Pseudomonas putida MnB1 as a whole-cell biocatalyst for the effective oxidation of β-keto ester with excellent yield. Differing from known chemical protocols toward this transformation that generally necessitate the use of organic solvents, stoichiometric oxygenating agents and complex chemical catalysts, our strategy can accomplish it simply under aqueous and mild conditions with higher efficiency than that provided by chemical manganese oxides. Moreover, the live MnB1 bacteria are capable of continuous catalysis for this C-O bond forming reaction for several cycles and remain proliferating, highlighting the favorable merits of this novel protocol for sustainable chemistry and green synthesis.Manganese oxidizing bacteria can produce biogenic manganese oxides (BMO) on their cell surface and have been applied in the fields of agriculture, bioremediation, and drinking water treatment to remove toxic contaminants based on their remarkable chemical reactivity. Herein, we report for the first time the synthetic application of the manganese oxidizing bacteria,  Developing sustainable biocatalytic processes for chemical synthesis has attracted considerable attention due to the ever-increasing environment concerns ,2,3. Con2) is a classic oxidant in organic synthesis with broad substrate scope and high reaction selectivity, as seen in alcohol oxidation, aromatization, oxidative coupling, and thiol oxidation  was cultured in the Lept medium , 0.5 g/L Casamino Acids , 5 mM D(+)-glucose , 10 mM HEPES , 0.48 mM CaC12 (Macklin), 0.83 mM MgSO4 (Macklin), 3.7 μM FeCl3 (Macklin), and 1 mL of trace element solution ) containing 1mM MnCl2  at 30 °C and shaken at 150 rpm for 5 d [g for 20 m and the supernatant was discarded, sediments of bacteria and BMO were diluted three times with deionized water by means of centrifugation (20 m at 8000× g), then the mixture of precipitates were collected for freeze-drying to obtain the dried BMO sample.The bacterial strain 3 overnight and heated at 70 °C for 30 min, then was added H2O2 . The resulting mixture was treated to evaporate at 200 °C for 1 h and then dissolved in deionized water to 4 mL solution to be measured by Inductive Coupled Plasma Optical Emission Spectrometry  to quantify the amount of Mn2+. ICP-OES combined with a water cross-flow nebulizer and Ar was run as the carrier gas; auxiliary energy flow, coolant flow, and nebulizer flow were set as 0.7 L/min, 13.00 L/min, and 0.7 L/min, respectively. The analyses were calibrated by gravimetric standards with different concentrations  that were measured before sample quantification.The 25 μg dry m-MnB1 was treated with 200 μL 90% HNO2O = 1:1) containing 50 mM β-keto ester 1, 5.0 mg dry m-MnB1 or 1.75 mg CMO was shaken at 150 rpm, 30 °C for 3 h. A control experiment was conducted without manganese oxide under otherwise identical reaction conditions. All reaction mixtures were monitored by TLC (hexanes/ethyl acetate = 3:1). Then, the resulting samples were centrifuged at 8000× g for 5 m, supernatant was extracted with dichloromethane and evaporated under reduced pressure. The dried residues were purified by silica gel chromatography with hexanes/ethyl acetate (20:1–10:1) to get desired products for the NMR analysis. Finally, the purified samples were used as standards for the following HPLC detection.A reaction mixture  containing 50 mM β-keto ester (1) , 5.0 mg1 (0.02 mmol) and dry m-MnB1/CMO   were added to test tubes and dissolved in MeCN (40 μL) and H2O (360 μL). The reaction samples were stirred at 150 rpm, 30 °C for 1.5 h. Then, the reaction systems were centrifuged at 8000× g for 5 m and supernatants were analyzed using HPLC to obtain the yield of the oxidation product.Substrate 1 (0.02 mmol), dried-BMO/CMO (0.02 mmol), MeCN (40 μL) and H2O (360 μL) were stirred at 150 rpm, 30 °C, and the reaction progress was monitored by yield of the oxidation product.To further compare the reaction efficiency of dry m-MnB1 and CMO, kinetic parameters were detected. The reaction mixtures  at 150 rpm, 30 °C and transferred into fresh Lept medium containing 1 mM MnCl2 and various concentrations of 1 . The colony forming units (CFU) were quantified by using the standard spread plate method [600 nm level of bacteria was performed with microplate reader .The bacterial strain 4 was used as the standard to confirm the absorbance at 620 nm of supernatant.The oxidized Mn content generated in the suspension that was described in the Pseudomonas putida MnB1 was cultured in the Lept medium containing 1 mM MnCl2 at 150 rpm, 30 °C for 5 d to generate the BMO. The concentration of BMO was quantified by LBB method before adding the 5 mM substrate 1 to the medium. The mixture was then continued to shake at 150 rpm, 30 °C to get target product and the reaction progress was monitored by HPLC analysis. After the first-round reaction, the bacteria with BMO were collected by centrifugation and diluted three times with deionized water. The precipitates were added to fresh Lept medium containing 1 mM MnCl2 to allow the continuous mineralization of manganese at 30 °C and 150 rpm. The mixture was used for the second-round reactions under comparable conditions when the concentration of BMO was similar with the first-round initial content. Four consecutive rounds of biocatalysis by whole-cell were conducted and yield of product in each round was measured. All procedures were performed under rigorous aseptic conditions.The bacterial strain For HPLC detection, all of the collected supernatant samples were filtered over a 0.22 μm filter  and analyzed by HPLC system containing a LC-UV 100 absorbance detector . The compounds were separated on a reverse phase HPLC C18 column  at a constant flow rate of 1.0 mL/min and analysed by UV/Vis detection at 254 nm. Solvent A was deionized water containing 5% trifluoroacetic acid  and solvent B was acetonitrile containing 5% TFA. A gradient from 0% to 70% solvent B was applied from 0 to 5 min, the solvent B decreased 70% to 55% from 5 to 20 m, then dropped to 5% during 5 m. All analyses were calibrated by the absorption of substrate and product standards with different concentrations.All data were presented as means ± standard deviation of three replicates. Statistical analysis was completed using SPSS 16.0 .


Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive impairment that increasingly afflicts the elderly population. Soluble oligomers (AβOs) has been implicated in AD pathogenesis: however, the molecular events underlying a role for Aβ are not well understood. We studied the effects of AβOs on mitochondrial function and on key proteins that regulate mitochondrial dynamics and biogenesis in hippocampal neurons and PC-12 cells. We find that AβOs treatment caused a reduction in total Mfn1 after a 2 h exposure (42 ± 11%); while DRP1 increased at 1 and 2 h , correlating to changes in mitochondrial morphology. We also observed that SIRT1 levels were reduced after acute and chronic AβOs treatment ; while PGC-1α levels were reduced with the same time treatments . Interestingly, we found that chronic treatment with AβOs increased the levels of pSIRT1 (24 h: 157 ± 18%), and we observed changes in the PGC-1α and p-SIRT1 nucleus/cytosol ratio and SIRT1-PGC-1α interaction pattern after chronic exposure to AβOs. Our data suggest that AβOs induce important changes in the level and localization of mitochondrial proteins related with the loss of mitochondrial function that are mediated by a fast and sustained SIRT1/PGC-1α complex disruption promoting a “non-return point” to an irreversible synaptic failure and neuronal network disconnection. Alzheimer's disease (AD) is a type of dementia characterized as a progressive brain disorder with high prevalence in elderly people. The etiology of AD has not been completely determined. The time course of the disease involves the loss of cognitive abilities and behavioral changes related to progressive neuronal failure and death, mainly in the hippocampus and cortex . There a2+ overload and dysfunction; this could be as consequence of plasma membrane pore formation or by direct interaction of AβOs to mitochondrial cristae , supplemented with 5% fetal bovine serum, 5% serum horse , and 1% streptomycin-penicillin . The cells were plated in 12-well plates at a density of 2.5x105 cells/ml. Cultures were maintained at 37°C with 5% CO2. All experiments are performed in triplicate for three independent experiments.Eighteen to nineteen days pregnant C57BL/J6 mice were treated in accordance with regulations recommended by NIH  and the ethics committee at the University of Concepción. Mice were deeply anesthetized by CO2 inhalation before being sacrificed by cervical dislocation. Primary cultures of embryonic hippocampi were prepared as previously published  and platBrain slices were obtained from C57BL/6J mice . Brain slices (120 µM) were obtained on a Leica VT1200S vibratome following the Káradóttir & Attwell method . Once cuData was analyzed with GraphPad Prism Software  where all statistical calculations  were done. All experiments a performed in triplicate for three independent experiments.1–40  was reconstituted in DMSO at a concentration of 2.3 mM. 2 µl of this solution was diluted in sterile distilled water to reach a concentration of 80 µM. Subsequently, the peptide was aggregated at 500 rpm for 4 h at room temperature. Soluble Aβ oligomers (AβOs) were used to treat the cells and tissues for 1, 2, 5, and 24 h at a final concentration of 0.5 µM. The identity of oligomeric Aβ species (AβOs) were checked by silver staining and electronic microscopy (were performed as previously published (A lyophilized stock of Aβublished ).1–40 at a concentration of 80 µM was applied to carbon-coated Formvar grids. Samples were fixed with a 2% glutaraldehyde solution for 5 min was exposure to colloidal beta amyloid antibody  and gold particles (10 nm) conjugated with a secondary antibody . The aggregates were stained with 5 µl of 0.2% (w/v) phosphotungstic acid (PTA) and the grid was air-dried. Samples were examined using a JEOL 1200 EX II electron microscope.Ten microliters of AβTo study the expression of mitochondrial biogenesis and dynamics proteins, we prepared whole cell lysates with a buffer containing 10 mM Tris, 10 mM EDTA, 100 mM NaCl, 0.5% Triton, 10% glycerol, and protease inhibitors. Culture lysates were mixed with 4X loading buffer and denatured at 95°C for 10 min. Then, samples were subjected to SDS-PAGE in 10% acrylamide gels at 100 V for 100 min and transferred to nitrocellulose membranes at 250 mA for 120 min. Membranes were blocked with 5% non-fat milk in TBS 1X-Tween for 1 h. The primary antibodies SIRT1 , PGC-1α , DRP1 , Mfn1 , and β-actin  were incubated overnight. Anti-rabbit-HRP  and anti-mouse-HRP  were used as secondary antibodies and were incubated for 1 h. Immunoreactive signals were detected using the Western Lighting kit  andquantified with an Odyssey imaging system .To evaluate the interaction between SIRT1 and PGC-1α, we prepared lysates from PC12 cells control and treated 24 h with AβOs. Protein concentration was normalized for the different samples. Then, the lysates were precleared with 10 µl of Protein A/G PLUS Agarose Immunoprecipitation Reagent  and the resulting mixture was incubated at 4°C for 1 h. Subsequently, the precleared samples were incubated for 1 h with 1 µg of an anti-PGC-1α rabbit monoclonal antibody  or a rabbit control IgG , per mg of protein. Finally, we added 50 µl of Protein A/G PLUS Agarose Immunoprecipitation Reagent to each sample and incubated the mixture overnight. Proteins were eluted in 2X loading buffer at 100°C for 8 min, analyzed by SDS-PAGE, and visualized by western blot using anti-PGC-1α and anti-SIRT1 antibodies .1–40 toxic effects on mitochondrial biogenesis, dynamics proteins, organization, and distribution, PC-12 cell cultures were treated with 0.5 µM AβOs 1–40 for 1, 2, and 24 h. The cells were washed with PBS1X and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). Later, cells were permeabilized with 0.1% Triton X-100 and blocked with 10% horse serum for 30 min at RT. Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 , PGC-1α , Ser-46 SIRT1 , DRP1 , and Mfn1 . Subsequently, the corresponding Cy3 or Alexa Fluor 488 conjugated were incubated for 45 min. Then, samples were incubated with 300 nM DAPI nuclear staining for 10 min and mounted using DAKO immunofluorescence mounting media . Images were acquired using a LSM780 NLO confocal microscope , a Nikon Eclipse TE2000-U epifluorescence microscope, and Zeiss SR-SIM equipment (Elyra S1 model), which was equipped with a DPSS Diode laser (488 and 561 nm of excitation). The 63X/1.4 NA oil immersion objective with an additional 1.6X lens was used which explains why the total increase was 1008X. Images were processed, and quantification was performed using Image J . Images were deconvolved considering the following parameters: immersion oil refractive index (1.518), objective numerical aperture (1.4), and wavelength (510 nm). Immunoreactivity for mitochondrial biogenesis and dynamics proteins was quantified. Additionally, nuclear and cytoplasmic immunoreactivity was analyzed using three aleatory areas in both cases and quantifying the mean intensity for each cell in three independent experiments.To study AβTo study AβOs1–40 toxic effects on mitochondrial morphology, PC-12 cells and hippocampal neurons were stained with a specific primary antibody for TOM 20 . Images were acquired using a LSM780 NLO confocal microscope  or superresolution microscopy (see above). After acquisition, images were processed with Image Processing and 3D reconstructions were performed using BitPlane IMARIS 9.1 software to evaluate the shape and area of MTG-stained cells. Briefly, binary objects were obtained by image segmentation, and subsequently an automatic measurement tool was used to calculate the volume and elongation of each mitochondrion.Images by structured illumination were obtained using a Zeiss SRSIM model Elyra S1 superresolution microscope equipped with a DPSS diode laser (561 nm excitation). An oil immersion 63x/1.4 NA objective was used with an additional 1.6x lens for a total magnification of 1008x. The emission filters used were a BP570-650 and LP750 for the fluorophores FITC and Cy3, respectively. For all captured images a configuration with five grid positions was used (28 μm for the 488 nm laser and 34 μm for the 561 laser), with a z-stack spacing of 110 nm between each plane captured. Images were processed using Image J software .The obtained data was analyzed using GraphPad Prism 6 Software. Normality was verified using the Kolmogorov-Smirnov test or Shapiro-Wilk normality test depending on sample size. In the case of normal data, statistical significance was determined using unpaired Student's t-test (for two groups) orone-way ANOVA followed by the Dunnett's multiple comparisons test (for multiple group comparisons). In the case of non-normal data, statistical significance was determined using Mann-Whitney test (for two groups) or Kruskal-Wallis test followed by the Dunn's multiple comparisons test (for multiple group comparisons). All experimental data are expressed as the mean ± SEM (unless otherwise indicated). P-values < 0.05 were considered statistically significant. Exact n for each experiment is reported in Figure legends.All procedures related with animal management and tissue isolation were done following NIH (USA) and CONICYT guidelines and with protocols approved by the bioethical committee of the Universidad de Concepcion. 2971–40 aggregates, we performed transmission electron microscopy and silver staining experiments to confirm the presence of Aβ1–40 oligomers  on two of the most important proteins related with mitochondrial dynamics: DRP1 and Mfn1 with chronic AβOs treatments (24 h). Using western blot, we evaluated total protein levels of Mfn1 (3).To confirm our previous observations, we studied the morphology of the mitochondrial network, based on our previous work, where we observed that ATP leakage induced by AβOs causes a decrease on neuronal energetic availability . We evalAdditionally, mitochondria with shorter size were significantly increased with chronic Aβ treatment (24 h), compared to control conditions  or metabolic activity (in the case of common cells). Thus, there is a possibility that the irreversible effects of AβOs could be associated with an impediment of the mitochondria to repair and/or restore their intracellular network. Therefore, to evaluate this aspect we examined the status of the key mitochondrial biogenesis regulators: PGC-1α , which may also help delineate the underlying causes in age-related dysregulation of mitochondria in damaged neurons that are not available to support the metabolic demand of the synapse . In the Taken together, any alterations on its role as fine tuner of mitochondrial biogenesis could be crucial for neuronal functions. Therefore the possibility to understand the early changes in this factor that can induce the loss of balance between fusion and fission may be considered as early market of neuronal damage by toxic agents, in this case amyloid β peptide. Several studies have reported that the level of PGC-1α evidently decreases in association with diminished mitochondrial density in the brains of AD patients . Indeed +/NADH ratio increases, promoting SIRT1 activation and transcriptional activity of PGC-1α by deacetylating at least one of its 13 acetylated lysine residues  and CONICYT guidelines and with protocols approved by the Bioethical Committee of the Universidad de ConcepcionJP and JF designed the study. JP, PG, OR-M and JG contributed to the sample preparation, data collection and analysis. JP, JF, CM-M, GY, GM-C, PC, LG, JS, and ET contributed to write the manuscript. JP, TS-G, and MC directed the experiments. All authors reviewed the manuscript.This work was supported by Fondecyt 1161078 (JF), 1200908 (JF), and Conicyt 21141247 (JP).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacillus subtilis spore antigens. We find that Bac5 can trigger the release of nitric oxide from murine macrophages and upregulate surface marker expression including CD86, MHC-I and MHC-II, in the absence of additional agonists. When coupled with mycobacterial Ag85 and B. subtilis spores, Bac5 also enhanced IFNγ secretion. We provide evidence that B. subtilis spores, but not the Bac5 peptide, act as strong adjuvants in promoting antigen-specific immunoglobulin production in Ag85B-vaccinated mice. Our findings suggest that Bac5 is an important regulator of the early cell-mediated host immune response.Bactenecin (Bac) 5 is a bovine antimicrobial peptide (AMP) capable of killing some species of bacteria through the inhibition of protein synthesis. Bac5 and other AMPs have also been shown to have chemotactic properties and can induce inflammatory cytokine expression by innate immune cells. Recently, AMPs have begun to be investigated for their potential use as novel vaccine adjuvants. In the current work, we characterise the functionality of Bac5 in vitro using murine macrophage-like cells, ex vivo using human tonsil tissue and in vivo using a murine model of vaccination. We report the effects of the peptide in isolation and in the context of co-presentation with mycobacterial antigen and whole, inert  Antimicrobial peptides (AMPs) are conserved components of innate immunity that can be found across the taxonomic kingdoms . They arUnlike many other AMPs that lyse bacteria through assembly in the inner-membrane , prolineWe and others have shown that the native Bac5 sequence exhibits species-restricted antimicrobial activity ,8. HowevH2-bias that supports IgG production and improves IFNγ response upon restimulation.AMPs represent potential alternatives to the use of antibiotics in human medicine and in farming, which may be drivers of antimicrobial resistance . InnovatBacillus subtilis spores, combined with a fusion protein (heparin binding domain only protein) directs their trafficking to lung epithelial cells and supports the generation of mucosal immunity in a murine model when used as a booster for the Bacillus Calmette-Guérin (BCG) vaccine [We previously published findings that adsorption of early- and late-stage mycobacterial antigens  to  vaccine . Novel v vaccine .H2 type immune outcome with associated cytokines such as IL-4 and IL-10 [H2 bias is associated with antibody production, whereas TH1 bias is linked with the activation of cell-mediated killing effectors and associated cytokines such as IFNγ. Arguably, a TH1 response may be particularly important in TB vaccine/adjuvant strategies, and several approaches utilise toll-like receptor (TLR) agonists to achieve this outcome  vaccines are the subject of much debate and ongoing research [The most widely used types of adjuvant are aluminium salts. Alum-compounds tend to elicit a Tnd IL-10 . Broadlyt et al. ). Suitabresearch ,20. Alumresearch . We prevresearch .There are, currently, several novel subunit vaccines in development for the next generation of tuberculosis vaccines . SubunitDanio rerio) model we have used previously [In our previous work, we identified that Bac5 was a potent chemokine for macrophages in vivo, and was able to activate them in vitro . We thereviously , since teviously .M. tuberculosis infection. Although we observed a titratable effect, whereby the maximal dose of Bac5 (100 µg mL−1) reduced total intracellular bacterial burden, this was not statistically significant (−1)  to control nificant A. We connificant . Consistant (−1) B. IntereWe furthermore analysed the expression of cell-surface markers on MH-S cells after treatment with Bac5 . We founB. subtilis spores coated with mycobacterial Ag85 antigen. We utilised this approach as it has previously been validated as having adjuvant properties in support of BCG vaccination in murine models [9 spores using 25 µg mL−1 Bac5. When ex vivo isolated human tonsils were stimulated with Bac5 in combination with spore-Ag85, a significant increase in IFNγ was observed  through adsorption onto inert e models . We confobserved . This wa+ and CD8+ T-cells sub-populations. Bac5 did not induce IL-12, Ki67, IFNγ or TNFα upregulation in either of the T-cell subsets, alone, or in combination with spores-Ag85  or TNFα (tonsil cells) secretion. We previously reported a similar effect whereby human THP-1 cells optimally synthesised IL-1b when costimulated with both Bac5 and M. marinum in combination [M. tuberculosis infection, in which it has been demonstrated to be a crucial innate immune effector in controlling disease progression [When Bac5 was coupled with PAMP in the form of gression .+ or CD8+ T-cells, additional cell types in the whole tonsil cell population may be responsible for the observed IFNγ release. Although activated T-cells and natural killer cells are regarded as the major source of IFNγ during early infection, macrophages are also capable of secreting this cytokine in the presence of IL-12 and IL-18 [Since the same conditions did not induce IFNγ production in either CD4nd IL-18 . We did B. subtilis spores, which induced a mixed T-cell cytokine profile, supported humoral immunity upon vaccination with mycobacterial antigens [Spore-adsorbed Ag85 was able to significantly increase the magnitude of Ag85B-specific IgG titre compared with Ag85 alone. This effect was observed in total IgG as well as IgG1 and IgG2 subclasses, and supports our previously published finding that antigens . BesidesBy contrast, Bac5 peptide failed to demonstrate an adjuvant effect, either alone or when adsorbed onto the spore surface alongside vaccination with Ag85B. We considered whether adsorption of the peptide to the spore may either degrade the spore or mask adjuvenic epitopes. We confirmed by flow cytometry that FITC-labelled Bac5 was associated with the spores, and there was no change in the forward or side scatter of the spores, suggesting no physical degradation. Previously, we showed that the adjuvenic effect of the spores was not affected by spore viability . AdditioM. tuberculosis [H1 inflammation, improved protection against M. tuberculosis challenge [H1 type outcome. For instance, IFNγ and CD86, which are upregulated by Bac5, both support TH1 polarisation of CD4+ T-cells via enhanced IL-12 production by antigen-presenting cells [H17 cells, which contribute to expansion of antigen specific B-cells and enhanced antibody titres [H2 and TH17 immunity, linked with development of immune memory in mucosal tissue, is desirable [Peptide-based adjuvants have been explored previously, and some have demonstrated the ability to enhance the expression of protective immunological markers and improve the efficacy of certain vaccines, including against rculosis ,30. Recehallenge . While tng cells , while ny titres . It is lesirable .In conclusion, our data support a prominent, immunomodulatory role for Bac5 as a conductor of the early, cell-mediated immune response, capable of activating noninfected cells, enhancing IFNγ secretion alongside PAMP and altering surface marker expression in alveolar macrophage-like cells above and beyond its additional role as a directly antimicrobial peptide.Bac5 and Bac5-FITC were synthesised by Biomatik Corporation, Ontario, Canada. We used the active portion of the Bac5 sequence (Bac5 = RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLGPFP), representing the cleaved product from the inactive precursor sequence (UniProt entry: CTHL2_BOVIN). The peptides were prepared as acetate salt formulations, with >99% TFA removal and purity assessed by HPLC.M. bovis BCG and M. tuberculosis H37Rv) were grown in Middlebrook 7H9 media . All cultures were incubated at 37 °C until log phase (OD600 = 1.0–1.2).Mycobacterial strains (2 tissue culture flasks (Corning) in complete RPMI-1640  supplemented with 2 mM L Glutamine, 0.05 mM 2-mercaptoethanol, 10 U/mL Penicillin, 50 μg/mL Streptomycin and 10% (v/v) FBS, and maintained at 37 °C, 95% RH and 5% CO2.Murine alveolar macrophage MH-S cells were grown in 75 cmg for 5 min. The red blood cell (RBC) pellet was mixed with equal volume of 1X phosphate buffered saline (PBS) (pH 7.4) before recentrifugation. 