In this study, we evaluated the immunogenicity and protective capacity of full-length recombinant Plasmodium falciparum circumsporozoite protein administered with the novel cationic liposomal adjuvant system CAF09. Using newly developed transgenic rodent malaria parasites expressing the full-length Plasmodium falciparum circumsporozoite protein, we demonstrate that this liposome-based protein-in-adjuvant formulation is capable of inducing robust antibody and CD8+ T-cell responses that strongly inhibit parasite infection and development of liver stages, conferring durable sterilizing immunity. These findings underscore the potential of liposome-based adjuvants for inducing robust humoral and CD8+ T-cell responses and warrant further studies toward the development of novel subunit vaccine formulations with this adjuvant system.Despite several decades of extensive research, the development of a highly efficacious malaria vaccine has yet to be accomplished. While the RTS,S malaria vaccine candidate shows the potential to prevent a substantial number of clinical malaria cases, significant improvements in protective efficacy are still needed. Multiple studies have shown that RTS,S induces protective antibody and CD4 + T cells, greater protection is conferred against the disease. Fidel Zavala, of the Johns Hopkins University, United States, and an international group of researchers developed their vaccine by encapsulating proteins from the malaria-causing parasite Plasmodium falciparum in fat-based carriers called liposomes. In past experiments, killer T cells recruited via this vaccine-type have effectively protected against other diseases. In this study, the vaccine induced both CD8+ T cell and antibody responses and provided significant immunity against P. falciparum-instigated malaria. As a highly efficacious vaccine against malaria is not yet available, this research will likely prove invaluable in guiding further studies.A vaccine consisting of parasitic proteins enveloped by fatty molecules provides comprehensive protection against malaria in a rodent model, Previous and current malaria vaccines concentrate on priming antibodies to recognize malarial infection, despite evidence that, by activating ‘killer’ CD8 While the protective capacity of these T cells is firmly established, the development of vaccines designed to induce CD8+ T-cell-mediated immunity has not advanced significantly, likely due to a lack of safe and efficient vaccine platforms capable of inducing robust and long-lived T-cell responses in humans.1 Attenuated viruses expressing foreign antigens are highly immunogenic and capable of inducing protective T-cell responses in animal models; however, they may not be as efficient in human vaccination. This is probably due to limitations imposed by pre-existing immunity, species specificity of recombinant viruses, and safety concerns that limit the magnitude of immunizing doses.4CD81 And while humoral immunity is a highly efficient protective mechanism, it is reasoned that vaccines that are also capable of inducing CD8+ T cells will likely increase the protective efficacy of vaccine-induced antibody responses.6 Indeed, CD8+ T cells will complement the humoral protective activity by exerting their anti-microbial effects against intracellular stages of infection, which are not commonly affected by circulating antibodies.All vaccines currently in use, as well as those in advanced development stages, are designed to induce protective antibody responses.+ T-cell responses. RTS,S, a subunit malaria vaccine candidate that is currently moving through the policy and regulatory process, is based on the Plasmodium falciparum circumsporozoite protein , incorporating the repeat and C-terminal regions of this antigen hybridized with the viral envelope protein of the hepatitis B virus (HBsAg). RTS,S was adjuvanted with AS01, consisting of monophosphoryl lipid A and QS21 incorporated into liposomes comprising dioleoylphosphatidylcholine and cholesterol.7 Phase 3 clinical trials demonstrated that among children 5 months to 17 months old, who received three doses of RTS,S/AS01, vaccine efficacy against clinical malaria ranged from about 51% over 12 months of follow-up from dose 1 to about 26% after approximately 4 years of follow-up. Vaccine efficacy was enhanced by a fourth dose given 18 months after dose 3. Among those who received a fourth vaccine dose, efficacy against clinical malaria was around 39% over roughly 4 years of follow-up.9 The trials showed that RTS,S-induced immunity wanes over time, with significantly reduced efficacy 3 years post-immunization.10 Thus, while the results obtained with RTS,S are encouraging, it is generally accepted that improvements in the efficacy of this vaccine are still necessary.Malaria vaccines currently in preclinical and human clinical trials may greatly benefit from the development of new immunogenic constructs capable of inducing antibody and CD8+ T-cell responses that correlate with protection against sporozoite infection.14 Nevertheless, this vaccine does not appear to induce significant CD8+ T-cell responses among vaccinated individuals.18 This may be an important shortcoming of RTS,S as it has been extensively demonstrated in animal models that CD8+ T cells are highly efficient at eliminating parasite-infected hepatocytes.20 To improve this aspect, prime-boost approaches using RTS,S and viral vectors or DNA constructs have been evaluated in clinical trials. However, these strategies did not result in considerable gains in overall protective immunity and vaccine efficacy in humans.23Immunization with RTS,S induces robust anti-CSP antibodies and CD4+ T cells by soluble protein-in-adjuvant systems has remained a difficult task, as poor results are obtained in different models.24 However, a newly developed liposomal adjuvant (CAF09) has recently demonstrated remarkable capacity to induce strong antigen-specific antibody and CD8+ T cell responses.25 CAF09 is based on cationic surfactant dimethyldioctadecylammonium (DDA) incorporating immunostimulators monomycolyl glycerol (MMG) and polyinosinic:polycytidylic acid (poly I:C), which are ligands for Mincle and TLR-3 receptors, respectively. Importantly, antigen adsorbed to CAF09 liposomes efficiently induces cross-priming by dendritic cells (DCs), and the induction of antigen-specific CD8+ T cells has been documented with several different antigens on proteins or peptides.25 In addition, CD8+ T cells elicited by CAF09 in combination with the E7 antigen from HPV16 confer complete protection in a prophylactic skin tumor model and enhanced protection as measured by reduced tumor growth for all mice in a therapeutic tumor model.25The induction of CD8P. falciparum CSP (Pf rCSP) administered with CAF09. We demonstrate that immunizations with this protein-in-adjuvant formulation induce potent anti-CSP antibody responses as well as robust antigen-specific CD8+ T-cell responses. Using a newly developed transgenic Plasmodium berghei parasite strain expressing the entire P. falciparum CSP, we show that these immune responses are capable of strongly inhibiting sporozoite infection and confer sterile immunity in mice.In this study, we characterized the protective capacity of immune responses induced by a full-length recombinant 25 To determine the immunogenicity of Pf rCSP administered with CAF09 (Pf rCSP-CAF09), mice were immunized by intraperitoneal (i.p.) injection with this construct and immune responses evaluated after the 2nd and 3rd immunizations. In immunofluorescence assays (IFA), we found that mice immunized two or three times with Pf rCSP-CAF09 developed comparable antibody responses, capable of recognizing sporozoites from P. falciparum and P. berghei transgenic parasites expressing the entire P. falciparum CSP  is not recognized as a murine Class I MHC epitope.29 This new transgenic parasite strain, P. berghei-P. falciparum CSP-full-length CD8 C-terminus (P.b.-P.f. CSP-FL CD8CT), develops normally in Anopheles stephensi mosquitoes and can efficiently infect naïve mice through mosquito bites  P. falciparum 3D7 CSP (i.e. with the unmodified 359DYANDIEKKI368 epitope) , compared to mice receiving adjuvant only or naïve controls. To establish whether Pf rCSP-CAF09 could confer sterile immunity, we challenged immunized mice with sporozoites delivered by infectious mosquito bites and monitored the development of blood-stage parasites . Remarkably, none of the Pf rCSP-CAF09-immunized mice developed blood-stage infection, while parasitemia in all of the age-matched naïve controls became patent by day 5 after challenge Fig. . Serum s10) compared to the adjuvant-only control -in-adjuvant formulation can induce some level of protective humoral immune responses capable of preventing infection of hepatocytes. However, improvements in vaccine efficacy are still needed, and this may be accomplished by developing vaccines capable of inducing wide-breadth antibody responses and CD8+ T cells that can exert their protective effect against intracellular liver stages of P. falciparum infection. In this study, we demonstrate that immunizations with a single construct, consisting of full-length Pf rCSP in combination with the cationic liposomal adjuvant system CAF09, induce robust humoral and CD8+ T-cell responses that are capable of conferring durable sterilizing immunity against sporozoite infection.The development of a fully-efficacious protective vaccine is a top priority in the fight against malaria. In the RTS,S phase 3 trial, the vaccine candidate demonstrated a reproducible -yet partially- protective effect, ranging from 51% after 12 months follow-up from dose 1 to 39% after a fourth dose and approximately 48 months follow-up.P. falciparum CSP as well as P. falciparum sporozoites and transgenic P. berghei sporozoites expressing the P. falciparum CSP. Interestingly, mice immunized two or three times with Pf rCSP-CAF09 developed comparable IgG titers against the P. falciparum CSP repeat region. However, three immunizations resulted in higher IgG titers against the N-terminal and C-terminal regions of the P. falciparum CSP. And while the potentially protective effect of antibodies against the C-terminal region of CSP is yet to be determined, we recently demonstrated that antibodies against the N-terminal region of this protein can significantly inhibit sporozoite infection.30 The fact that antibody titers persisted for several weeks after the last immunization of a 3-dose regimen is indicative of the enduring humoral responses elicited by Pf rCSP-CAF09. In this regard, findings from the recently completed RTS,S phase 3 clinical trial showed that anti-CSP antibody titers are indicative of the strength and duration of RTS,S/AS01-induced protective efficacy,31 thus underscoring the importance of robust and long-lived humoral responses. Additional studies should be useful to assess the kinetics of Pf rCSP-CAF09-induced anti-CSP antibodies over an extended follow-up period and to establish if comparable responses can be evoked by similar protein-in-adjuvant formulations. Nonetheless, our studies indicate that the antibody titers obtained with Pf rCSP-CAF09 are entirely comparable or better than those obtained with the same Pf rCSP antigen and immunization dose (20 μg) in combination with different adjuvant systems.32 Most importantly, only immunizations with CAF09 were capable of inducing strong CD8+ T-cell responses.Mice immunized with Pf rCSP-CAF09 developed high antibody titers, capable of binding different regions of the + T cells. Remarkably, these responses are comparable to those elicited by a prime-boost immunization using recombinant influenza virus for priming followed by recombinant vaccinia virus as booster, a protocol optimized to induce high-frequency antigen-specific CD8+ T cells against epitopes present in influenza virus, P. berghei and P. falciparum CSP.29 Differently, studies in our laboratory with recombinant CSPs together with other adjuvant systems have consistently shown that these immunizations induce poor or no CD8+ T-cell responses. In this regard, it is also worth mentioning that the RTS,S vaccine does not elicit CD8+ T cells among vaccinated individuals, thus underscoring the limited ability of protein-in-adjuvant formulations to induce cytotoxic responses.11Indeed, our results indicate that, in addition to eliciting strong antibody responses, immunizations with two or three doses of Pf rCSP-CAF09 induce strong antigen-specific CD8P. berghei strain expressing the full-length P. falciparum 3D7 CSP, incorporating the H-2Kk cytotoxic epitope DYENDIEKKI, naturally found in the C-terminal region of the CSP of different P. falciparum strains, such as 7G8 and T4, but not 3D7. The P.b.