function recovery was assessed by the Basso, Beattie, Bresnahan (BBB) scale after SCI. RESULTS The expression levels of P2X7, NLRP3, ASC, cleaved XIAP, caspase-1, caspase-11, IL-1ß, and IL-18 were increased significantly after SCI, and BBG administration inhibited this increase at 72 h after SCI. BBG administration significantly reduced EB leakage at 24 h after SCI. Furthermore, treatment with BBG significantly attenuated histological alterations and improved motor function recovery after SCI. CONCLUSIONS BBG administration promoted motor function recovery and alleviated tissue injury, and these effects might be related to the suppression of inflammasomes and the maintenance of BSCB integrity.

Effect of Photodynamic Therapy on Microorganisms Responsible for Dental Caries: A Systematic Review and Meta-Analysis.

The aim of this study was to perform a systematic review of the literature followed by a meta-analysis about the efficacy of photodynamic therapy (PDT) on the microorganisms responsible for dental caries. The research question and the keywords were constructed according to the PICO strategy. The article search was done in Embase, Lilacs, Scielo, Medline, Scopus, Cochrane Library, Web of Science, Science Direct, and Pubmed databases. Randomized clinical trials and in vitro studies were selected in the review. The study was conducted according the PRISMA guideline for systematic review. A total of 34 articles were included in the qualitative analysis and four articles were divided into two subgroups to perform the meta-analysis. Few studies have achieved an effective microbial reduction in microorganisms associated with the pathogenesis of dental caries. The results highlight that there is no consensus about the study protocols for PDT against cariogenic microorganisms, although the results showed the PDT could be a good alternative for the treatment of dental caries.

Mapping the Site of Action of Human P2X7 Receptor Antagonists AZ11645373, Brilliant Blue G, KN-62, Calmidazolium, and ZINC58368839 to the Intersubunit Allosteric Pocket.

The P2X7 receptor is a trimeric ligand-gated ion channel activated by ATP. It is implicated in the cellular response to trauma/disease and considered to have significant therapeutic potential. Using chimeras and point mutants we have mapped the binding site of the P2X7R-selective antagonist AZ11645373 to the known allosteric binding pocket at the interface between two subunits, in proximity to, but separated from the ATP binding site. Our structural model of AZ11645373 binding is consistent with effects of mutations on antagonist sensitivity, and the proposed binding mode explains variation in antagonist sensitivity between the human and rat P2X7 receptors. We have also determined the site of action for the P2X7R-selective antagonists ZINC58368839, brilliant blue G, KN-62, and calmidazolium. The effect of intersubunit allosteric pocket "signature mutants" F88A, T90V, D92A, F103A, and V312A on antagonist sensitivity suggests that ZINC58368839 comprises a binding mode similar to AZ11645373 and other previously characterized antagonists. For the larger antagonists, brilliant blue G, KN-62, and calmidazolium, our data imply an overlapping but distinct binding mode involving the central upper vestibule of the receptor in addition to the intersubunit allosteric pocket. Our work explains the site of action for a series of P2X7R antagonists and establishes "signature mutants" for P2X7R binding-mode characterization.

Exploring the influence of brilliant blue G on amyloid fibril formation of lysozyme.

Evidence suggests that amyloid fibril mitigation/inhibition is considered a promising approach toward treating amyloid diseases. In this work, we first examined how amyloid fibrillogenesis of lysozyme was affected by BBG, a safe triphenylmethane compound with nice blood-brain-barrier-permeability, and found that shorter fibrillar species were formed in the lysozyme samples treated with BBG. Next, alterations in the features including the secondary as well as tertiary structure, extent of aggregation, and molecular distribution of lysozyme triggered by the addition of BBG were examined by various spectroscopic techniques, right-angle light scattering, dynamic light scattering, and SDS-PAGE. In addition, we have investigated how BBG affected the lysozyme fibril-induced cytotoxicity in SH-SY5Y cells. We found that a large quantity of shorter fibrillar species and more lysozyme monomers were present in the samples treated with BBG. Also, the addition of BBG rescued SH-SY5Y cells from cell death induced by amyloid fibrils of lysozyme. Finally, information about the binding sites and interacting forces involved in the BBG-lysozyme interaction was further explored using synchronous fluorescence and molecular docking approaches. Molecular docking results revealed that, apart from the hydrophobic interaction(s), hydrogen bonding, electrostatic interactions, and van der Waal forces may also be involved in the binding interaction.