140 µL of 5.14% RBC solution was added to the first row of a 96 well plate (Corning). 90 µL 4% RBC solution was subsequently added to all the other wells. 40 µL of Bac5 was then added to the top row and serially diluted two-fold across the plate to get a concentration range of 100 to 0.78 µg/mL in triplicates. PBS and RBC only were used as negative controls, while 0.2% Triton-X was used as a positive control. The plate was incubated for 1 h at 37 °C. It was then centrifuged at 1000× g for 5 min at 4 °C. Following the formation of RBC pellets, 70 µL of supernatant was removed from each well and transferred into clear, flat, round bottom 96 well plate (Nunc), ensuring no pellets were disturbed. The absorbance was read using TECAN Sunrise spectrophotometer at 450 nm.Fresh blood was obtained from St Georges Hospital blood testing department. Blood was centrifuged at 1000× 5 cells per mL) were added and the plates were incubated in a CO2 incubator at 37 °C. Following a 48-h incubation, 30 µL 0.01% freshly prepared resazurin solution was added to each well and plates were further incubated overnight in the CO2 incubator. The absorbance was then read at 590 nm using TECAN Sunrise spectrophotometer.The cytotoxicity assay was performed in a 96-well plate based on Resazurin Microtitre assay (REMA) as described previously ,35. Brie9 spores were centrifuged and pellets were suspended in 0.2 mL of 1X PBS (pH 7.4) in 1.0 mL tubes. Bac5 conjugated to FITC at 1, 10, 25, 50 µg/mL were added to the spore suspensions, and the binding mixture was incubated for 1 h at room temperature. Spore only was taken as spores were centrifuged at 400× g for 5 min, and the pellet was washed twice with 1X PBS. Finally, 200 µL of spore suspension was added to 1X FACS buffer . A further 1/20 dilution was carried out using 1X FACS buffer for each tube to limit the number of spores to 5 × 106 spores/mL. Binding of Bac5 to spores was evaluated by flow cytometry using a BD FACSCalibur on the basis of their scatter/fluorescence characteristics and the presence of FITC signal.To determine the binding of Bac 5 to spores, a FITC conjugated Bac 5 was generated. Dead and live spores were prepared according to Copland et al. . For the5 cells/mL) prepared in phenol-free RPMI/FBS media, was added to the plate. The plate was incubated for at 37 °C for 24 h. After incubation, the cells were centrifuged at 400× g for 5 min, and washed twice in 1X PBS. The cells were then resuspended in 200 µL of 1X FACS buffer. The cells were analysed by flow cytometry and FITC signal intensity measured. To confirm the viability of MH-S cells in presence of Bac5, an identical plate as above was prepared and incubated at 37 °C for 24 h. After incubation and washes in 1X PBS, the cells in each well were resuspended in 50 µL of eFluor780 dye (Invitrogen). The plate was incubated at 4 °C for 15 min. The cells were then resuspended in 200 µL of 1X FACS buffer, washed twice and then analysed by flow cytometry.Similarly, for evaluating binding of Bac5 to MH-S cells, two-fold serial dilutions of FITC conjugated Bac5 were performed between 100 mg/L and 0.15 mg/L (in triplicates) with phenol-free RPMI media (Sigma) in 96-well U-bottom plates (Corning). A 100 µL of MH-S cells  was added at a multiplicity of infection (MOI) of 1. Plates were sealed and incubated in a CO2 incubator at 37 °C for 18 h. The plate also contained MH-S cells, M. bovis BCG and media- only controls. Post incubation, 100 µL of sample from each well was mixed with 100 µL of Griess reagent and incubated in the dark at room temperature for 6 h. Griess reagent was prepared as per manufacturer’s instructions (Sigma-Aldrich). The absorbance was then read using TECAN Sunrise spectrophotometer at 560 nm.Serial dilutions of Bac5 were made between 50 mg/L and 0.39 mg/L (in triplicates) with phenol-free RPMI media (Sigma) in 96-well U-bottom plates . 1 × 10g for 10 min and resuspended in fresh RPMI/FBS media and 5 × 105 cells/mL were then added to a 24-well plate. To this, Bac5 at concentrations of 100, 50, 25, 1 µg/mL were added in duplicates, and the plates were incubated at 37 °C, 5% CO2 for 16 h. After 16 h, 1 × 106 cells/mL of M. tuberculosis H37Rv prepared in RPMI/FBS were added to the wells to achieve a MOI of 2. The plates were incubated at 37 °C, 5% CO2 for 4 h. After incubation, the cells were centrifuged at 1200× g for 10 min, and resuspended in fresh RPMI/FBS media. 200 µg/mL of amikacin was added to test wells to kill extracellular bacteria, and the plate was incubated further for 2 h at 37 °C, 5% CO2. Post incubation, the cells were lysed by adding 0.1% Triton-X and centrifuged at 16,000× g for 10 min at RT. The pellet was resuspended in 200 µL of Middlebrook 7H9 media and two dilutions were plated on Middlebrook 7H11 agar containing 10% (v/v) of Middlebrook Oleic Albumin Dextrose Catalase Growth Supplement (OADC). The plate was incubated at 37 °C for 14 days to determine intracellular survival of M. tuberculosis.The in vitro infection assay was carried out in a Category III laboratory. To set up the infection model, MH-S cells were centrifuged at 1200× 6/mL and stimulated with Bac 5 at 10 μg/mL or 100 μg/mL for 72 h in triplicate wells. Cells were then stained for 10 min at 4 °C with the viability dye, eFluor_780. Cells were then washed and stained with anti-mouse CD40_FITC; MHC II_PE; CD86_APC; PDL1_PE Cy7; CCR7_PerCP Cy5.5; MHC I_BV421; CD80_BV510  for 30 min at 4 °C. Cells were washed and resuspended in FACS buffer. Cell-surface marker expression was assessed using a Beckton Dickinson Canto II flow cytometer. Cells were gated by size and doublets and nonviable cells were excluded. Flow cytometry analysis was performed using FlowJo software version 10.6.2, with 30,000 events recorded for each condition.MH-S cells were used at a density of 1 × 10Human tonsil tissue used in this study was obtained as part of a clinical study entitled “Vaccine immunogenicity using tuberculosis and dengue fever as models” (IRAS project ID 239627). This study is run by Prof Reljic’s group in collaboration with the ENT department at St Georges Hospital NHS Trust. Ethical approval for this project was provided by the SGUL JREO and the South Central Oxford-C NRES Committee.g without brake. The supernatant was removed and cells resuspended in R1O media. Cells were counted and cell viability was determined using trypan blue. Cells were spun at 400× g for 10 min and the supernatant discarded. Frozen stocks were prepared by resuspending cells in freezing media at a concentration of 1 × 107 cells/mL. Cells were frozen at −80 °C and then stored in liquid nitrogen.Tonsil mononuclear cells (TMCs) were generated from tonsil tissue and used to determine the immunogenicity of vaccine candidates. To prepare TMCs, tonsil tissue was aseptically cut into fragments using a sterile scalpel. Cells were passed through a 70 µM strainer using R1O media and the sterile plunger of a 10 mL pipette. Cells were then suspended in 50 mL R1O media and 20 mL gently pipetted on to 20 mL Ficoll  and the cells spun for 30 min at 700× 6 cells per mL in 96-well plates, with 1 × 105 cells in each well, cultured in RPMI 1640 media at 37 °C. They were stimulated with either Bac 5 (10 ng/mL), B. subtilis spores (100:1 ratio to cells) or Ag85B (5 μg/mL) and supernatant collected for ELISA. Human TNFα or Human IFNγ uncoated ELISA kits from Invitrogen were used according to the manufacturer’s instructions. Anti-TNFα or Anti IFNy capture antibody was diluted 1/250 in 1× coating buffer. 100 µL were incubated overnight in wells of a 96-well ELISA plate . The plate was washed three times with PBS with 0.05% Tween. Wells were blocked with assay diluent for 1 h and then washed. Cytokine standards were prepared in 1× assay diluent and 100 µL added to wells. Supernatant from tonsil cultures was added to wells and then serially diluted 1 in 2 in assay diluent. These plates were incubated for 2 h and then washed. 100 µL of Anti TNFα or Anti IFNy detection antibody diluted 1/250 in assay diluent was added to each well. The plates were incubated for 1 h at room temperature and subsequently washed. 100 µL of Avidin HRP diluted 1/250 in assay diluent was added. The plate was incubated for 30 min and washed five times. 100 µL of TMB substrate solution was added. After 1 min, the colour developed and 100 µL of 0.2 M sulphuric acid was added to wells. The plate was then read in a TECAN Sunrise absorbance microplate reader at 450 nm.ELISAs were used to determine the production of IFNy and TNFα by stimulated tonsil cells. Cells were used at a density of 1 × 10+ and CD8+ T cells through intracellular expression of Ki67. Tonsil mononuclear cells were used at a density of 1 × 106 cells per mL in 96 well plates, with 1 × 105 cells in each well. Cells were cultured in supplemented RPMI 1640 media grown at 37 °C, 5% CO2, 95% humidity. A range of vaccine formulations were tested. Bac 5 was added at either 10 ng/mL, 100 ng/mL or 1 μg/mL. B. subtilis spores were added at a ratio of 100:1 to cells. Ag85B was added at a concentration of 5 μg/mL.This assay established the direct stimulatory effect of different vaccine formulations on the production of IL-2, IFNy and TNFα by T-cells. This assay also investigated the proliferation of CD4Cells were washed lysed with permeabilization buffer consisting of FACs buffer with 0.5% saponin. Intracellular staining was performed on the cells for 45 min at 4 °C with the following antibodies; CD3_FITC (Biolegend), CD4_PerCP Cy5.5 (Biolegend), CD8_BV510 (Biolegend), IL-2_PE (Biolegend), IFNy_BV421 (Biolegend), TNFα_PE-Cy7 (Biolegend) and Ki67_APC (Biolegend). Expression levels of CD3, CD4, CD8, Ki67, IL-2, TNFα, and IFNy were measured by flow cytometry. Cells were gated on size and linearity. Nonviable cells were excluded. T cells were gated by expression of CD3 and then CD4 and CD8. Fluorescence minus one (FMOs) were used to set the appropriate gates and determine positive populations.All animals were used with approval from St. George’s University of London Ethics Committee under an approved UK Home Office animal project license and used in accordance with the Animals (Scientific Procedures) Act 1986. Eight- to ten-week-old female C57BL/6 mice were used for this study obtained from Charles River, Kent, UK. Animal work was conducted at St. George’s University of London Biological Research Facility in accordance with local guidelines, including approval from the St. George’s University of London Research Ethics Committee and national legislation, the Animals in Scientific Procedures Act, 1986. All procedures were performed under the approved UK Home Office animal project license.9 spores were in each dose. Animals received two doses, three weeks apart. A further three weeks after the second dose, the animals were culled, and blood was collected for quantification of anti-Ag85B IgG in serum by ELISA.C57BL/6 mice were divided into five groups, each containing three mice. The mice were immunised with; PBS only, Ag85B only, Ag85B and Bac 5, Ag85B and spores or Ag85B with spores and Bac5. 50 µg of Ag85B and Bac 5 was administered per dose, whilst 1 × 1096 well ELISA plates  were coated with 100 µL of 5 µg/mL Ag85B  in coating buffer overnight. The plate was washed three times with 1 × PBS with 0.05% Tween 20. Wells were then blocked with assay diluent  and incubated for 1 h at RT. The plate was then washed three times. Serum was serially diluted 1:2 in assay diluent and transferred to 96 well plate precoated with Ag85B and incubated at RT for 2 h. The plate was then washed five times. To detect total IgG, α-mouse IgG (Fc Specific) conjugated to horseradish peroxidase  was used. Anti-Ag85B IgG1 was detected using α-mouse IgG1 (γ chain specific) conjugated to horseradish peroxidase (Sigma). These detection antibodies were diluted 1/250 final concentration in assay diluent and 100 µL added to each well and incubated for 1 h at RT and then washed five times. 100 µL substrate solution was added and after 1 min, the colour developed and 100 µL of 0.2 M sulphuric acid was added to all wells. The plate was then read in a TECAN Sunrise absorbance microplate reader at 450 nm.


Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide–protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from  These molecules specifically target bacterial pathogens by interfering with bacterial cell wall synthesis, which will eventually cause cell lysis . However2+ metal ions as co-factor for catalysis. B1, B3 and B4 MBLs require two Zn2+ ions for their catalytic activity, while B2 MBLs require only one Zn2+ ion for their activity [AMR among bacteria can be acquired through various means, i.e., through: (1) hydrolysis or inactivation of antibiotics by synthesizing enzymes ; (2) redactivity ,14,15,16activity ,18,19, bactivity . Hence, activity .Bacillus lehensis G1 alkaliphile. Its predicted in silico structure revealed similarity to the αββα fold and global topology of MBLs. Analysis of its active site and metal-binding ligands revealed that similarity to B3 MBL. Biochemical characterization of purified recombinant Bleg1_2478 further confirmed that it was indeed capable of degrading β-lactam antibiotics, with ampicillin as the preferred substrate [Previously, a hypothetical protein termed Bleg1_2437 (currently renamed as Bleg1_2478), which has a comparable sequence identity to MBL in the range of 43–65%, was discovered from the pool of hypothetical proteins of ubstrate . Howeverubstrate . In viewubstrate ,24.Through both fixed and random docking analysis of ampicillin onto previously built dizinc Bleg1_2478 protein model  using YASeveral peptides were retrieved from the Collection of Anti-Microbial Peptides (CAMPR3) online database  and wereAs a result, RYWPRF and RYTPRF were derived from RRAARF, while RSWNWH, RSWCWH and RSWPWH were derived from RRWFWR, and SRWWDR, SRWWYR and SSWWDR were derived from RRWWFR. Most of these peptides exhibited better binding energy and affinity to ampicillin when docked to Bleg1_2478 via both fixed and random docking . CompariEscherichia coli BL21(DE3) in LB media containing 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and 100 µM ZnSO4 after cultivation for 18 h at 20 °C. Most of the recombinant protein (corresponding to protein size of 26 kDa in the sodium dodecyl sulphate polyacrylamide electrophoresis (SDS PAGE) gel) was expressed in the soluble fraction  was recorded to be 25 U/mL . With th50) is 0.90 µM for RSWPWH and 0.50 µM for SSWWDR  and strength (Kd) towards Bleg1_2478 compared to ampicillin , Arg159  and Arg163 (on loop 15)  . The hydloop 15)  helps to50) at only 0.90 and 0.50 μM, respectively  , transformed with previously constructed pET28b(+):Bleg1_2478 recombinant plasmid harbouring bleg1_2478 open reading frame (ORF) , was used as the expression host in this study.Previously built dizinc Bleg1_2478 protein model  was usedShort antibacterial peptides (AMP) were retrieved from the Collection of Anti-Microbial Peptides (CAMPR3) online database  and dockE. coli BL21 StarTM (DE3)::Bleg1_2478 and cultivated for 18 h at 37 °C. Following this, 2 mL of the starter culture was transferred to a fresh 200 mL of LB medium containing 50 µg/mL of kanamycin and 100 µM of ZnSO4. This culture was cultivated until its optical density at 600 nm (OD600) reached 0.6–0.8. At this time, isopropyl-β-d-thiogalactopyranoside (IPTG) was added into the culture to a final concentration of 0.1 mM to induce the production of the recombinant protein. After induction, the culture was further cultivated at 20 °C for 20 h. Cells were then harvested by centrifugation at 9000× g for 20 min at 4 °C. The cell pellet was resuspended in 5 mL of binding buffer  (pH 7.4). Cells were then disrupted via sonication with 30% amplitude, 15 s ON and 15 s OFF pulses for 2 min 30 s on ice. The lysate was centrifuged at 9000× g for 20 min at 4 °C. The soluble and insoluble fractions were collected and analysed with 12% (w/v) sodium dodecyl sulphate polyacrylamide electrophoresis (SDS PAGE). Soluble fraction containing the N-terminal His-tagged protein was filtered through a 0.45 µM hydrophilic membrane before subjected to purification via affinity chromatography. The filtrate was loaded into charged nickel-nitriloacetic acid (Ni2+-NTA) column, pre-equilibrated with 5 column volumes (CV) of Buffer A at 1 mL/min, before sample injection. Recombinant Bleg1_2478 protein was then eluted with elution buffer (Buffer B: 20 mM sodium phosphate buffer (Na2HPO4-NaH2PO4), 0.5 M sodium chloride (NaCl) and 0.5 M imidazole) (pH 7.4). Subsequently, the protein was dialysed against storage buffer  glycerol) (pH 7.4) at protein to buffer ratio of 1:100. Snakeskin dialysis tube (Thermofisher) with a cutoff size of 10 kDa was used for the process. Protein samples were analysed with SDS-PAGE and subjected to Bradford assay [Heterologous production and purification of Bleg1_2478 recombinant protein were performed following the method by  with slird assay  using bo2HPO4-NaH2PO4) (pH 7.0), 20 µg/mL BSA, 100 µM ZnSO4, 10 µM Bleg1_2478 and 100 µM ampicillin. The reaction was monitored by recording the absorbance at 235 nm every 1 min for a total duration of 6 min using a 50 Bio UV Visible Spectrophotometry. For inhibitory assay of the designed peptides, the assay above was performed with the addition of the peptides into the assay mixture at concentrations of 1, 10 and 20 µM. All assays were done in triplicates. Following this, peptides that showed significant inhibition on Bleg1_2478 were further assayed at a concentration below 1 µM to determine the least concentration needed to exert the slightest inhibition of Bleg1_2478.The ability of the peptides in inhibiting Bleg1_2478 was investigated via inhibitory assay. Firstly, enzymatic assay of purified Bleg1_2478 recombinant protein using ampicillin as the substrate was performed, serving as the positive control. The assay was carried out based on the method by . All ass2HPO4-NaH2PO4) (pH 7.0). Bleg1_2478 protein sample, the selected peptide inhibitors and 20 mM sodium phosphate buffer (Na2HPO4-NaH2PO4) (pH 7.0) were firstly degassed by using Themovac Unit. The experiment was carried out at 30 °C with stirring speed of 300 rpm. The calorimetry data were subsequently analysed using NanoAnalyze Software v3.8.0. ITC measurement was performed at 30 °C with nanoisothermal titration calorimetry, TA Instrument, USA at Malaysian Genome Institute. The peptides were dissolved in 20 mM sodium phosphate buffer  [http://www.calctool.org/CALC/prof/bio/protein_length) [Online tools were used to predict the physicochemical properties of the peptides. Genescript tool (culator)  was used_length)  was used_length)  to predi_length)  was used_length)  was used_length)  was used


To evaluate the potency of potential helicase modulators, we developed an assay of helicase enzyme activity. Using a DNA or RNA biotin labelled oligonucleotide and after the addition of a recombinant helicase, the nucleic acid unwinds, causing the emission of luminescence, which is quantified with a particular antibody. In our assay, one of the DNA oligos was biotinylated, while the other was labelled with digoxygenin (DIG), both in their 5’ termini. The biotin molecule immobilises the DNA duplex on a neutravidin-coated plate and the helicase activity is measured through the unwinding of DNA, due to ATP activation. The subsequent release of DIG-labelled oligos results in a luminescence signal measured with a chemiluminescence antibody. Our goal was to provide a high throughput screening method for potential helicase inhibitors. The method described in this paper has been demonstrated to be fast, easy and reproducible and doesn’t use radiochemicals. Helicase activity assays include analysis of ATPase activity, but it was shown that measuring the helicase unwinding activity is the best method for evaluating modulators of this class of enzyme  was expressed and isolated through recombinant protein methods and later used in our assay.All solutions were prepared using ultrapure water obtained by purifying deionised water. All reagents were stored at room temperature unless indicated otherwise.Escherichia coli induction: Prepare 4 conical flasks with 750 mL of LB and add 34 μg/mL chloramphenicol and 25 μg/mL kanamycin to each;cell lysis buffer: 20 mM sodium phosphate pH 7.5, 300 mM NaCl;buffer S: 20 mM sodium phosphate pH 7.4, 500 mM NaCl;exchange buffer: 25 mM Tris–HCl pH 7.5, 0.05% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate), 20% glycerol, 5 mM DTT (dithiothreitol).oligonucleotides  1:1 molar, HEPES 2mM, NaCl 0.05M, EDTA 0.1 mM, SDS 0.01% w/v;Oligonucleotide mix:neutravidin stock solution: add neutravidin in final concentration 1 mg/ml in phosphate buffered saline (1 M PBS - pH 7.0);BSA solution: 0.1% w/v BSA;substrate solution: mix 2.5 ng partially annealed DNA duplex and 75μL 1 M PBS containing 1M NaCl, for each well;substrate wash solution: 50 mM Tris HCl pH 7.5, 50 mM NaCl;helicase reaction mix: 11 nM purified full-length HCV NS3 protein, 25 mM 4-morpholinepropanesulphonic acid (MOPS) pH 7.0, 2 mM DTT, 3mM MnCl2, 100 μg/ml of BSA and 5 mM ATP. The reaction mix for the negative control lacks ATP;reaction wash solution: 150 mM NaCl;detection washing buffer: 0.1 M maleic acid, 0.15 M NaCl, 0.3%, Tween20, pH 7.5;blocking solution: 10% BSA w/v, 0.1 M maleic acid, 0.15 M NaCl, pH 7.5;antibody solution: 1:10.000 solution of the anti-Dig antibody (75 mU/mL) in Blocking solution;detection buffer: 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5;chemiluminescence substrate solution: CSPD – 0.25 mM.All procedures have to be carried out at room temperature unless specified otherwise.Insert the full-length HCV helicase coding region in a pET28a vector, with a N-terminal 6xHis-Tag region;verify the intactness of the gene before inducing the protein production;transform Escherichia coli cells  with the helicase plasmid and inoculate the prepared LB flasks with them;induce the recombinant helicase production, adding 0.5 mM IPTG to each flask and then allow the cultures to grow for 3 hours at 18°C;resuspend the cell pellet from the 4 cultures in 30 mL Lysis buffer and then add lysozyme 100μg/mL and Triton X-100 0.1%;incubate on ice for 30 min and then sonicate four times for 20 sec with 15 sec intervals;centrifuge the suspension at 15.000 x g for 20 min;adjust the clarified homogenates to 10 mM imidazole and filter them through a 0.45 μm membrane;load the homogenates twice on nickel affinity columns (Ni-NTA);wash each column with five times the column volume of buffer S, containing 10 mM imidazole;elute the NS3 helicase with buffer S containing 300 mM imidazole;immediately after the elution, exchange the buffer in the helicase-containing fractions for the Exchange buffer, via dialysis; this step is critical in order to avoid precipitation;evaluate the protein concentration using the Bradford assay with BSA as standard;create aliquots of the NS3 helicase and store at −80°C.This recombinant protein preparation is estimated to be more than 85% pure by SDS gel electrophoresis and Coomassie blue staining, yielding almost 1.6 mg of HCV NS3 per 3 litres of E. coli cultures .Chemiluminesence readings were taken using all different control combinations  of the experiment to ensure the reliability of the measurements .We demonstrated that the NS3-mediated unwinding is proportional to the amount of DNA substrate in the well, but also to the HCV helicase concentration in the reaction. The reactions were ATP-dependent .Prepare for annealing by heating the oligonucleotide mix at 100°C for 5 min;incubate at 65°C for 30 min and then at 22° for 4 h, to allow gradual annealing;store the annealed NS3 helicase substrate at −20°C See .Coat each of the 96 wells overnight at 4°C with 100 μl/well of a 5 μg/ml neutravidin solution in 0.5 M sodium carbonate buffer pH 9.3;wash the plates three times with 100μl/well of PBS and air-dry at room temperature.Add 100 μL of the 0.1% w/v BSA solution;incubate at 22°C for 2 h;wash the plate three times with PBS, 200 μl/well and air-dry at room temperature;store the plate at 4°C with desiccant See .Pre-warm all solutions to 37°C See ;mix 75 μl of the Substrate solution with 2.5 ng of the partially annealed DNA duplex for each well;incubate at 22°C for 4 h;wash each well twice with 200 μl PBS per well and once with Substrate Wash solution.Add 90 μL of the Reaction mix per well;incubate the reactions at 37°C for 1 hour;wash the plate twice with 200 μL Reaction Washing Buffer per well and let dry for 15 minutes at room temperature.Wash all wells for 5 min with the Detection washing buffer;then fill up each well with Blocking solution for 30 min and then incubate for 30 min in 20 μl Antibody solution;wash twice the plate with 100 μl of Detection buffer;apply 20 μL of Detection buffer for equilibration and 1 μL of chemiluminescence substrate working solution per well and incubate for 5 min at 17°C;drain the wells and incubate the plate at 37°C for 30 min to allow any remaining solution to evaporate;the luminescence has a constant intensity for about 24 hours and continues for approximately 48 hours. The remaining DIG in each well is counted for 10 min against controls (one of which lacks protein and the other lacks ATP) in a luminescence plate reader See .


RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C  and CACNA1 G  were the most abundant voltage-gated Ca2+ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca2+ signalling in VSMCs with Kv7.5 playing a dominant role.Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation-contraction coupling in muscle cells is mediated by Ca Thus, depolarisation of VSMCs membrane potential (Em) results in the opening of voltage-gated Ca2+ channels (VGCCs), predominantly L-type i) via the influx of Ca2+ from L-type Ca2+ channels [Em in A7r5 rat aortic smooth muscle cells [2+]i in VSMCs still remain to be clarified i in VSMCs could identify novel approaches for the treatment of cardiovascular disease, including pulmonary hypertension. In the present study, we examined the influence of Kv7 channel activity on [Ca2+]i in rat and primary human arterial SMCs.The clinical use of AVP has increased significantly in recent years , but its22.12+ regulation i and Em by microfluorimetry. For fluorescence measurements, confluent cell monolayers grown on glass coverslips were used 2–5 days after plating.Rat A7r5 VSMCs derived from rat thoracic aorta were obtained from the European Collection of Cell Cultures. The A7r5 cell line retains many characteristics of VSMCs, which provides a convenient model system to study mechanisms of Cagulation . Human Vgulation ,31. Vess2.22). Once the cell monolayer was confluent, the coverslips were transferred to a 35 mm petri dish and washed with the Ca2+ containing buffer twice. The Ca2+ containing buffer was composed of (in mM) 135 NaCl, 5 KCl, 1.2 MgSO4, 2.5 CaCl2, 5 HEPES, and 10 glucose (pH7.4 with NaOH). The osmolality was adjusted to 300 mOsm with sucrose. The cells were loaded with 4 μM Fura 2-AM  dissolved in Ca2+ containing buffer without pluronic acid for 40 min in the dark at 37 °C. Loading solution was pipetted out and the cells were washed with Ca2+ containing buffer. Coverslips were then incubated in Ca2+ containing buffer to de-esterify for 10 min. For simultaneous recordings of Ca2+ transients and action potentials, rat A7r5 cells were also loaded with the voltage-sensitive dye FluoVolt  in Ca2+ containing buffer supplemented with the dye (1:1000), PowerLoad Concentrate (1:100) and Background Suppressor (1:10) in the dark at 37 °C. Before recording, each coverslip was broken into fragments with a diamond pencil, and fragments were then transferred to a perfusion chamber. Cells were constantly perfused with standard bath solution at 2−3 ml/min using gravity perfusion system via lengths of Tygon tubing . The composition of standard bath solution was (in mM): 135 NaCl, 5 KCl, 1.2 MgSO4, 2.5 CaCl2, 5 HEPES, and 10 glucose (pH7.4 with NaOH); the osmolality adjusted to 300 mOsm with sucrose. The 50 mM K+ bath solution was made with isotonic Na+ substitution; all drugs were applied in standard bath solution via the perfusion system. Intracellular Ca2+ levels were measured ratiometrically using a Cairn Research ME-SE Photometry system . Changes in [Ca2+]i were evaluated as the ratio of intensities of fluorescence emitted at 510 nm following alternating excitation at 340 and 380 nm  using a monochromator. For measurement of Em, cells were sequentially excited at 340, 380 and 488 nm (Fura 2-AM and FluoVolt loaded samples). Acquisition Engine 1.6.1 software was used to record and analyse the traces. Magnitude of changes in [Ca2+]i was calculated either as peak F340/F380 ratio or as the area under the curve . Change of Em was presented as ΔF/F0 in cells.Cells were plated on circular glass coverslips  in 24-well plates and kept in a humidified incubator . A cDNA template was generated using the iScript cDNA synthesis instructions  with 1 μg RNA. End-point PCR and RT-PCR were performed using TaqMan gene expression assays . The probes used in this study are listed in the Supplemental Table I. All samples consisted of 1 μl of cDNA and 19 μl of RT-PCR reaction mix (as per manufacturer’s instructions). Quantitative analysis of mRNA expression was determined by using a CFX Connect System . The reaction profile was 2 min at 50 °C, 10 min at 95 °C, 15 s at 95 °C for 50–60 cycles, then 1 min at 60 °C. PCR amplification products were separated by the 2.0 % agarose gel electrophoresis with gels containing SYBR safe DNA stain . Quantitative analysis of mRNA expression was carried out using a CFX Connect System , and relative gene expression calculated using the 2−ΔΔCt method with hypoxanthine phosphoribosyltransferase 1 (Hprt1) as the housekeeper gene. Statistical analysis was performed on the ΔCt values i followed by a slow decline, presumably due to VGCCs inactivation and extrusion of Ca2+ from cytosol by Ca2+ ATPases and/or Na+/Ca2+ exchanger. A limited number of rapid oscillations was often observed during the transient. Smooth muscle cells mainly express L- and T-type VGCCs, while other isoforms are not significantly expressed [2+ channel blocker [2+ channels [+-induced [Ca2+]i transients by 95.9 ± 2.3 % (n = 5) and by 85.0 ± 13.3 % (n = 5), respectively  and T-type  VGCC subunits. We found that Cacna1c (Cav1.2) L-type and Cacna1g (Cav3.1) T-type Ca2+ channel genes were the predominant subtypes in A7r5 cells  induced a sustained [Ca2+]i elevation and evoked Ca2+ oscillations persisting for the duration of XE991 application; both effects recovered upon XE991 washout  was applied first to induce oscillations, then nifedipine (2 μM) was applied in the continued presence of XE991. Nifedipine virtually abolished the oscillations and reverted [Ca2+]i, elevation induced by XE991 . We then performed a triple-wavelength  simultaneous recordings of [Ca2+]i and Em changes in response to XE991. 2+ trace (black) and FluoVolt Em trace (red); XE991 induced slow Em depolarisation followed by a burst of sharp spikes which were temporally well-correlated with [Ca2+]i (upper panel). Peak cross correlation between the FluoVolt Em and the ratiometric Ca2+ signal was 0.6 ± 0.18, occurring at a time lag of 3.0 ± 0.5 s (n = 5) (lower panel). Both, [Ca2+]i oscillations and Em spikes were abolished by retigabine.We next asked what is the nature of i elevation followed by a period of repetitive Ca2+ oscillations lasting throughout the AVP application; washout of the AVP was followed by a gradual return to the baseline ; peak cross correlation between the FluoVolt Em and the ratiometric Ca2+ signals was 0.84 ± 0.01, occurring at a time lag of 2.5 ± 1.4 s (n = 4) (lower panel). These [Ca2+]i oscillations and Em spikes were abolished by retigabine. Collectively, these data indicate that Kv7/KCNQ channels are present and functional in A7r5 cells and that treatment with a physiological concentration of AVP (100 pM) leads to depolarisation and Ca2+ influx through both L- and T-type Ca2+ channels; these effects were abolished by retigabine and were qualitatively very similar to these produced by the XE991.To investigate if the control over the currents . AVP is  pathway . Exposurbaseline A. Retigabaseline B,F. To tal level C,G. In tt manner D,H. We ae to AVP E. Simila3.42+]i and Em, suggesting a common mechanism, a Kv7 channel inhibition, the action of these two agents on the Kv7 channels is different. While XE991 is a direct ion channel blocker, the action of AVP on the Kv7 channels depends on the PLC-mediated signalling cascade, which inhibits Kv7s by a combination of PIP2 hydrolysis, PKC activation and ER Ca2+ release [2+]i response to AVP (100 pM) in A7r5 cells (p = 0.051) in peak Ca2+ levels between the control (untreated) and edelfosine-treated groups  significantly attenuated the amplitude of the sustained [Ca2+]i elevation induced by AVP (100 pM); it did not completely abolished oscillations but reduced their amplitude. RyRs inhibitor, tetracaine, effectively inhibited the amplitude and frequency of Ca2+ oscillation caused by AVP, and when applied together, 2-APB and tetracaine significantly attenuated [Ca2+]i response to AVP (2+ oscillations evoked by Kv7 channel inhibitor. A7r5 cells were pre-treated (10 min) with edelfosine and the effect of XE991 (10 μM) was explored with Ca2+ imaging. Edelfosine (10 μM) did not inhibit Ca2+ oscillations: neither the amplitude of sustained [Ca2+]i elevation nor spike frequency were different from control  receptors i, however, there were no oscillations  L-type and CACNA1 G (Cav3.1) T-type Ca2+ channel genes were the predominant subtypes in IMA SMCs  L-type Ca2+ channels and low voltage-activated (LVA) T-type Ca2+ channels [2+]i through the L-type Ca2+ channels i were completely blocked by the Kv7 opener, retigabine, indicating a strong control over the excitability and Ca2+ signalling in VSMCs exerted by the Kv7 channels. The inhibitory effect of retigabine was even stronger than that of the L-type VGCC blocker nifedipine, which is widely prescribed to treat cardiovascular disease i that are very similar to these of XE991. Indeed, the main mechanism of action of such hormones to stimulate rhythmic constrictions of arteries has been recognized as depolarisation of the Em to open VGCCs i oscillations. The effect of AVP took longer to develop but resulted in a similar spike frequency to that of XE991  i elevation and mean spike frequency induced by AVP. Moreover, inhibition of IP3Rs and RyRs with 2-APB and tetracaine, respectively, also attenuated the AVP-induced Ca2+ oscillations  and late (sustained) phases of constriction via IPectively . Higher r5 cells ,74. PLC-led GPCR . Within epletion ,19, IP3-modulin)  and actirolysis)  are the rolysis) . Our finllations , suggest neurons ,76. As p2+ signalling in primary human VSMCs as well. AVP has marked vasoconstrictor effect and gained attention as a possible tool against septic shock. However, it has been reported to induce graft vessel spasm during open-heart surgery [2+]i which may be a characteristic feature in human IMA SMCs. Importantly, nifedipine, NNC 55-0396 and retigabine all significantly supressed the AVP-induced [Ca2+]i transients, with retigabine being the most efficacious. Notably, there were no obvious oscillations in in human IMA SMCs in response to AVP or XE991. The reason for this difference between A7r5 and human primary SMCs cells remains to be elucidated, but it could be due to the more pronounced depolarisation and Ca2+ overload in the latter cell type. Despite this difference, these results indicated that Kv7 channels may have important clinical implications in cardiovascular disease and retigabine or its successors (e.g. drugs tailored to selectively activate Kv7.5) could have a potential in containment of the AVP-related perioperative vasospasm.Finally, we present evidence that Kv7 channel activity exerts a strong control over the intracellular Ca surgery . Here weYuan-Ming Tsai: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Visualization, Writing - original draft. Frederick Jones: Methodology, Software, Validation, Formal analysis. Pierce Mullen: Software, Formal analysis. Karen E. Porter: Conceptualization, Resources, Writing - review & editing. Derek Steele: Methodology, Writing - review & editing, Supervision. Chris Peers: Conceptualization, Methodology, Resources, Supervision. Nikita Gamper: Conceptualization, Methodology, Supervision, Resources, Funding acquisition, Writing - review & editing, Supervision.The authors have no conflicts of interest to report pertaining to this study.