-P.f. CSP-FL CD8CT transgenic parasites develop normally in both mice and mosquitoes, and produce highly infectious sporozoites. Naïve mice exposed to the bites of three P.b.-P.f. CSP-FL CD8CT-infected mosquitoes develop blood-stage parasitemia within 5 days after exposure. Critically, CD8+ T cells induced by FluPf and VacPf viruses inhibited the development of liver stages of P.b.-P.f. CSP-FL CD8CT parasites expressing the cytotoxic epitope DYENDIEKKI but had no effect on parasites that expressed the P. falciparum CSP containing the sequence DYANDIEKKI. These data indicated that CD8+ T cells are protective only against the parasite expressing the CSP with the cytotoxic pertinent epitope, providing the rationale for using P.b.-P.f. CSP-FL CD8CT sporozoites for the assessment of Pf rCSP-CAF09-induced protective immune responses. The low level of antibodies induced after immunization with recombinant viruses, which are also known to be protective, likely explains the absence of protection in virus-vaccinated mice challenged with the parasite strain that does not express the DYENDIEKKI epitope.For this study, we developed a new transgenic 33 is considerably lower than the amounts required to boost CD8+ T-cell responses and to overcome T-cell self-regulation. As described in our previous studies, tens of thousands sporozoites are needed to boost established CD8+ T-cell responses and this booster dose needs to be administered at least 4 weeks after the last immunization.34 Furthermore, the fact that antibody titers against the different regions of CSP did not increase after sporozoite challenge, as shown in Fig. P. falciparum CSP immunizations in combination with different adjuvants did not achieve the same degree of protection and enduring immunity as Pf rCSP-CAF09.35Mice immunized with either two or three doses of Pf rCSP-CAF09 were able to significantly inhibit P.b.-P.f. CSP-FL CD8CT sporozoite infection and/or liver stage development with comparable efficiency, underscoring the potency of the immune response elicited by this protein-in-adjuvant formulation. To establish if Pf rCSP-CAF09 could confer sterilizing immunity against sporozoite infection, mice were challenged by infectious mosquito bites. In the first of these experiments, 90% of immunized mice did not develop blood-stage parasitemia. Most important, all sterilely protected mice were fully protected against an additional mosquito bite challenge 5 weeks later. This robust protection cannot be attributed to a boosting effect by sporozoites during the first experiment because the number of parasites delivered by mosquito bites, which is estimated to be just a few dozen,+ T cells against pre-erythrocytic stages of malaria, we sought to determine the relative contribution of cytotoxic CD8+ T cells induced by Pf rCSP-CAF09 immunizations against parasite liver stages. Treatment of Pf rCSP-CAF09-immunized mice with anti-CD8 antibody significantly decreased the vaccine-induced protective effect. In contrast, the depletion experiments suggest that CD4+ T-cell responses are not critical effectors, as treatment with anti–CD4 did not affect the overall anti-parasite effect in immunized mice. The fact that in mice treated with anti-CD8 antibody, the parasite burden is 1 log10 higher than in CD4+ T-cell depleted and control mice indicates that Pf rCSP-CAF09-induced CD8+ T cells have a major anti-parasite effect. However, it is worth mentioning that the magnitude of the antibody responses induced by Pf rCSP-CAF09 may still understate the strength of the role of T-cell responses induced by these immunizations.Given the importance of CD8+ T-cell responses.36 This has to do with the ability of CAF09-adjuvanted vaccines to rapidly drain to the lymph nodes and spleen, which is required for cross-priming of CD8α+ DCs residing in the lymphoid tissues.36 Nevertheless, novel liposome systems incorporating poly I:C are currently under development and aimed to facilitate lymph node targeting after s.c./i.m. immunizations.The vaccination strategy used in the present study involves i.p. immunizations, which for a human malaria vaccine may not be the preferred administration route. However, previous research indicated that this route of immunization is superior than subcutaneous (s.c.) or intramuscular (i.m.) routes for the induction of CD8+ T-cell responses. In general, this is not observed upon immunization with subunit vaccines based on recombinant proteins, which are known to mainly induce antibodies or CD4+ T-cell responses, or with recombinant viruses, which preferentially induce T-cell responses.1 These findings highlight the potential of liposome-based approaches for inducing robust humoral and CD8+ T-cell responses, and warrant further research for vaccine development.In conclusion, the most prominent finding of this study is that Pf rCSP-CAF09 was capable of inducing both strong antibody and CD8Five-week old to 8-week old female C3H/HeNCr MTV and C57BL/6 mice were purchased from Charles River . Animals were tagged and distributed randomly in different cages following procedures recommended by the Institutional Animal Care and Use Committee (IACUC) of The Johns Hopkins University. The size of experimental and control groups to allow proper statistical analysis was based on estimates of anticipated variation of rodent models of malaria infection and on prior experience with this experimental system. The investigators were not blinded to analysis. All experimental procedures involving animals were approved by the IACUC of The Johns Hopkins University.P. falciparum CSP from the Indian strain IND637HDD1 (GenBank: AAN87606.1) and it contains the murine H-2Kk cytotoxic epitope 373DYENDIEKKI383, which is also present in other P. falciparum reference strains, including 7G8 and T4. These strains express the same epitopes in the repeat domain and the amino acid sequence of this recombinant protein is over 90% identical to the CSP sequences of the P. falciparum reference strains 3D7 and 7G8. Its use in pre-clinical studies has been previously reported.32 The cationic adjuvant formulation CAF09 was developed by the Statens Serum Institut  and it consists of DDA-liposomes stabilized with MMGs-1 in combination with Poly(I:C). The use of this formulation for animal immunizations has been previously reported.25The full-length Pf rCSP used for all immunizations was manufactured by Gennova Biopharmaceuticals  and kindly provided for this study by the PATH Malaria Vaccine Initiative. This protein is based on k) were immunized with Pf rCSP in combination with CAF09. For each immunization, 20 µg of Pf rCSP were mixed with CAF09 , 0.5 mg/ml) and administered by i.p. injection on study days 0 and 14 (2-dose regimen) or on days 0, 14 and 28 (3-dose regimen). The injection volume of each immunization was 0.2 ml.C3H/HeNCr MTV mice  expressing the peptide KPKDELDYENDIEKKICKMEKCS encoding a region of the P. falciparum CSP C-terminus and boosted with 5 × 106 plaque forming units of recombinant vaccinia (VacPf) expressing the full-length P. falciparum 7G8 CSP gene. Immunizations were administered by i.p. injection on study days 0 and 14.Recombinant influenza and vaccinia viruses expressing selected sequences of the 7 or the C-terminal region (SDKHIKEYLNKIQNSLSTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYENDIEKKICKMEKHHHHHH) of P. falciparum CSP. Briefly, MaxiSorp® ELISA plates  were coated with 100 µl of synthetic peptide (1 µg/ml) and incubated overnight at room temperature. Plates were then washed and incubated with a 1% (w/v) bovine serum albumin (BSA) /1× phosphate buffered saline (PBS) solution (1% BSA-PBS) for an hour at room temperature. After another washing step, wells were incubated for 1 h at room temperature with serial dilutions of polyclonal sera. Plates were then washed and incubated for an hour at room temperature with a peroxidase-labeled goat anti-mouse (IgG H+L) secondary antibody  at 0.5 µg/ml in 1% BSA-PBS. The assay was developed using a 2,2′-Azino-di(3-ethylbenzthiazoline-6-sulfonate) horseradish peroxidase substrate kit , according to the manufacturer’s specifications. Antibody titers are reported as the corresponding sera dilution at which the optical density at 405 nm (OD405) was 1.0. Values were determined using a nonlinear regression with an asymmetric sigmoidal curve (GraphPad Prism 6 software).To assess antibody responses-induced Pf rCSP-CAF09 immunizations, polyclonal sera from immunized mice were evaluated 10 days after the last dose of each immunization regimen. We used an ELISA against synthetic peptides representing the N-terminal region (YGSSSNTRVLNELNYDNAGTNLYNELEMNYYGKQENWYSLKKNSRSLGENDDGNNEDNEKLRKPKHKKLKQPADHHHHHH), the repeat region [NANP]P. falciparum or transgenic sporozoites. In brief, poly-L-lysine-treated slides  were coated with a sporozoite suspension (4–6 × 105 parasites/ml) and allowed to air dry at room temperature. Polyclonal serum samples were then diluted in 1% BSA-PBS and incubated on slides for 30 min at room temperature in a humidity chamber. Slides were then washed with PBS-1% BSA and a secondary-antibody solution  was added for 30 min at room temperature. Fluorescent sporozoites were visualized using an upright fluorescence microscope (Nikon Eclipse 90i).Indirect IFA were used to assess anti-CSP titers in + T-cell responses in mouse spleens were assessed 2–3 weeks after the last immunization using an ex vivo stimulation assay. Single-cell suspensions (2 × 106 cells/well) were incubated with LM1 (H-2Kk) cells (5 × 105 cells/well),37 pulsed with or without the synthetic peptide DYENDIEKKI (8 µM). Incubations were performed for 5–6 h at 37 °C in the presence of Brefeldin A and Monensin . Cells were surface stained with APC-anti-CD8  and then treated with Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences). FITC-anti-IFN-γ  was then used for intracellular staining.CD8+ T cells or CD8+ T cells were depleted by i.p. injections with 150 μg of anti-CD4 antibody (clone GK1.5) or anti-CD8 antibody (clone YTS 169.4), respectively. Antibody injections were administered on two consecutive days.To evaluate the anti-parasite effect of Pf rCSP-CAF09-induced T cells upon sporozoite infection, CD4P. berghei strain in which the . This portion was then replaced with a 943-bp fragment, which was released using the same restriction enzymes from the plasmid pHZ-PfCSP. Thus, the csp gene (1188 bp) in the resulting plasmid, pIC-CSPfFL-CD8CT, consists of a full-length P. falciparum 3D7 CSP in which the signal sequence has been replaced with the one of the P. berghei CSP (base pairs 1 to 69). In addition, a single nucleotide in the csp gene of plasmid pIC-CSPfFL-CD8CT was replaced to incorporate a cytotoxic epitope that is not present in the P. falciparum 3D7 strain. This change was introduced in position 1079 by site-directed mutagenesis using a QuikChange II XL Site-Directed Mutagenesis Kit . We then excised the csp gene from pIC-CSPfFL-CD8CT as a KpnI-PacI fragment and inserted it into the transfection plasmid, pR-CSPfFL-CD8CT. Lastly, XhoI and KasI were used to linearize pR-CSPfFL-CD8CT prior to transfection of GFP-Luciferase P. berghei ANKA parasites,38 as previously described 39  followed by residues 25 to 397 from the P. falciparum 3D7 CSP.We developed a transgenic P.b.-P.f. CSP-FL CD8CT transgenic parasites were selected in Swiss Webster mice by treatment with pyrimethamine  in drinking water (0.07 mg/ml). Drug-resistant parasites were cloned by limiting-dilution. The successful recombination at the 5′ and 3′ ends of the modified locus was verified by PCR. The primers used to confirm 5′ integration were PbCS5′-F (TGCCCTATTCTCATATTTACCAC) and hDHRF5′ UTR-R (CACCATTTTTGAAAAGATTAATTTGA); the primers to verify integration at the 3′ end were PfCSP-F (TGAATGGTCCCCATGTAGTG) and PbCS3′UTR-R (CGCCTTAATGTGCATTGCT)  with 2 × 10A. stephensi mosquitoes. Starting on day 4 after challenge, daily blood smears were taken and observed under a microscope. Smears were fixed with methanol (for 30 s) before staining with 10% Giemsa stain solution  for 15 min.To evaluate sterile protection, mice were challenged by mosquito bites using five infected All sporozoite challenge experiments were performed 2 weeks after the last immunization.U test for post hoc analysis. Analysis of flow cytometry data was performed using FlowJo software (TreeStar). No animals or samples were excluded from statistical analyses.Data were plotted using GraphPad Prism 6.0 software. Data were analyzed for significance using Kruskal–Wallis test, with Mann–Whitney Supplmental material


This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F420 (two glutamates) from a methanogen isolate and long-chain F420 (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F420 purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F420-dependent oxidoreductases. However, long-chain F420 bound to these enzymes with a six- to ten-fold higher affinity than short-chain F420. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F420 increasing the substrate affinity (lower Km) but reducing the turnover rate (lower kcat) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F420 makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F420 to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F420 will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F420-dependent oxidoreductases.F Short-chain F420 was extracted from a thermophilic methanogen strain, Methanothermobacter marburgensis A60. We isolated the strain by repeated serial dilution of geothermally heated sediments from Ngatamariki, New Zealand. 16S rRNA gene sequencing of genomic DNA extracts  using the archaeal-specific primer set 109f/912r confirmed the strain shared 99% sequence identity with the well-studied laboratory strain Methanothermobacter marburgensis MarburgT . Cultures were grown to stationary-phase in a rotary incubator (100 rpm) at 60°C for 3 days with periodic gas feeding before harvesting.Long-chain Fand cofE . CultureMarburgT . For F42 formate  and a H2420 was harvested from the mycobacterial and methanogen cultures through variations on an existing protocol  at a ratio of 1 g per 10 mL. The cells were autoclaved at 121°C to release F420, a heat-stable cofactor, into the buffer. The cell debris was removed by centrifugation at 18,000 × g for 20 min and the supernatant was decanted and vacuum-filtrated through 0.45 μm filter paper. The F420 was isolated by FPLC (fast protein liquid chromatography) with a Macro-prep High Q Resin anion exchange column (Bio-Rad). A gradient of buffer A  and buffer B  was applied, with buffer B increasing from 0 to 100% over 10 column volumes. Fractions containing F420 were identified via analysis of absorbance spectra on a SpectraMax® M3 Multi-Mode Microplate Reader . Fractions containing F420 were pooled. The F420 solution was further purified and concentrated by hydrophobic interaction chromatography through a high capacity C18 column equilibrated in H2O. F420 was eluted in 2 mL fractions in 20% methanol, dried by rotary evaporation, and stored at -20°C.Fprotocol . The cel420 purified from mycobacterial and methanogen sources. An Agilent 1200 series system equipped with an Agilent Poroshell 120 EC-C18 2.1 × 50 mm 2.7 μm column and diode array detector was used. The F420 species were separated at a flow rate of 0.3 mL min-1 using a gradient of two buffers, namely A  and B (100% acetonitrile). A gradient was run from 25 to 40% buffer B as follows: 0–1 min 25%, 1–10 min 25–35%, 10–13 min 35%, 13–16 min 35–40%, 16–19 min 40–25%. F420 absorbance was measured at 420 nm using a diode array detector. This system was also used to execute an absorbance scan from 400 to 600 nm on the sole F420-2 peak of the methanogen F420 and the prominent F420-6 peak of the mycobacterial F420. The length of the F420 oligoglutamate tails were verified by a reverse phase LC/MS (liquid chromatography / mass spectrometry) protocol with an Agilent 1100 series LC/MSD TOF equipped with a Poroshell 120 EC-C18 2.1 × 100 mm 2.7 μm column. A gradient protocol comprising of Buffer A (20 mM ammonium acetate pH 6.8) and Buffer B (100% acetonitrile) was applied as follows: Held from 0 – 1 min at 5% B; 1 – 10 min from 5 – 20% B. Negative mode ESI was used with a capillary voltage of 2500 V and gas temperature of 300°C. The system was run at a flow rate of 0.2 mL min-1 and chemical species were scanned from 150 – 1500 m/z.An ion-paired reverse phase HPLC (high-performance liquid chromatography) protocol was used to determine the oligoglutamate chain length of F420-reducing glucose 6-phosphate dehydrogenase  and two F420H2-dependent reductases  from M. smegmatis mc2155 were recombinantly overexpressed in Escherichia coli BL21(DE3) using previously described vectors and protocols  until use in assays. The high purity of the proteins was confirmed by running the fractions on NuPAGE Novex 10% Bis-Tris gels  and staining with Coomassie Brilliant Blue. We measured the activities of the enzymes by monitoring the rates of F420 reduction or F420H2 oxidation in the presence of different substrate concentrations; this serves as a reliable measure of substrate transformation given F420-dependent oxidoreductases directly mediate hydride transfer between cofactor and substrate in an equimolar manner (® M3 Multi-Mode Microplate Reader (Molecular Devices); loss of absorbance was observed due to Fgd-mediated reduction of F420, whereas gain of absorbance occurred due to FDOR-mediated reoxidation of F420H2. Prior to measurement of FDOR activity, F420 was enzymatically reduced with 1 μM Fgd in a nitrogen glovebox for 4 h and purified by spin filtration as previously described  with a quartz cuvette containing 500 nM of protein in 20 mM TrisHCl, pH 8.0 at 24°C was used. Samples were excited at 290 nm and emission was monitored at 340 nm. One microliter aliquots of F420 standards in the same buffer were added to produce a solution with final concentrations ranging from 0 to 12.3 μM F420, with the concentration recalculated for the incremental increase in volume. The fractional saturation (F/Fmax) was plotted against the concentration of free F420, and the Kd derived from fitting the data points to the function: F/Fmax = Fmax∗[Free F420] / (Kd + [Free F420]).Fescribed . A SpectM. tuberculosis Rv3547/Ddn  and 1.2PDB: 3RZ ] as the PDB: 3RZ  and calcPDB: 3RZ . The Antock Vina . For MSMock Vina ]. Diglut0H- form  and all 420-dependent oxidoreductases from different subgroups within the LLHT, FDOR-A, and FDOR-B superfamilies were retrieved from the NCBI database. Multiple sequence alignments were constructed with Clustal Omega . To confirm chain length, we separated the purified F420 on a HPLC equipped with an anion-exchange column and detected the cofactor at 420 nm using a diode array detector. F420 purified from M. smegmatis contained between five to eight glutamates , consistent with previous mass validation . The mass of the dominant chemical species was validated by LC/MS , the F420H2-dependent quinone reductase MSMEG_2027 (FDOR-A family), and a promiscuous F420H2-dependent reductase of unknown function MSMEG_3380 (FDOR-B family). We observed that long-chain mycobacterial F420 bound the enzymes with nanomolar affinities  of 650 nM, 190 nM, and 54 nM respectively , though the kinetic parameters differed depending on the length of the F420 oligoglutamate chain. Observed substrate turnover rates (kcat) were between 1.9 and 3.7 times greater in the presence of short-chain F420 compared to long-chain F420 (Table 1); such enhancements of initial rate were observed irrespective of the F420 concentration used . Hence, high-affinity cofactor binding results in both enhanced substrate binding and decreased reaction turnover.We compared the reaction kinetics of the three enzymes in the presence of the different Ffindings , reactio420-6) and short-chain (F420-2) variants of F420 to the available crystal structures of the FDOR-A MSMEG_2027 . These glutamate residues also interacted with Arg205, Arg23, and Arg54 of MSMEG_3380, but these interactions were more transient . In both cases, the terminal four glutamates of F420-6 made multiple transient electrostatic interactions with surface cationic residues, but these residues were highly dynamic and displayed little specificity in the various interactions they formed. These additional interactions also appeared to stabilize the core interactions made by the cofactor, for example between the phosphate group and Lys53 of MSMEG_3380 . The multiple alternative tail conformations observed remained favorable even though the headgroup was weakly restrained within the active site . Such observations are consistent with the lower Kd values determined in the tryptophan fluorescence quenching experiments . The internal bond, angle, and dihedral energies of the cofactor were essentially unaffected by the various binding modes . Conserved cationic surface residues are also proximal to the F420 oligoglutamate chain in the LLHT superfamily , the main contacting residues of MSMEG_3380  all lie on highly stable β-sheet and helical secondary structural regions. Multiple sequence alignments and comparative structural analysis suggest that these cationic residues were highly conserved within their respective FDOR-A and FDOR-B superfamilies  and hence reduced substrate turnover. It is also plausible that conformational changes caused by the electrostatic interactions are transmitted to the adjoined isoalloxazine- and substrate-binding sites, thereby modulating substrate affinity; this may be particularly important in FDOR-A proteins, where interactions between the terminal glutamate residues and the lysine-rich loop region may stabilize the split β-barrel fold and in turn the substrate-binding site. The finding that substrate turnover of F420-dependent oxidoreductases is accelerated in the presence of short-chain F420 is important for biotechnological reasons: it suggests that F420 purified from methanogen sources will result in higher turnovers in the various bioremediation and biocatalysis processes for which F420-dependent oxidoreductases have been advocated , is required for optimal functionality of F420 in mycobacterial cells, though is less important than the other F420 biosynthetic enzymes  and in turn the production of short-chain F420 (420 producers but is fused with an FMN reductase domain in most long-chain producers (420-dependent oxidoreductases co-evolved with CofE, resulting in higher-affinity cofactor-enzyme interactions.The observed differences between bacterial and archaeal Fancestor . We receain F420 . We proproducers ; recent roducers . It is pCG and AW conceived the study. CG, AW, BN, CC, RS, TJ, JO, and MS designed experiments. BN, TJ, AW, CG, CC, RS, and MS performed experiments. CG and CJ supervised students. CG, AW, BN, JO, TJ, and CJ interpreted data. CG, AW, and BN wrote the paper.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.