However, during electrochemical reactions, the large volume change of Cu2Se results in poor rate performance and cycling stability. To solve this issue, nanosized-Cu2Se is encapsulated in 1D nitrogen-doped carbon nanofibers (Cu2Se-NC) so that the unique structure of 1D carbon fiber network ensures a high contact area between the electrolyte and Cu2Se with a short Na+ diffusion path and provides a protective matrix to accommodate the volume variation. The kinetic analysis and DNa+ calculation indicates that the dominant contribution to the capacity is surface pseudocapacitance with fast Na+ migration, which guarantees the favorable rate performance of Cu2Se-NC for SIBs.Cu Howformance ,5. Thus, of SIBs ,7.2 microboxes with high reversible capacity of 492 mAh g−1, excellent rate capability and long cycling life [2/C) as freestanding electrodes with high areal capacity (4.0 mAh cm−2) and promising cycling stability [2 with high Se content, M2Se such as Cu2Se with an inactive Cu component has the advantages of high electronic conductivity, structural stability and low volume variation during cycling [+ insertion [Among various potential electrode material candidates for SIBs, metal selenides have attracted particular attention because of good electronic conductivity and high reversible capacity based on multielectron conversion reactions ,9. Lou eing life . Yang antability . Compare cycling ,13,14. H cycling ,16. As a cycling . Neverth cycling . Nanosiznsertion ,20,21.2Se nanoparticles are embedded in 1D nitrogen doped carbon nanofibers (Cu2Se-NC). The unique structure of 1D carbon fiber can shorten the Na+ diffusion path with a high contact area between Cu2Se and the electrolyte. Meanwhile, carbon conductive network provides a buffering matrix for the effective accommodation of volume variation and alleviates the aggregation of Cu2Se nanoparticles to maintain the structural integrity during repeated cycling. Based on the these favorable effects, Cu2Se-NC displays a promising rate capability and long-term cycling life for efficient sodium storage.In this paper, Cu3COO)2·H2O (Aladdin), and 0.8 g polyacrylonitrile  was vigorous stirred for about 12 h, which was then loaded into a 20-mL syringe with 19-G needle for electrospinning. The electrospinning process was applied voltage of 1 kV cm−1 with flow rate of 10 μL min−1. After electrospinning, the collected Cu(AC)2/PAN nanofibers were selenized with Se powder (amass ratio of 1:2) in tube furnace at 500 °C for 4 h  to obtain Cu2Se-NC. The Cu2Se and nitrogen doped carbon nanofibers (NC) were acquired through the similar treatment procedure by using Cu(AC)2 with Se powder and PAN nanofibers, respectively.A solution containing 10 mL dimethylformamide , 0.4 g Cu(CH−1).Field emission scanning electron microscope  and transmission electron microscope  were used to observe morphology and structure. X-ray diffraction patterns  and X-ray photoelectron spectroscopy  were conducted to analyze the phase structure and chemical composition, respectively. Thermal gravimetric analysis  was performed from room temperature to 600 °C  was cast onto copper foil as electrode with active material loading of about 0.8~1.0 mg cm−2. The electrolyte was 1 M NaClO4, which dissolved in a solution of ethylene carbonate (EC) and dimethyl carbonate (DMC) with volume ratio of 1:1 as well as additive of 5 wt% fluoroethylene carbonate (FEC). The electrochemical performances tests were measured by a Neware battery testing system in a voltage window of 0.1~3.0 V. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) conducted on an electrochemical workstation .The slurry composed of 80 wt% active material, 10 wt% Super P, 10 wt% polyvinylidene fluoride (PVDF) dispersed in appropriate 2/PAN is composed of highly uniform and smooth nanofibers with diameter of ~200 nm. The selenized nanofibers maintained the 1D structure, where Cu2Se nanoparticles are uniformly distributed in the carbon nanofibers  image reveals the crystal phase of Cu2Se possess good crystallinity, and the lattice spacing of 0.204 nm is ascribed to the (220) crystal plane of Cu2Se , which proves successfully synthesis of Cu2Se phase. In addition, a sharp and strong (220) peak indicates significantly crystallinity and in good agreement with the HRTEM and SAED characterization for Cu2Se–NC. As shown in the thermogravimetric analysis  and 54.2 eV (Se 3d5/2) are ascribed to Cu-Se bonds [2Se–NC, respectively [2Se–NC composite [3 C–C (285.2 eV) and sp2 C=C bonds (284.6 eV) [The X-ray diffraction (XRD) pattern of as-prepared Cusis TGA, b, the wed strong 0 peak inanalysis . In the analysis c, the peized CuO ,23. For Se bonds . The O 1ectively . The N 1omposite . The C 184.6 eV) . The exi2Se-NC. At around 0.21 and 0.85 V of reduction process in the initial CV curve, there are obviously irreversible peaks, corresponding to the irreversible reduction of electrolyte and the formation of solid electrolyte interphase (SEI) film [2Se, and the two oxidation peaks at 0.45 and 1.55 V are assigned to the extraction of Na+ from Na2Se. The CV curves are well overlapped from the second cycle, demonstrating excellent electrochemical reversibility of the Cu2Se–NC electrode. 2Se–NC electrode at a current density of 0.1 A g−1. The Cu2Se–NC electrode yields a first discharge/charge specific capacity of 412/192 mAh g−1, which was higher than those of Cu2Se (−1) and NC (−1). The initial Coulombic efficiency of Cu2Se–NC electrode is 46.6%, which is consistent with the CV test results [2Se–NC, Cu2Se and NC anode for SIBs at a current density of 0.1 A g−1 is shown in 2Se–NC maintains a reversible capacity of 172 mAh g−1 after 200 cycles. However, the capacity of Cu2Se is sharply decreased to 10 mAh g−1 from 189 mAh g−1 after 10 cycles, suggesting 1D carbon fiber network provides more stable structure networks for accommodation of volume expansion to ensure cycling stability. The specific capacity of NC is only 70 mAh g−1 due to the sluggish reaction kinetics of NC for SIBs.EI) film . In the  results . The cyc2Se–NC, Cu2Se and NC anode is illustrated in 2Se–NC electrode exhibits a high specific capacity of 193, 176, 148, 126, and 99 mAh g−1 at the current densities of 0.1, 0.2, 0.5, 1, and 2 A g−1. The specific capacity of 188 mAh g−1 can be obtained, when current density is returned to 0.1 A g−1, revealing the outstanding reversibility of the Cu2Se-NC. While the capacity of Cu2Se and NC decreases to only 3 and 24 mAh g−1 at 2 A g−1, indicating a significant enhancement of the electrochemical performance resulting from nanosized-Cu2Se embedded 1D nitrogen doped carbon nanofibers. Moreover, as depicted in 2Se–NC also exhibits favorable long-term cycling performance. After activation for five cycles, the Cu2Se–NC anode shows capacity retention of 99% (101 mAh g−1) after 2000 cycles, further implying the strong stability of the 1D nanofiber structure.The rate performance of the Cu2Se–NC. −1). The relationship between the peak current  and sweep rate  and obeys the equation of bi = av that b value of 0.5 or 1.0 implies diffusion-controlled or capacitive-controlled processes, respectively [b value is identified by the slopes of log (i) vs. log (v) in 2Se–NC possesses high b values for both cathodic  and anodic  peaks, indicating the surface capacitive-controlled processes. This is responsible for the fast reaction kinetics and ensures considerable rate performance. To qualify the predominated storage capacity contribution, the capacity can be divided into capacitive (k1) and diffusion (k2) according to 1υ1/2 + k2υi = k (1/2 = k1 + k2υ1/2i/υ) [2Se–NC at 0.6 mV s−1 are calculated as 64.9%, which indicates that Cu2Se–NC possesses fast Na+ intercalation/deintercalation. Furthermore, 2Se–NC increases to 72.6% as the sweep rate elevates to 1.0 mV s−1. The high ratio of surface capacitive contribution is beneficial to the rate performance, which agrees with the favorable rate capability of Cu2Se–NC  ,33. As sCu2Se–NC d.2Se–NC and Cu2Se are shown in ctR) of Cu2Se–NC and Cu2Se is 1225 and 242 Ω, which decreases to 130 Ω and increases 466 Ω after 100 cycles, respectively. The decrease in resistance of Cu2Se-NC electrode is due to the activation process during repeated cycling, resulting in faster electron transfer for SIBs. In contrast, the ctR of the Cu2Se increased significantly ctR after cycles, which might be ascribed to cracking and pulverization of Cu2Se caused by volume expansion. The diffusivity coefficient of sodium ions (Na+D) is obtained by EIS measurements based on 2T2/D = R(4A2F4C2b22n) and e + Rct + bω−1/2Z′ = R equations [ω is the low range angular frequency of EIS, and b is the Warburg factor that is the slope of the line −1/2Z′ ~ ω. Obviously, the slope b of Cu2Se–NC is much lower than that of Cu2Se after cycled, and the sodium-ion diffusion coefficient of Cu2Se–NC and Cu2Se are calculated as ~3.4 × 10−21 and 5.4 × 10−22 cm2 s−1, respectively. The higher Na+D of Cu2Se–NC electrode indicates fast diffusion kinetics for sodium storage.The EIS curves and equivalent electrical circuit of Cuquations . Here, ω2Se–NC and Cu2Se electrode before and after 100 cycles. As illustrated in 2Se–NC electrode is flat with regular nanofibers, which is similar to their original morphology  peak could be detected in the pristine Cu2Se-NC electrode. When it is discharged to 0.1 V, two new peaks at 43.3° and 44.1° ascribe to (111) plane of Cu (JCPDS No. 04-0836) and (042) plane of Na2Se (JCPDS No. 47-1699), which indicates the insertion of Na+ into Cu2Se and the formation of Cu and Na2Se. After recharging to 3 V, the XRD pattern displays similar signals for Cu peak, which might be attributed to the irreversible reactions. Moreover, the Cu2Se (220) peak could not restore to the pristine state that could be due to crystalline Cu2Se to amorphous state, providing more active sites and fast reaction kinetics.To further prove the structural stability of the electrode, we investigate the morphologies of the Curphology . Meanwhiechanism . The Cu22Se–NC was successfully fabricated and evaluated as anode materials for sodium storage. Corresponding CV kinetic analysis and DNa+ calculation indicates the Cu2Se–NC possesses dominant surface pseudocapacity contribution and fast Na+ migration, which ensures favorable rate performance for SIBs. Moreover, the carbon fibers networks afford a conductive and protective layer for the effective accommodation of volume changes to ensure their superior long cycle life. Thus, the as-prepared Cu2Se–NC electrode displays enhanced sodium storage properties with the high reversible capacity of 172 mAh g−1 (0.1 A g−1), considerable rate capability of 99 mAh g−1 (2 A g−1) and ultra-long cycling stability (99% capacity retention over 2000 cycles).In summary, Cu