We found that a therapeutic course of AAL-R reversed experimental AHR in the methacholine challenge test, which was not replicated by dexamethasone or the non-phosphorylatable isomer AAL-S. AAL-R efficiently interfered with ASM cell proliferation in vitro, supporting the concept that immunomodulation is not necessary to interfere with cellular mechanisms sustaining AHR. Moreover, the sphingosine-1-phosphate lyase inhibitor SM4 and the sphingosine-1-phosphate receptor antagonist VPC23019 failed to inhibit proliferation, indicating that intracellular accumulation of sphingosine-1-phosphate or interference with cell surface S1P1/S1P3 activation, are not sufficient to induce cytostasis. Potent AAL-R-induced cytostasis specifically related to its ability to induce intracellular AFD-R accumulation. Thus, a sphingosine analog that possesses the ability to be phosphorylated in situ interferes with cellular mechanisms that beget AHR.In asthma, excessive bronchial narrowing associated with thickening of the airway smooth muscle (ASM) causes respiratory distress. Numerous pharmacological agents prevent experimental airway hyperresponsiveness (AHR) when delivered prophylactically. However, most fail to resolve this feature after disease is instated. Although sphingosine analogs are primarily perceived as immune modulators with the ability to prevent experimental asthma, they also influence processes associated with tissue atrophy, supporting the hypothesis that they could interfere with mechanisms sustaining pre-established AHR. We thus assessed the ability of a sphingosine analog (AAL-R) to reverse AHR in a chronic model of asthma. We dissected the pharmacological mechanism of this class of agents using the non-phosphorylatable chiral isomer AAL-S and the pre-phosphorylated form of AAL-R (AFD-R)  Asthma is a prevalent and complex syndrome . Alarminex vivo is determined by the amount of ASM    that occigure 1) , which aigure 1)  and inhiigure 1) , which iigure 1) . Importaid bases . In agreD. pteronyssinus; Greer, Lenoir, NC, USA), three times a week, for 5 weeks. Mice were then left untouched during week 6 . Sphingosine analogs  randomly assigned to experimental groups were anesthetized with isoflurane and instilled intranasally with 50 μl of saline or with saline containing 50 μg of HDM extract , the length of the basal membrane and the area of ASM for individual circular bronchi was measured. ASM thickness was defined as area of ASM divided by the length of the basal membrane. Two to five bronchi were measured blindly per lung and the average size of bronchi did not differ between groups.4 cells per cm2 in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum (complete medium) for 24 h, and then incubated with sphingosine analogs, the S1P lyase inhibitor  antagonist  for 24–72 h. Cells were either incubated with tetrazolium dye -2,5-diphenyltetrazolium bromide; Sigma Aldrich, Oakville, ON, Canada) to determine the viable cell numbers; or incubated with bromodeoxyuridine  overnight to assess proliferation. Flow cytometric analyses were performed as described  were plated at a density of 10tor SM4;  or the described .Cell pellets were spiked with internal standards  and lipids were extracted using a published method . Liquid t-test was employed. The results were considered significant with p-values ≤ 0.05. Data were analyzed in collaboration with a professional biostatistician at the IUCPQ biostatistics core service using the statistical package program SAS v9.4 .Data were expressed using mean ± SEM. Homogeneity of variance and normality of the data were verified. When appropriate, variables were log-transformed. Rank transformation and the ordinary F test from ANOVA were used when appropriate. Data from mice were analyzed using ANOVAs or two-way ANOVAs with an interaction effect. When heteroscedasticity was significant, the statistical analyses were performed using separate residual variance per group. The Satterthwaite’s degree of freedom statement was added for unequal variance structure. Posteriori comparisons were performed using Tukey’s comparison. When appropriate, bi-directional unpaired Figure 2), we first evaluated the in vivo potential of AAL-R to reverse AHR, when administered at well-tolerated doses, yet sufficient to induce biological effects. We found that AAL-R (1 mg/kg/day) reversed HDM-induced AHR to a level 70% lower than the vehicle treatment . Contrary to AAL-R, corticosteroid intervention  failed to reverse AHR, furthering the concept of usefulness for sphingosine analogs in the context of asthma. On that note, we previously showed that AAL-R efficiently interfered with the inflammatory cascade in response to HDM soon after its instatement in the airways . Mice receiving saline and vehicle were used as baseline controls. Planimetry on transverse lung slices revealed an average bronchial smooth muscle thickness of 4.1 ± 0.6 μm in the group receiving saline and vehicle. In the group receiving HDM and vehicle, ASM thickness was increased to 5.6 ± 0.5 μm. This 36% increase in ASM thickness  was reduced to a near-baseline level of 4.3 ± 0.3 μm by AAL-R. In agreement with reduced ASM thickness, AAL-R again robustly reversed AHR caused by chronic exposure to HDM (not shown). Thus, these three series of experiments revealed that AAL-R retains its ability to inhibit TH2 inflammation in the remodeled airways and efficiently reverses ASM thickening as well as the ensuing AHR.Since ASM thickening is central to the development of AHR, we performed a new series of experiments where lungs of mice receiving either AAL-R or vehicle after chronic HDM exposure were stained with Masson’s trichrome, in order to visualize the ASM layer located between the epithelium and the parenchyma , when compared to the vehicle. We then set out to determine whether apoptosis also contributed to the inhibition of ASM accumulation under mitogenic conditions. We found that, at concentrations of 1 μM or lower, AAL-R mildly modulated apoptosis in vitro , while being a potent cell death inducer at 10 μM with nearly 100% dead cells. On the other hand, AFD-R failed to induce apoptosis, even at the concentration of 10 μM , and AAL-S only induced partial apoptosis at 10 μM. Our results thus unravel a clear potential for AAL-R to interfere with mechanisms that sustain ASM cell proliferation.Because AAL-R reverses AHR when administered at a phase where acute inflammation is resolved, we next addressed the idea that AAL-R could act directly on ASM cells to impair underlying features of ASM thickening. We show that AAL-R, but not its pre-phosphorylated/cell impermeant form (AFD-R), nor its non-phosphorylatable isomer (AAL-S), efficiently interfered with the proliferation of ASM. For instance, AAL-R induced a 26% inhibition of bromodeoxyuridine incorporation at 1 μM . AAL-R increased absolute levels of intracellular S1P (2.0 ± 0.13 picomoles) when compared to the vehicle (0.5 ± 0.05 picomoles), to AAL-S (0.6 ± 0.06 picomoles), and to AFD-R (0.9 ± 0.05 picomoles). This resulted in a significant twofold increase in the S1P/sphingosine balance, when compared to the vehicle treatment .Given the putative impact of sphingosine analogs on the balance of sphingolipidic species, we tested the concept that intracellular S1P levels could explain the differential potency of sphingosine analogs in inducing ASM cytostasis. We first characterized which S1P pathway proteins were expressed in ASM cells . The S1P lyase inhibitor SM4 potently increased intracellular S1P level (2.8 ± 0.08 picomoles), which resulted in a 33% increase of the S1P/sphingosine ratio compared to the AAL-R-treated cells . This occurred in the absence of an effect on the C16-ceramide content . Also, we found no evidence that AAL-R, AAL-S, or AFD-R (at the concentration of 1 μM) impacted on cellular levels of C16-ceramide, arguing against a major role for a shift in the sphingolipid rheostat to justify AAL-R-induced cytostasis. In spite of potent enhancement of intracellular S1P content, SM4 failed to inhibit ASM cell proliferation , revoking the sufficiency for increased intracellular S1P content to induce ASM cell cytostasis.To resolve whether or not increase of intracellular S1P was sufficient to induce cytostatis, cells were incubated with an inhibitor of S1P lyase  . Similar to AFD-R and AAL-S, dual inhibition of S1P1 and S1P3 failed to significantly affect the accumulation of proliferating ASM cells in vitro, when compared to the vehicle.Since intracellular accumulation of S1P might reduce extracellular S1P levels and the subsequent impairment of S1PSphK2 . This is demonstrated by the retrieval of relatively high amounts of AAL species in cells incubated with AAL-S (263 ± 17 picomoles) and AAL-R (126 ± 15 picomoles), compared to an equimolar concentration of AFD-R (32 ± 4 picomoles) . Most importantly, in ASM cells, the intracellular balance between the non-phosphorylated and the phosphorylated form of AAL-R favored the accumulation of AFD-R by up to twofold . Of note, this AFD-R accumulation (263 ± 16 picomoles) represents more than 100 times the cellular amount of S1P under the same experimental conditions. Thus, ASM cells express SphK2 and display a massive intracellular accumulation of AFD-R upon incubation with AAL-R.Since ASM cells express in situ phosphorylation of the sphingosine analog, we ruled out the ability of the non-phosphorylatable sphingosine analog AAL-S to reverse AHR in our in vivo model. Figure 9 depicts normoresponsiveness to graded methacholine doses in mice receiving saline and vehicle. In this group, peak resistance was recorded at 2.89 ± 0.27 cmH2O/ml/s in response to an aerosolized dose of 30 mg/ml of methacholine. AHR was present in the group receiving HDM and vehicle, with a threefold increase of respiratory system resistance at 30 mg/ml of methacholine, when compared to mice receiving saline and vehicle. AAL-R again inhibited by more than 70% the HDM-induced AHR at the methacholine dose of 30 mg/ml. In stark contrast, the non-phosphorylatable AAL-S enantiomer failed to alleviate AHR in HDM-exposed mice. Of note, neither AAL-R nor AAL-S affected airway responsiveness to methacholine in mice without experimental asthma  fails to reverse AHR in vivo, despite its documented anti-inflammatory effects .Current literature suggests that likely mechanisms underlying the atrophic effects of sphingosine analogs include activation of protein phosphatase 2A , interfef asthma . Yet, thf asthma . Yet, on1 (We observed increased intracellular S1P in cells treated with AAL-R. This intracellular S1P likely resulted from interference with S1P lyase activity and/or inhibition of S1P export . In T ce1 . Alterna1 , and mig1 . Still, It remains possible that differential subcellular localization of accumulating S1P between the current study and previous studies , 2011 miin vitro, when compared with FTY720 (SphK2 and display a nearly 2:1 AFD-R to AAL-R ratio. In face of a preferential distribution of SphK2 at the endoplasmic reticulum (The fact that SphK2-metabolized sphingoid bases preferentially interfere with cell accumulation also supports the concept that, as described in Jurkat T cells , intraceh FTY720 . In agreeticulum  and sinceticulum , we conceticulum .It is well-documented that a prolonged daily administration of sphingosine analogs at low doses can increase the risk of pulmonary side effects , which lin vitro. This effect depends on the propensity of this sphingosine analog to be phosphorylated in situ and likely results from an increase of the intracellular aminophosphate content. Given the central role of ASM thickening in AHR and asthma, this experimental therapeutic strategy needs to be further explored.We found that a punctual treatment with AAL-R resorbs the thickening of ASM in the context of asthma, while not aversively affecting the airways within a short time window. Moreover, we determined that AAL-R could directly interfere with ASM cell accumulation Participated in research design: DG, PL, A-ML, M-JB, NF, AD, EB, M-RB, ML, SB, YB, DM. Conducted experiments: DG, PL, A-ML, C-AH, AL-G. Contributed new reagents: SB. Performed data analysis: DG, PL, A-ML, C-AH, AL-G, DM. Wrote or contributed to the writing of the manuscript: DG, PL, A-ML, M-JB, C-AH, AL-G, NF, AD, EB, M-RB, ML, SB, YB, DM.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.