Pinctada fucata martensii (P. f. martensii) and designated as PmC1qDC-1. The full-length sequence of PmC1qDC-1 was 902 bp with a 534 bp open reading frame (ORF), encoding a polypeptide of 177 amino acids. Quantitative real-time PCR (qRT-PCR) result showed that PmC1qDC-1 was widely expressed in all tested tissues, including shell formation-associated tissue and immune-related tissue. PmC1qDC-1 expression was significantly high in the blastula and gastrula and especially among the juvenile stage, which is the most important stage of dissoconch shell formation. PmC1qDC-1 expression was located in the outer epithelial cells of mantle pallial and mantle edge and irregular crystal tablets were observed in the nacre upon knockdown of PmC1qDC-1 expression at mantle pallial. Moreover, the recombined protein PmC1qDC-1 increased the rate of calcium carbonate precipitation. Besides, PmC1qDC-1 expression was significantly up-regulated in the mantle pallial at 6 h and was significantly up-regulated in the mantle edge at 12 h and 24 h after shell notching. The expression level of PmC1qDC-1 in mantle edge was significantly up-regulated at 48 h after LPS stimulation and was significantly up-regulated at 12 h, 24 h and 48 h after poly I:C stimulation. Moreover, PmC1qDC-1 expression was significantly up-regulated in hemocytes at 6 h after lipopolysaccharide (LPS) and poly I:C challenge. These findings suggest that PmC1qDC-1 plays a crucial role both in the shell formation and the innate immune response in pearl oysters, providing new clues for understanding the shell formation and defense mechanism in mollusk.The C1q protein, which contains the globular C1q (gC1q) domain, is involved in the innate immune response, and is found abundantly in the shell, and it participates in the shell formation. In this study, a novel gC1q domain-containing gene was identified from  Originally, C1q is involved in the classical complement pathway as the ligand-binding unit of the C1 complex4. As a versatile recognition protein, C1q recognizes many other ligands such as bacteria, viruses, parasites, mycoplasma, apoptotic cells, and pathogen-associated molecular patterns8. gC1q contains the globular subunit A, globular subunit B and globular subunit C9. Each subunit consisting of two five-stranded β-sheets is made of anti-parallel strands10. The Ca2+ ion binding site is present at the top of the C1q globular domain10 affecting the binding of C1q to diverse ligand11.C1q protein, a versatile recognition protein, binds to a wide variety of immune and non-immune ligands12, to participate in the immune process. They can also be synthesized from specific tissues and organs such as microglial cells, glomerular and tubular cells, osteoclasts, and trophoblasts13 and are involved in the regulation of multiple cellular functions such as cell growth, clearance of the apoptotic cells, and promotion of cell adhesion14. When C1q is immobilized on a substrate or within a soluble immune-complex, it will enhance phagocytic function15. In humans, C1q deficiency causes systemic lupus erythematous16. During embryonic development, when the extravillous trophoblasts start to invade the decidua, C1q binds to the extracellular matrix and by interacting with cell surface-expressed receptors for the globular head of C1q and the associated α4β1 integrin, delivers an activation signal and promotes trophoblast migration17. C1q can regulate osteoclast development18. C1qDC proteins of bivalve mollusks can also recognize versatile ligands. MgC1q gene is involved in host defense in Mytilus galloprovincialis19. HcC1qDC6 is involved in innate immunity by directly binding to peptidoglycan (PGN) and LPS in Hyriopsis cumingii20. In Crassostrea gigas, CgC1qDC-6 mediates hemocyte phagocytosis and migration and can bind to various pathogen-associated molecular patterns (PAMPs), including LPS, PGN, mannose and poly I:C, and microorganisms21.C1q domain-containing (C1qDC) proteins can be synthesized and secreted locally by various cell types, including macrophages, dendritic cells, fibroblasts, and mast cells that are ubiquitously distributed throughout the body23, the C1qDC gene of bivalve mollusks has undergone a massive expansion. The number of C1qDC genes in humans24, mice25, zebrafish26, and amphioxus27 ranges from 29 to 52. Strikingly, in mollusks, the number of C1qDC genes ranges from 168 to 47630. The large expansion of C1qDC causes its functions to have diversified. Many C1qDC genes of P. f. martensii are highly expressed in the mantle and pearl sac31, which is the shell formation associated tissue32. Wang discovered that PFMG4, which is highly homologous to C1q, could enhance osteoblast differentiation and serves as evidence of its participation in biomineralization33. Beside, shell matrix proteins (SMPs) containing C1q domain have been identified from many mollusks35. Although the C1q domain is an immune-related functional domain, growing body of evidence shows that in addition to immune function, C1qDC protein also contributes to shell formation. In most cases, the function of C1qDC only focuses on immunity or biomineralization. In Hyriopsis cumingii, a kunitz proteinase inhibitor participated in antimicrobial process duiring pearl sac formation and induced the overgrowth of calcium carbonate36. This show that there is no adversative relation in the roles between immune and biomineralization.Views on biochemical defense34, gastropod shell has been co-opted as a defense system against parasitic nematodes37. However, C1qDC protein, which has both immune function and biomineralization function, has not been reported yet. Therefore, it is necessary to further study the functions of C1qDC in the formation and defense of mollusk shell, which will help to understand the relationship between them.Related to the evolution of shells and the bivalve mollusk lifestylePmC1qDC-1 in different tissues, various larvae development stages, and shell damage repair stages to expose its biomineralized function and temporal response to immune challenge. Investigating the biomineralized and immunological function of PmC1qDC-1 in P. f. martensii may provide new insights into the roles of C1qDC proteins in shell formation and defense mechanism of mollusks.In the present study, we aimed to examine the effect of PmC1qDC-1 on calcium carbonate precipitation rate and the expression pattern of PmC1qDC-1 gene was 902 bp, containing an ORF of 534 bp, a 5′-untranslated region (UTR) of 288 bp, and a 3′-UTR of 50 bp with a 30 bp poly (A) tail . PmC1qDC-1 gene encodes 177 amino acids with a theoretical isoelectric point of 9.11 and a predicted molecular weight of 19.6 kDa. A signal peptide of 17 amino acid residues was predicted in the N-terminus of PmC1qDC-1 by using SignalP-5.0 server. A globular C1q domain was located in the C-terminus according to the result of SMART analysis.The full-length cDNA of Crassostrea virginica (28.57% identity with C1qDC), from C. gigas (26.20% identity with C1qDC), from Ostrea edulis (32.20% identity with C1qDC), and from Mizuhopecten yessoensis (29.7% identity with C1qDC). The gC1q region of these C1qDCs was aligned. The 10-stranded β-sandwich in the gC1q domain of PmC1qDC-1 and the eight conserved residues in the human gC1q domain had been labeled  and the lowest expression in hemocytes .We detected the expression level of tes Fig. a. We alsges Fig. b. The rePmC1qDC-1 in mantle  was performed to study the spatial distribution of tle Fig. . No obvid-galactopyranoside (IPTG) induction. The main band in the figure of SDS-PAGE gel  tag was expressed under 1 mM isopropyl 1-thio-β-gel Fig.  was consIn vitro, the effect of PmC1qDC-1 on the precipitation rate was determined via a calcium carbonate precipitation experiment. Compared with the control, PmC1qDC-1 increased the precipitation rate, and this effect was concentration dependent Fig. .Figure 6PmC1qDC-1 expression by injecting ds_PmC1qDC-1. The mRNA expression levels of PmC1qDC-1 in the mantle pallial significantly decreased after treatment   Fig. . β-actinLPS Fig. a. After ted Fig. b. We alsion Fig. c. After oup Fig. d.Figure 38. In the present study, we identified a novel gC1q gene from P. f. martensii and designated it as PmC1qDC-1. PmC1qDC-1 contains a typical C-terminal globular C1q domain with 10 β-stand sheets and is considered as a member of the C1qDC protein family. All invertebrate C1qDCs, including those from C. gigas, M. yessoensis, C. virginica, O. edulis, and A. irradians, were grouped into a single branch. Thus, PmC1qDC-1 and other invertebrate C1qDCs may have evolved from the same ancestral gene, and subsequent selection pressure then led to the diversification of those genes within the invertebrate lineage. Multiple sequence alignment also supports this hypothesis. PmC1qDC-1 has low sequence similarity to C1qDC in other mollusks (26.2–32.2%), and some of the eight conserved amino acids39 in the human C1q domain had been mutated in PmC1qDC-1; thus these amino acids may have mutated to accommodate versatile attributes of function.C1q is the key component of complement system containing the typical C-terminal globular C1q domain. C1qDC protein can recognize many ligands via the gC1q domain with the common binding sitesPmC1qDC-1 in seven tissues. Constitutive expression in all of these tissues suggested the possibility of PmC1qDC-1 performing various functions in different tissues. Although the C1q domain is an immune-related domain, it also functions as SMPs existing in shell35. The high expression of PmC1qDC-1 in the mantle suggests that it may be involved in shell formation. The highest expression of PmC1qDC-1 in juveniles indicats that PmC1qDC-1 participats in the formation of dissoconch shell, which is the normal adult shell structure40. The mantle is the most important tissue involved in biomineralization and mantle pallial is responsible for the formation of nacreous layer. The strong hybridization signals located in the outer epithelial cells of mantle pallial indicates that PmC1qDC-1 participated in nacreous formation. Moreover, in vitro, PmC1qDC-1 can increase the calcium carbonate precipitation rate. Especially, disordered crystal growth was observed in the nacre upon knockdown of PmC1qDC-1 expression at the mantle pallial. This result indicats that PmC1qDC-1 is directly involved in nacre formation. The C1q globular domain consists of spherical heterotrimeric with Ca2+ ion bound at the top2. Therefore, PmC1qDC-1 may participate in nacre formation by accumulating Ca2+ ions. However, the mechanism through which PmC1qDC-1 participates in nacre formation requires