Actinobacillus pleuropneumoniae S-8 genome sequence revealed the presence of pdxS and pdxT genes, which are implicated in deoxyxylulose 5-phosphate (DXP)-independent pathway of PLP biosynthesis; however, little is known about their roles in A. pleuropneumoniae pathogenicity. Our data demonstrated that A. pleuropneumoniae could synthesize PLP by PdxS and PdxT enzymes. Disruption of the pdxS and pdxT genes rendered the pathogen auxotrophic for PLP, and the defective growth as a result of these mutants was chemically compensated by the addition of PLP, suggesting the importance of PLP production for A. pleuropneumoniae growth and viability. Additionally, the pdxS and pdxT deletion mutants displayed morphological defects as indicated by irregular and aberrant shapes in the absence of PLP. The reduced growth of the pdxS and pdxT deletion mutants under osmotic and oxidative stress conditions suggests that the PLP synthases PdxS/PdxT are associated with the stress tolerance of A. pleuropneumoniae. Furthermore, disruption of the PLP biosynthesis pathway led to reduced colonization and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model. The data presented in this study reveal the critical role of PLP synthases PdxS/PdxT in viability, stress tolerance, and virulence of A. pleuropneumoniae.Pyridoxal 5’-phosphate (PLP) is an essential cofactor for numerous enzymes involved in a diversity of cellular processes in living organisms. Previous analysis of the  Pyridoxde novo PLP biosynthesis pathways, referred to as deoxyxylulose 5-phosphate (DXP)-dependent pathway and DXP-independent pathway ×100.eviously . The A. A. pleuropneumoniae WT S-8, S-8ΔpdxS mutant, S-8ΔpdxT mutant and complemented strains S-8ΔpdxScomp and S-8ΔpdxTcomp were cultivated in BHI medium supplemented with 10 μM PLP at 37°C for 16 h, then washed three times with un-supplemented BHI medium to remove residual PLP and diluted to optical densities at 600 nm (OD600) of 0.05. The fresh cultures were grown to mid-exponential growth phase in 10 ml of BHI medium with or without 10 μM PLP. The cells were washed three times with PBS and fixed for 2 h in 2% osmic acid. Dehydration was performed in upgraded ethanol, and then the samples were embedded in SPI-Pon 812 resin (emicron) for two days. Resin-soaked sample blocks were polymerized at 70°C, and samples were counterstained with uranyl acetate and lead citrate. Electron micrographs were recorded with a transmission electron microscope .The A. pleuropneumoniae infection [A. pleuropneumoniae WT S-8, S-8ΔpdxS mutant, and S-8ΔpdxT mutant strains. Briefly, all A. pleuropneumoniae strains were cultured in BHI medium supplemented with 10 μM PLP at 37°C, and harvested during the mid-exponential phase and washed three times with sterile PBS. A total of 160 mice were randomly divided into 16 groups (n = 10/group). For each strain, five experimental groups were inoculated intraperitoneally with 100 μL of PBS containing varying concentrations of bacterial suspension (105–109 CFU). Non-infected mice in the control group were inoculated with 100 μL of sterile PBS (pH 7.4). Animal suffering was reduced with buprenorphine (0.05 mg/kg), given subcutaneously for analgesia every 12 hours during the first 72 hours after infection. The health status and the weight of the mice were monitored twice daily for a 14-day period and humane endpoints used to determine if the mice met criteria to be euthanized [50) of A. pleuropneumoniae WT strain S-8, S-8ΔpdxS mutant, and S-8ΔpdxT mutant strains were calculated as previously described by Reed-Muench [BALB/c mouse has been acknowledged as an appropriate model for nfection , 25. Spethanized . These cd-Muench .7 CFU of WT S-8 strain, S-8ΔpdxS mutant, and S-8ΔpdxT mutant strains respectively. Three (3) days post-infection, mice from each group were humanely euthanized and the organs of lung, liver, and kidney were removed aseptically. Samples were weighed, and homogenized using a tissue homogenizer (100 mg weight/ml of PBS). Viable counts in serial dilutions of homogenates were determined following culture on BHI agar plates for 24 h at 37°C.A total of 15 specific-pathogen-free, six week-old female BALB/c mice were randomly divided into 3 groups (n = 5), and each group was intraperitoneally administered with 1.0×10P-values less than 0.05 were considered statistically significant.All statistical analyses were performed using GraphPad Prism version 5.01 . The data are expressed as the means +/- standard deviation. pdxS and pdxT genes are 888-bp and 576-bp in length, respectively, and encode the putative pyridoxal 5'-phosphate synthase subunits PdxS and PdxT. Searching the respective conserved domains in NCBI database revealed that PdxS has SOR_SNZ domain which represents a family of pyridoxal 5'-phosphate synthase and PdxT has SNO domain which represents glutamine amidotransferase family. Both PdxS and PdxT shared a high identity in the amino acid sequences with their orthologous sequences in Bacillus subtilis  and Streptococcus pneumoniae . In this study, to determine the activities of PdxS and PdxT in vitamin B6 formation in vitro, A. pleuropneumoniae pdxS and pdxT genes were overexpressed in E. coli BL21 (DE3) and the recombinant His6-tag fusion proteins were purified using Ni2+-affinity chromatography. SDS—PAGE analysis showed that the recombinant proteins, with the molecular weight of approximately 35.3 kD and 23.4 kD  than that of WT S-8 (75.68%)  and 47.16% (P < 0.01) survival rates, respectively. Similar results were obtained when the cells were exposed to osmotic stress. The viable counts of S-8ΔpdxS and S-8ΔpdxT were lower than that of the S8 cells (pdxS mutant and the S-8ΔpdxT mutant respectively exhibited 32.08% (P < 0.01) and 52.13% (P < 0.05) survival rate  than that of WT S-8 (86.09%); and the survival rate of S-8ΔpdxT mutant was 59.16%, which was also significantly lower (P < 0.05) than that of WT S-8  . To excle of PLP . As showS8 cells . In the val rate . In the f WT S-8 . These rpdxS mutant, S-8ΔpdxT mutant and complemented strains S-8ΔpdxScomp and S-8ΔpdxTcomp were observed using transmission electron microscopy. These data showed that the morphologies of the five strains were similar in the presence of PLP supplementation, and were consistent with the normal morphology of coccobacilli  was found to be only 15.5-fold lower than WT S-8. These results suggested that the loss of both pdx enzymes attenuates the pathogenicity of A. pleuropneumoniae, with a greater impact observed with the deletion of pdxS.To address whether pdxS mutant, and S-8ΔpdxT mutant strains to colonize mice was then tested. The A. pleuropneumoniae load in tissues of systemically infected mice was determined by culturing the lungs, livers, and kidneys homogenates 3 days post-infection. As shown in pdxS mutant in the lungs, livers and kidneys were significantly lower compared with those of WT S-8 (P<0.01). Similarly, significant differences (P < 0.05) in bacterial loads were also found between the WT S-8-inoculated mice and the S-8ΔpdxT-inoculated mice in lungs. In addition, the bacterial loads in the livers and kidneys of the S-8ΔpdxT-inoculated mice were decreased compared to the WT S-8-inoculated mice, although these differences were not statistically significant , suggesting that the levels of PLP in mice may be too low to support the optimal growth of these mutants, which further affected the colonization and virulence of the S-8ΔpdxS and S-8ΔpdxT mutants. Another probability is the involvement of PLP in the biosynthesis of surface-exposed structures of A. pleuropneumoniae. Indeed, it has been shown that PLP biosynthesis is essential for the synthesis of surface-exposed structures involved in adhesion and colonization in some pathogenic bacteria, such as lipopolysaccharide (LPS) and flagella [pdxS and S-8ΔpdxT mutants exhibited aberrant shapes in the absence of PLP  and PdxT (rPdxT) were separated by 12% SDS-PAGE. Lane M, molecular mass markers.(TIF)Click here for additional data file.S2 FigpdxS gene in WT S-8, S-8ΔpdxS, and S-8ΔpdxScomp strains. (B) Transcriptional levels of pdxT gene in WT S-8, S-8ΔpdxT, and S-8ΔpdxTcomp strains. (C) Transcriptional levels of downstream genes of pdxS in WT S-8 and S-8ΔpdxS strains. (D) Transcriptional levels of downstream genes of pdxT in WT S-8 and S-8ΔpdxT strains.(A) Transcriptional levels of (TIF)Click here for additional data file.S3 FigpdxS, S-8ΔpdxT, S-8ΔpdxScomp strain and S-8ΔpdxTcomp strains were diluted into fresh BHI broth and grown to OD600 0.8. Bacteria were then treated with 5 mM H2O2 in the absence (A) and presence (B) of PLP for 45 min, 0.4 M NaCl in the absence (C) and presence (D) of PLP for 45 min. Viable CFUs of A. pleuropneumoniae were counted. The data shown are the means of three independent assays.Overnight culture of WT S-8, S-8Δ(TIF)Click here for additional data file.S4 FigΔpdxS, S-8ΔpdxT, S-8ΔpdxScomp strain and S-8ΔpdxTcomp strains in the mid-log phase in the presence of PLP supplementation were carried out. The morphologies of the five strains were similar and were consistent with the normal morphology of coccobacilli.TEM of WT S-8, S-8(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


PRODH are linked to behavioral alterations in schizophrenia and as part of DiGeorge and velo-cardio-facial syndromes, but the role of PRODH in their etiology remains unclear. Here, we establish a Drosophila model to study the role of PRODH in behavioral disorders. We determine the distribution of the Drosophila PRODH homolog slgA in the brain and show that knockdown and overexpression of human PRODH and slgA in the lateral neurons ventral (LNv) lead to altered aggressive behavior. SlgA acts in an isoform-specific manner and is regulated by casein kinase II (CkII). Our data suggest that these effects are, at least partially, due to effects on mitochondrial function. We thus show that precise regulation of proline metabolism is essential to drive normal behavior and we identify Drosophila aggression as a model behavior relevant for the study of the mechanisms that are impaired in neuropsychiatric disorders.Mutations in the proline dehydrogenase gene  Editors' choice: A Drosophila model to study the role of PRODH, a schizophrenia-associated gene, in behavioral disorders. PRODH maps to 22q11, a chromosomal region associated with the most frequently observed interstitial deletion in humans and linked to different diseases, including DiGeorge and velo-cardio-facial syndrome (PRODH gene and schizophrenia were subsequently demonstrated (Loss of proline dehydrogenase (PRODH) has been linked to various behavioral defects. Human syndrome . Individsyndrome . Furthersyndrome . Multiplsyndrome . Associanstrated . Finallynstrated .PRODH is localized on the inner mitochondrial membrane, where it converts proline to delta-1-pyroline-5-carboxylate in the first, rate-limiting step of the two-step oxidation of proline to glutamate . This prIn addition to being a metabolic precursor of glutamate, proline acts as a co-agonist for the N-methyl-D-aspartate (NMDA) receptor . FurtherDespite the strong implication of PRODH in behavioral disorders, the exact mechanisms by which PRODH and altered proline metabolism contribute to these disorders are not well understood and their study would benefit from a genetically tractable model.Drosophila genome contains a single PRODH homolog, sluggish A (slgA)  . Previoubehavior . TherefoslgA, the DrosophilaPRODH homolog, is broadly expressed in the adult brain. Regions expressing slgA include the mushroom bodies and the lateral neurons ventral (LNv). Overexpression of human PRODH and knockdown and overexpression of slgA in the LNv result in changes in aggressive behavior, demonstrating the need for a careful balance of proline metabolism for normal behavior. We further use this model to show that different slgA isoforms have differential effects on aggression, with the D and E isoforms not increasing aggression upon overexpression. These isoforms are distinguished by the presence of a casein kinase II (CkII) phosphorylation site. RNAi-mediated knockdown of the catalytic casein kinase II alpha (CkIIα) subunit in LNv and pharmacological inhibition of casein kinase II result in the D and E isoforms also inducing aggression similar to the A, B and C isoforms. Furthermore, we provide evidence that CkII and SlgA interact directly. Finally, we show that the effects of SlgA on aggression can at least in part be explained by mitochondrial alterations. Our results define a role for PRODH in Drosophila aggressive behavior, thereby establishing a model in which to dissect further the role of proline metabolism and signaling in behavioral abnormalities.We here show that slgA was initially identified by and named for its role in locomotor behavior  calyx, consistent with the position of the MB neurons. Furthermore, we found prominent expression in the lateral neurons ventral (LNv), the main pacemaker cells of the Drosophila clock, which express Pigment-dispersing factor (Pdf), and in cells located in the suboesophageal ganglion (SOG) .To characterize possible roles of lt brain . We perfon (SOG) A-D. We che brain E-H. We aslgA could be involved in aggression . We excluded the possibility that this increase in aggression is due to increased locomotion by analyzing the locomotor behavior of these flies. We observed no significant changes in velocity or path length . These findings were confirmed with an independent overexpression line for human PRODH, ruling out insertional effects . Given that the tested flies were starved for 90 min prior to testing, we also excluded the possibility that the observed increase in aggression was due to a difference in starvation resistance .PRODH has been implicated in behavioral abnormalities in humans and mice, and our data suggest that gression . This le and fly . TherefoPdf-Gal4 to drive expression in a more restricted expression pattern compared with cry-Gal4.Because overexpression of PRODH in the LNv induced hyperaggression, we decided to focus on these cells in further experiments, using slgA in the LNv by RNAi-mediated knockdown. Interestingly, knockdown also resulted in an increase in aggressive behavior (slgA levels (and thus proline metabolism) must be tightly controlled to maintain normal behavior and that genetic disruption of proline homeostasis by up- and downregulation of SlgA and PRODH leads to similar increases in aggressive behavior. In Drosophila, alternative splicing of slgA mRNA leads to the generation of five different protein isoforms (Movie 5). These findings were confirmed with an independent overexpression line for each isoform, ruling out insertional effects . qRT-PCR showed strong overexpression of slgA upon ubiquitous overexpression of all constructs using tstubP-Gal4; tubP-Gal80 . Analysis of locomotor behavior showed no correlation between changes in locomotion and changes in aggressive behavior . We also excluded the possibility that the observed differences in aggression are due to a difference in starvation resistance .First, we confirmed an endogenous requirement for behavior B. We conisoforms C. ProteiDrosophila and vertebrates, including circadian rhythmicity, cell cycle regulation and neuronal development  in the two isoforms that have no effects on aggression (D and E). This 12-aa insertion introduces a putative phosphorylation site for Casein kinase II (SDDD) (CkII) C. CkII ielopment . These fJF01436CkIIα and GL0003CkIIα) in the adult LNv, using tsPdf-Gal4; tubP-Gal80, combined with overexpression of the two isoforms that had no effect on aggression. Knockdown of CkIIα in the LNv combined with overexpression of slgA-D or slgA-E in these neurons resulted in a significant increase in aggressive behavior compared with the control lines . To account for possible effects on aggression due to the changes in temperature, we tested tsPdf-Gal4; tubP-Gal80 flies as a control both on 18°C and switched to 25°C after eclosion and 4 days before testing. This shift in temperature had no significant effect on aggressive behavior. We also tested the knockdown efficiency of both CKIIα RNAi lines by qRT-PCR. Both lines result in a significant knockdown of approximately 50% .We first addressed this possibility with a genetic approach. We performed RNAi-mediated knockdown of CkIIα . Lower concentrations of TBBz (50-100 µm) had no effect on aggression in flies overexpressing slgA-D or -E . The combined results demonstrate that the effects of CkII and SlgA in the adult brain are sufficient to modulate aggressive behavior.We next investigated whether pharmacological inhibition of CkII by 4,5,6,7-tetrabromo-benzimidazole (TBBz) would induce a behavioral change in these lines. TBBz has been shown to inhibit specifically the CkII holoenzyme by ATP competition with effective concentrations in yeast of 10-200 µm . We obseol flies C. This tts/UAS-RNAi-CkIIαtubP-Gal4; tubP-Gal80) resulted in significantly reduced quantities of bound CkIIα .Finally, we determined whether SlgA and CkII can interact directly by means of co-immunoprecipitation experiments using the anti-human PRODH2 antibody to pull down SlgA and subsequent immunoblotting to detect CkIIα, the catalytic subunit of the CkII complex. We find that CkIIα is co-immunoprecipitated in an isoform-specific way with SlgA bound to the PRODH2 antibody and that knockdown of CkIIα by means of two independent RNAi knockdown constructs (slgA and PRODH affect circadian rhythms in light-dark (LD) and subsequently in dark-dark (DD) conditions. Knockdown of slgA and overexpression of the different slgA splice variants or PRODH does not lead to alterations in circadian rhythmicity compared with the control . All tested genotypes are rhythmic. Flies show similar day-night rhythms in 12 h:12 h LD conditions and show the same ability as the control flies to maintain these in DD conditions . Based on these results, we conclude that it is very unlikely that release of neuropeptides by the LNv is altered.We next investigated how disruption of SlgA/PRODH results in aberrant aggressive behavior. First, we determined whether the effect could be mediated at the level of neurotransmitter production and release. Alterations in PRODH have been shown to influence multiple neurotransmitter signaling pathways, including GABA, glutamate and acetylcholine . HoweverslgA in the LNv. We did not observe changes in spontaneous activity and other physiological properties when modulating slgA  overexpression of which has an effect on aggression and one isoform (SlgA-E) overexpression of which has no effect, and knockdown of ing slgA . In concslgA affect mitochondrial morphology. For this analysis, we focused on the mitochondria in the sLNv terminal arbor area as previously described  resulted in changes in ethanol-related behaviors whereas mutations in the NADH dehydrogenase subunit 2 (ND2) led to abnormal bang-sensitive behavior . Flies were collected in 1 ml of TRI reagent  and ground with a plastic disposable pestle. Total RNA was isolated using standard procedures. Two replicates per genotype were analyzed.RNA was isolated from ten whole flies per replicate from the following genotypes: 18 primer according to the manufacturer's instructions . qRT-PCR was performed on an ABI7000 instrument with qPCR Mastermix Plus for SYBR Green I  with the following primers: slgA-F, ACGCTGGGCGACAATAAGG; slgA-R, GGAACAAATGCAAAATTCCCTCC; RpII140-F, TTCCCCGATCACAATCAGAGT; RpII140-R, ATATAAACGCCCATAGCTTGCTTAC. Expression levels of transcripts from the various samples were normalized to RpII140 expression.cDNA was generated from 1 μg of RNA of each sample by using an anchored oligo(dT)slgA (LD10578) was obtained from the Drosophila Genomics Resource Center . In situ hybridization on adult brains and subsequent imaging was performed as described by cDNA for Drosophila slgA protein isoforms (LGIPLDGTVCFGQLLGMCDHVSLALGQAGY VVYKSIPYGSLEEVIPYLIR). The sequence of the synthetic peptide serving as immunogen for anti-human casein kinase II alpha is fully conserved in Drosophila. Confocal imaging was performed using an Olympus FV1000 microscope.Immunohistochemistry was performed as described by tstubP-Gal80, 12 h:12 h light/dark cycle). CO2 was used as an anesthetic. NP4104slgA, JF01436, CkIIα GL0003, slgAGL01514, TubPUAS-mCD8-gfp, UAS-mito-tomato, UAS-RFP, CkIIαts, TubP-Gal4, OK107-Gal4-Gal80 and 201y-Gal4 were obtained from the Bloomington Drosophila Stock Center, Bloomington, IN, USA. P{cry-Gal4.E39} and P{pdf-Gal4.P2.4} were a gift of Dr Bassem Hassan . All fly stocks were isogenized by mating females to Canton-S males for ten generations to exclude effects due to differences in genetic background. The RNAi lines used were predicted to have no off-target effects  and velocity and path length were compared amongst the different genotypes. Twenty replicate measurements per genotype were performed and replicate tests were spread over multiple days to account for possible environmental alterations. Data showing a Gaussian distribution were analyzed by a one-way fixed-effects ANOVA with a subsequent post-hoc Holm–Sidak's multiple comparisons test to determine significant mean differences among the lines. Data not showing a Gaussian distribution were analyzed by a non-parametric Kruskall–Wallis test with Dunn's multiple comparison test.Three- to seven-day-old male flies were transferred without anesthesia to vials containing a wet cotton ball to prevent dehydration. Survival was observed until all flies were dead. Fifteen males were tested per genotype. Survival analyses were performed using Prism 6 (Graphpad). Significance was determined using Mantel–Cox and Gehan–Breslow–Wilcoxon tests.Drosophila Activity Monitoring (DAM) system (TriKinetics) at 25°C. Three- to seven-day-old socially experienced flies were loaded into tubes containing 1% agarose and 5% sucrose food. Flies were kept at 12 h:12 h LD for 5 days, the first day was excluded from the analysis. Subsequently, flies were kept under DD conditions for 7 days. Circadian locomotor rhythmicity was analyzed using FaasX . Sleep behavior was analyzed using Counting Macro 5.19.9 . Further statistical analyses were performed in Graphpad Prism 6.Circadian locomotor behavior was analyzed using the PRODH1 cDNA (Image clone 40108133) was obtained from Source BioScience . Drosophila slgA isoform A cDNA (LD10578) was obtained from the Drosophila Genomics Resource Center . The Kozak sequence for PRODH was generated with the following primers: F, CGTGCGGCCGCCAACATGAAGATGACCTTCTATGGGC; R, GAAGGCCCGGTGGGCCTGGTATTG. This region was directionally cloned into the PRODH cDNA using NotI and BglI. The Kozak sequence for the A, B, D and E isoforms was generated with the following primers: F, CGTGAATTCCAACATGGCTCTACTCCG; R, ATAAGGCCTGCAGCGGCCGGTCGCCG. This region was cloned into slgA isoform A cDNA (LD10578) using EcoRI and StuI. The region specific for the B and D isoforms (CTGGCGCGCAACCTGCTCGGCCAGAAGCTCTTCGTCCTGCTGATGAAGTCCAGCTTCTACGGACACTTTGTGGCCGGCGAGAATCGTCACACGATCGTGCCCGCC) was generated using the following primers: F1, ATTAG GCCTCCACTCTGGTCCAAC; R1, AGTGTCCGTAGAAGCTGGACTTCATCAGCAGGACGAAGAGCTTCTGGCCGAGCAGGTTGCGCGCCAGTTTCATAAGCGTCATGTTGT; F2, TGCTGATGAAGTCCAGCTTCTACGGACACTTTGTGGCCGGCGAGAATCGTCACACGATCGTGCCCGCCCTGGAAAGGCTAAGATCCTT; R2, GCATGCTGCCCTCCTCCTTTTTG. The region specific for the B and D isoforms was cloned into slgA isoform A cDNA using StuI and SphI. The region specific for the C, D and E isoforms (GATGATGATCGCAAGGCGCCCCGGGCAGTGGCCACG) was generated using the following primers: F1, ATCGCATGCCGCAGTACCATGTG; R1, CACTGCCCGGGGCGCCTTGCGATCAT CATCCGAAACAGCCTCCAGACACT; F2, GATCGCAAGGCGCCCCGGGCAGTGGCCACGGGCGCCACCTTTGGAACTGG; R2, GACGTCCTTATTGTCGCCCAG. The region specific for the C, D and E isoforms was cloned into slgA isoform A cDNA using SphI and AatII. Isoform C, including the Kozak sequence was generated using the following primers: F, TGGGAATTCCAACATGCGCACACGCAAGTACATGG; R, ATTGCGGCCGCTTAGATGGGCACGTAATTGCC. All slgA isoforms were cloned from pCR™-Blunt II-TOPO®  to pUAST using EcoRI and NotI. PRODH was cloned from pCR™-Blunt II-TOPO®  to pUASt using NotI and BamHI. Injections to generate transgenic flies were carried out as a service by Model Systems Genomics, Duke University, Durham, NC, USA.Alignments were performed using ClustalW . Analysi® 60 HG  to bring it in solution. 0.5% Methocel® 60 HG solution was prepared by adding Methocel® 60 HG to H2O at 70°C. This solution was stirred overnight. TBBz was mixed with 100 µl Tween20 and subsequently added to the Methocel® 60 HG solution while stirring. Four milliliters of the Methocel® 60 HG/TBBz  solution with 100 µl Tween20 was added to 1 g of Formula 4-24 Drosophila Medium, Blue . Flies were kept on this food for 3 days prior to testing.4,5,6,7-Tetrabromobenzimidazole (TBBz) was purchased from Sigma-Aldrich, Diegem, Belgium. TBBz is insoluble in water, hence we used MethocelCo-immunoprecipitation (Co-IP) was performed following the instructions of the Thermo Scientific Pierce Co-Immunoprecipitation kit . In the first step, the anti-human PRODH2 antibody  was immobilized onto an agarose support. For each genotype analyzed, an antibody column was made by mixing 10 µg of the anti-human PRODH2 with 50 µl of the Coupling Resin. All the details concerning the antibody immobilization are described in the Thermo Scientific Co-IP protocol.g) and protein concentration of the supernatant was quantified with a Bradford protein assay .For the sample preparation, ten flies of each genotype were homogenized in 100 µl of the IP lysis/wash buffer . After 15 min incubation on ice, the homogenate was cleared . Alternative washing was performed using PBS. Finally, bound proteins were eluted from the antibody by incubating the column with 50 µl of Thermo Scientific elution buffer (pH 2.8). Twenty microliters of each sample was loaded on a 4-12% Bis-Tris SDS-PAGE gel  and transferred to nitrocellulose membranes. For western blot analysis, anti-human casein kinase II alpha , rabbit HRP-conjugated secondary antibody  and standard ECL detection were used. Images were obtained using the LAS-3000 imaging system (Fuji) and analyzed with AIDA Imaging Analyzer software.Pdf-Gal4, UAS-mito-gfp. sLNv axon termini were imaged using an Olympus Fluoview 1000 confocal microscope using identical setup parameters. Number of mitochondria and mitochondrial size were analyzed using ImageJ  1 and 4, where ZT0 corresponds to lights-on. Whole fly brains were acutely dissected in extracellular saline solution containing (in mM): 101 NaCl, 1 CaCl2, 4 MgCl2, 3 KCl, 5 glucose, 1.25 NaH2PO4 and 20.7 NaHCO3 at pH 7.2. After removal of the photoreceptors, lamina, air sacks and trachea, a small incision was made over the position of the l-LNv neurons in order to give easier access for the recording electrodes. The brain was then placed ventral side-up in the recording chamber, secured using a custom-made anchor and neurons were visualized using a 63× lens on an upright Zeiss microscope . l-LNv neurons were identified on the basis of their fluorescence, size and position. Whole-cell current clamp recordings were performed at room temperature (20-22°C) using glass electrodes with 8-18 MΩ resistance filled with intracellular solution  and an Axon MultiClamp 700B amplifier, digitized with an Axon DigiData 1440A  and recorded using pClamp 10 . Chemicals were purchased from Sigma .We visualized the l-LNv using in) was calculated by injecting hyperpolarizing current steps and measuring the resulting voltage change.The liquid junction potential was calculated as 13 mV and subtracted from all the membrane voltages. A cell was included in the analysis if the access resistance was less than 50 MΩ. Resting membrane potential (RMP) and the spontaneous firing rate (SFR) were measured after stabilising for 2-3 min. The membrane input resistance (R