further investigation.We detected the expression pattern of Mediterranean mussel are gC1q proteins28. Numerous invertebrate gC1q proteins are involved in the immune process. For example, MgC1q acts as a pattern-recognition molecule that can recognize pathogens during innate immune responses in M. galloprovincialis19. AiC1qDC-1 of scallops can agglutinate fungi and has mannose and PGN binding sites at its gC1q domain41. PmC1qDC-1 is extremely highly expressed at the mantle edge, indicating that it may be involved in resisting bacterial and pathogenic invasion. Besides the key roles of the mantle edge in shell formation, it also plays important roles in direct immune defense during exposure to seawater condition and even in shell damage and parasitic invasion in bivalves. For example, the mantle edge of C. gigas can secrete defensin in response to pathogen colonization42. The expression level of PmC1qDC-1 in mantle edge was significantly up-regulated at 48 h after LPS stimulation. After challenge with poly I:C for 12 h, 24 h, and 48 h, PmC1qDC-1 was significantly-high expressed in mantle edge compared with control group. The expression fluctuation of PmC1qDC-1 after LPS and poly I:C stimulation indicates that it is involved in immune defense. PmC1qDC-1 was significantly up-regulated in the mantle pallial at 6 h and 12 h post of shell notching while it was significantly up-regulated at mantle edge at 12 h and 24 h after shell damage. At the shell regeneration stage, shell damage will accelerate SMPs secretion43, and the organic membrane where the initiation of crystal deposition occurrs has formed near the nick at 6 h after shell notching44. Moreover, the mantle tissue will retract and increase the zone of tissue exposed to seawater due to the “V” nick, which will easily result in a second injury due to bacterial and pathogen infection43. Thus, PmC1qDC-1 may participate in shell regeneration and in the immune process of shell damage.gC1q protein is common in bivalves. For example, 167 of the 168 C1qDC gene models in 45. The shell notching experiment indicates that PmC1qDC-1 participates in the immune process. The hemocytes, the most critical immune organ, play a central role in the recognition of exogenous agents and in the defense against bacterial invasion in mollusks46. However, the expression level in hemocytes was relatively low. We speculate that, similar to HcC1qDC620, PmC1qDC-1 expression in hemocytes may be induced under stress. As such, the challenge experiment of LPS and poly I:C was performed to further understand the immune-related functions of PmC1qDC-1 in pearl oysters. PmC1qDC-1 was significantly up-regulated in the hemocytes at 6 h after injecting LPS, and the same expression pattern was present in the poly I:C treatment group. This finding indicates that PmC1qDC-1 is involved in the immune response against pathogen invasion.C1qDC proteins, which are pattern recognition molecules, rely on their gC1q domain to recognize a variety of self and non-self ligands, including a vast range of PAMPs of bacteria, yeast, viruses, and parasitesPmC1qDC-1. PmC1qDC-1 participates in nacre formation by increasing the calcium carbonate precipitation. PmC1qDC-1 may be involved in shell regeneration and immune response after shell injury. PmC1qDC-1 gene can quickly respond to LPS and poly I:C challenge. These findings revealed that it participates in shell formation and the innate immune response in P. f. martensii.In this work, we identified a novel gC1q gene named Adult pearl oysters (2 years old) were obtained from Houhong Xuwen, Zhangjiang,Guangdong Province, China. The animals were cultured at 25–28 ℃ indoor breeding tanks with seawater for 2 days before the experiment.32.The sample of larvae at different development stages was the same used in previous genomic researchhttps://dx.doi.org/10.17504/protocols.io.9qgh5tw). Absorbance at OD260/OD280 was measured using a NanoDrop ND 1000 spectrophotometer  to confirm the RNA quantity. RNA integrity was determined by fractionation on 1.0% agarose gel. cDNA was synthesized using an M-MLV reverse transcriptase . In brief, 500 ng of RNA and 1 μL of random primers were mixed, and RNase free water was added until 6 μL was reached, and the mix was incubated at 70 ℃ for 10 min. During this time, 2 μL of 5× M-MLV buffer, 0.5 μL of 10 mM dNTPmixture, 0.25 μL of RNase inhibited, and 0.5 μL of RTase M-MLV were mixed and added with RNase free water to reach 4 μL. Finally, the two reaction solutions mixed well and incubated at 42 ℃ for 1 h and 70 ℃ for 15 min. The first-strand cDNA was synthesized using SMARTer Rapid amplification of cDNA ends (RACE) 5′/3′ kit  in accordance with the manufacturer’s instructions.Various tissues containing the mantle central, mantle pallial, mantle edge, foot, hemocytes, adductor muscle, and gill were separated from the pearl oysters. Total RNA was isolated through TRIzol method following the protocol we submitted to protocols.io before . The base sequence was obtained via Sanger sequencing. All the primers used in the study were listed in the Table In the present study, the full-length cDNA of https://www.ncbi.nlm.nih.gov/orffinder/) and SignalP-5.0 Server  to obtain the ORF region and signal peptide of PmC1qDC-1. Domain information of PmC1qDC-1 was obtained through the Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de/smart/show_motifs.pl). Clustal Omega website tool  was used to align the protein sequences. Evolutionary relationship of PmC1qDC-1 and other orthologs was build up by MEGA 7.We used the ORF Finder tool (PmC1qDC-1 expression levels. The mix reagent was from DyNAmo Flash SYBR Green qPCR kit (ThermoFisher Scientific). The qRT-PCR experiment of PmC1qDC-1 was carried out on Applied Biosystems 7500/7500 Fast Real-Time System . The PCR program was as follows: 95 °C for 5 min, 40 cycles at 95 °C for 30 s, 60 °C for 15 s, 72 °C for 15 s. The relative expression levels of reference genes (β-actin and GAPDH) and PmC1qDC-1 were calculated through the 2−ΔCT method.We used the qRT-PCR to test PmC1qDC-1 at the tissues, different development stages, and different time points of shell notching were analyzed using one-way ANOVA. Differences in PmC1qDC-1 expression between the two groups were evaluated using the T-test. The significant level for these analyses was set at P < 0.05.Significance was analyzed using SPSS 22.0 . The expression levels of PmC1qDC-1 were synthesized in vitro by using T7 RNA polymerase and digoxigenin (DIG) RNA Labeling Mix. Integrity of PmC1qDC-1 RNA probes was confirmed by using 1% agarose gel electrophoresis. Concentration and purity of PmC1qDC-1 RNA probes were detected by using a nucleic acid quantifier. The mantle of P. f. martensii was separated and fixed 2 h in 4% paraformaldehyde containing 0.1% diethyl dicarbonate (Sangon Biotech). Then the mantle tissues were washed with phosphate buffer saline (PBS) for three times and embedded in the medium called optimal cutting temperature (O. C. T.). Finally, the embedded samples were made into slices with 10 μm thickness via the instrument of LEICA CM3050 S. Fluorescent in situ hybridization was constructed through the instructions of Enhanced Sensitive ISH Detection Kit IV (CY3) (BOSTER). The detailed protocol was referred to the file we submitted to protocols.io before (10.17504/protocols.io.9qhh5t6). The fluorescence signals were observed under microscope .RNA probes of PmC1qDC-1 following the protocol of Hao et al.47. In brief, double-stranded RNA (dsRNA) of PmC1qDC-1 (ds_PmC1qDC-1) and Red Fluorescent Protein gene (ds_RFP) were synthesized and purified by using a T7 High-Efficiency Transcription kit  and EasyPure RNA Purification kit  correspondingly. 100 µL ds_PmC1qDC-1 were injected into the adductor muscle of P. f. martensii, while 100 µL ds_RFP were injected at the control group. Mantle pallial was collected, put in liquid nitrogen at the 4 days post second injection and then frozen samples were transferred in − 80 °C freezer. The shells of all groups were cut into a small piece (0.5 cm × 1.5 cm) containing the transition region of the nacre and prismatic layer. The shell samples were clean with ultrapure water and air dried. The inner surface of nacre layer near the transition was observed under a scanning electron microscope (JSM-6300 LV) to obtain the image of ultrastructure.RNAi was used to knockdown the expression level of PmC1qDC-1 gene sequence without signal peptide was subcloned to the vector modified from (pET30a) by Abmart, and the recombinant plasmid was transformed using Rosetta Competent Cell for expression. Transformed cells were cultured in Luria–Bertani medium (with 50 mg/mL kana+) at 37 ℃ 200 rpm, induced with 1 mM IPTG when OD600 reached 0.5–0.8, and cultured at the same condition for another 4 h. The cell pellet was collected after centrifugation at 6000g for 5 min at 4 ℃ and washed three times with PBS. The harvested cells were resuspended with PBS and subjected to ultrasonication. The protein was purified using Amylose Resin (NEB). The wash fractions with 10 mM maltose were collected, boiled and loaded onto an SDS-PAGE gel. The purified protein was desalted by dialysis and freeze dried for storage. The protein concentration was measured by using BCA assay kit (Sangon Biotech).Recombinant PmC1qDC-1 protein was prepared in cooperation with Abmart. 48. The control was 20 μg/mL MBP. The 10 μL of sample solution and 100 μL of calcium chloride  were added to 96-well plates and mixed completely. Then, 100 μL of 100 mM sodium bicarbonate was added to the mixed solution quickly. The formation of calcium carbonate precipitate was tested by recording the absorbance at 570 nm every 1 min by using a multimode plate reader .The effect of PmC1qDC-1 on the rate of calcium carbonate precipitation was tested following the method of Dong et al.The notching assays were performed on 35 normal pearl oysters. Thirty normal pearl oysters were selected and a “V” shaped notch was cut on the shell until the nacreous layer was reached. The mantle edge and mantle pallial of every five pearl oysters were collected at 2 h, 4 h, 6 h, 12 h, and 24 h after damage, and the mantle edge of five pearl oysters (no notching) was harvested at 0 h.The pearl oysters were randomly divided evenly into three groups , and 100 μL (10 μg/mL) each of LPS, poly I:C, and PBS were injected into the adductor muscle. The pearl oysters were cultured in an indoor container with sea water at approximately 25 ℃. Hemocytes and mantle edge were harvested at 6 h, 12 h, 24 h, and 48 h after stimulation. In blank group, hemocytes and mantle edge were collected from the pearl oyster without stimulation at 0 h.Supplementary Figure 1.