With the in vivo and in vitro I/R stroke models, we found that miR-375 mimic provides significant protection from injury to cerebral I/R, which is reflected by reduced infarct volumes and cell apoptosis, and increased proliferation and migration of PC12 cells. Mechanistically, our findings showed that miR-375 binds to 3′-UTR region of Ctgf mRNA, subsequently leading to the decreased expression of Ctgf in the I/R brain. Furthermore, we showed that miR-375/Ctgf-mediated protective effects are associated with p21/PI3K/Akt signaling pathways. Our findings thus provide a new insight into the mechanism of cerebral I/R injury and pave a potential new way for the therapy of cerebral I/R injury.Ischemic stroke is the leading cause of disability and deaths worldwide. MiRNAs have been shown to play an important role in development and pathogenesis of the nervous system. However, the precise function and mechanism of miRNAs are not fully understood in the brain injury induced by ischemia/reperfusion (I/R). Herein, our study showed that  Ischemic stroke is the main cause of permanent injury, disability, and deaths in the world. Ischemic stroke is usually treated by intravenous thrombolytic with alteplase or intra-arterial mechanical thrombectomy with stent retriever devices. However, this therapy, also known as cerebral ischemia/reperfusion (I/R), can lead to increased brain injury. Accumulating evidence shows that apoptosis and death of neural cells after I/R are the leading causes of aggravated brain injury ,2. Thus,MiRNAs are a class of small noncoding RNA molecules (~22 nts) found in most eukaryotes, including humans . Its dismiR-145 has recently been shown to regulate neural stem cell differentiation through the Sox2-Lin28/let-7 signaling pathway [MiR-455-3p has been proven to be a potential peripheral biomarker for Alzheimer’s disease [miR-375 which is specifically expressed in the pancreatic islets, brain, and spinal cord. Wang et al. first showed that miR-375 expression was down-regulated in the cerebral I/R rat [miR-375 expression is related to injury of I/R. Recently, Bhinge et al. [miR-375 was essential for motor neurone development and degeneration. Therefore, we hypothesized that down-regulation of miR-375 might be an important contributor to increased injury induced by I/R.In neural system, a large number of studies has suggested that miRNA plays an important role in the development and pathogenesis of the nervous system –6. For e I/R rat . Howevere et al.  showed tmiR-375 was identified using bioinformatics software. With middle cerebral artery occlusion (MCAO) rat model and in vitro hypoxia/reoxygenation (H/R) PC12 model, we provided direct evidence that miR-375 shows a protection from injury of cerebral I/R through targetting Ctgf mRNA and decreasing Ctgf protein expression. Our findings provide a new insight into the mechanism of cerebral I/R injury and pave a potential new way for the therapy of cerebral I/R injury.In the present study, a new target Ctgf of miR-375 and the mutant Ctgf 3′-UTR DNA sequence were amplified and inserted into psi-CHECK2 Renilla/firefly Dual-luciferase expression vector , with the following primers: Ctgf 3′-UTR-WT: 5′-CCGCTCGAGAAACTGATAGCCTCAAACTCC-3′ (forward) and 5′-ATTTGCGGCCGCTAAACTGCCTCCCAAACC-3′ (reverse). Ctgf 3′-UTR-MUT: 5′-TAAAATCACTGTTGGATCTGTCATGGCCTTTATTAAG-3′ (forward) and 5′-CTTAATAAAGGCCATGACAGATCCAACAGTGATTTTA-3′ (reverse). MiR-375 mimic, a chemically synthesized exogenous miR-375 mature sequence mimic and scramble negative control (NC) miRNA were supplied by Guangzhou RiboBio, China. HEK-293T cells were co-transfected with the reporter constructs and miR-375 mimic or NC with Lipofectamine 2000 . Luciferase activities were determined after 48 h using the Dual-luciferase reporter assay system (Promega) on Enspire . Data are presented calculated as the ratio of Renilla luciferase activity in cell lysates to firefly luciferase.The human Ctgf 3′-UTR DNA sequence that was predicted to interact with Male adult Sprague–Dawley rats (250–280 g) were obtained from Animal Center of Southern Medical University . All procedures conducted with rats were in accordance with standard procedures approved by the Institutional Ethical Committee. Rats were anesthetized by 10% chloral hydrate (350 mg/kg) and subjected to MCAO. Briefly, after vessel isolation, a 3-0 monofilament nylon suture  was inserted into the internal carotid artery and anterior cerebral artery through the external carotid artery to occlude the MCA. After 2 h of operation, the suture was removed to induce reperfusion, and the rats were kept in intensive care incubator under 37°C for 24 h.n=5); (ii) I/R group: 2 h ischemia and 24 h reperfusion (n=5); (iii) I/R + NC: rats were administrated with scramble miRNA  after ischemia treatment (n=5). (iv) I/R + mimic: rats were administrated with mimic  after ischemic treatment (n=5). (v) I/R + FNS: fastigial nucleus electrostimulation (FNS) of rats was electrically prestimulated for 1 h, and rats were treated in the same way as I/R group 24 h later (n=5). MiR-375 mimic and NC were resolved in artificial cerebrospinal fluid , while Sham, I/R group, and I/R + FNS were intracerebroventricularly injected with artificial cerebrospinal fluid.Rats were randomly divided into five groups: (i) Sham: threading without occluding, persisting perfusion  at 37°C for 30 min followed by immersion in 10% formalin overnight.After I/R treatment, some rat brains were fixed in 10% neutral buffered formalin (NBF) and embedded in paraffin. Paraffin sections were stained with Hematoxylin and Eosin (H&E) or terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling  or antibody against Ctgf . The images of H&E and immunohistochemistry were measured through phase-contrast light microscope , while the images of TUNEL were examined under the inverted fluorescence microscope (Leica). For TUNEL assay, the rate of apoptotic events in total cells was calculated.miR-375 mimic in inflammation caused by I/R. The rat brain tissue samples were homogenized for 10 min, and the intracellular ROS was determined using 2′,7′-dichlorofluorescin diacetate . After centrifugation at 4000 rpm for another 10 min, MDA and SOD activities in the supernatant of brain tissue lysates were measured according to its provider’s instructions.We employed intracellular reactive oxygen species , malondialdehyde  and superoxide dismutase  measurement to indicate the protective role of 2.H2O, 4 mM HEPES, 10 mM deoxyglucose, and 20 mM sodium lactate (pH 6.2)), and cells were transferred to an anoxic chamber containing 1% O2, 94% N2, and 5% CO2 at 37°C to simulate hypoxia. Cells were subjected to simulated hypoxia injury for 6 h, after which the hypoxia buffer was replaced with DMEM containing 1% FBS and cultured in a normoxic incubator containing 95% O2 and 5% CO2 at 37°C (simulated reperfusion) for 48 h.P12 cell culture medium was replaced by ischemic buffer  by an ABI PRISM 7500 Fast Real-time PCR instrument . It was carried out with the following procedures: 94°C for 2 min, 94°C for 20 s, followed by 40 cycles of 58°C for 20 s and 72°C for 20 s. The specific primers used in quantitative real-time RT-PCR were displayed as follows: miR-375: forward, 5′-ACACTCCAGCTGGGTTTGTTCGTTCGGCTCGC-3′; reverse, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCACGCGA-3′; Ctgf: forward: 5′-CTTCTGCGATTTCGGCTCC-3′; reverse, 5′-TACACCGACCCACCGAAGA-3′; GAPDH: forward, 5′-GGTGGTCTCCTCTGACTTCAACA-3′; reverse, 5′-GTTGCTGTAGCCAAATTCGTTGT-3′; U6: forward, 5′-CTCGCTTCGGCAGCACA-3′; reverse, 5′- AACGCTTCACGAATTTGCGT-3′. The data were analyzed according to the 2C−ΔΔt method.Total RNA from rat brains and cells was extracted with RNAprep pure tissue kit  and RNeasy Mini Kit , respectively. The expression levels of For whole-cell extract preparation, the brain tissue and cells were lysed in tissue protein extraction reagent (T-PER) and mammalian protein extraction reagent (M-PER) containing halt protease inhibitor cocktail , respectively. Equal amount of proteins was subjected to SDS/PAGE and then transferred on to PVDF membrane . After being blocked by 5% milk, the membranes were incubated with antibodies against Ctgf, PI3K, AKT, p-AKT, p21, and GAPDH . Last, corresponding secondary HRP conjugated antibodies were applied and the signals of targetted proteins were visualized using the ECL chemiluminescent detection kit .miR-375 mimic for 24, 48, and 72 h. After washing with PBS three times, a 1:10 diluted CCK-8 solution was added to the medium and incubated at 37°C for 1 h. The absorbance was measured by microplate reader at 450 nm.Cell viability was measured using Cell Counting Kit-8 . H/R PC12 cells were treated with scramble NC miRNA or miR-375 mimic were cultured in six-well plates, and Hoechst 33258  was added to the culture medium and incubated for 10 min, cells were then washed with PBS three times, and the counts of Hoechst-positive nuclei were detected by inverted fluorescence microscope using a 365-nm filter for Hoechst 33258.H/R PC12 cells treated with NC or miR-375 mimic were fixed with 4% paraformaldehyde and permeabilized in 0.5% Triton X-100. After being blocked with 3% BSA, cells were incubated with primary antibodies against Ctgf and DAPI. Next, cells were stained with the FITC-conjugated and Alexa Fluor 488-conjugated secondary antibodies . Finally, images were taken under the inverted fluorescence microscope.Immunofluorescence staining was performed to evaluate the expression of Ctgf. H/R PC12 cells treated with NC or 2, the invading cells through the membrane were fixed with 95% ethanol, stained with 0.1% Crystal Violet, and imaged under the microscope. The percentage of migrated cells was quantitated by counting five independent visual fields.Cell invasion was determined by the transwell assay. After transfection for 48 h, PC12 cells in serum-free medium were transferred into the upper Matrigel-coated invasion chambers (BD Biosciences) and medium containing 10% FBS was added to the lower chamber. After 24 h of incubation at 37°C with 5% COmiR-375 mimic and pcDNA3.0-Ctgf or pcDNA3.0-vector for 24 h. After removing the culture medium, the cells were fixed with 1 ml of 3.7% formaldehyde in PBS for 15 min and incubated with 0.5% Triton X-100 in PBS for 20 min. Then, the cells were incubated with 0.5 ml of Click-iT reaction cocktail at room temperature for 30 min in the dark. After removal of the reaction cocktail, cells were washed with 3% BSA in PBS followed by PBS. After addition of DAPI in PBS, cells were incubated for 20 min at room temperature in the dark. The cells were then washed with PBS, and images were obtained under the inverted fluorescence microscope.For EdU staining assay, cells were permeabilized and stained with Click-iT EdU Imaging Kit  according to manufacturer’s instructions. Briefly, the H/R cells were incubated with P<0.05.Statistical analyses were performed using GraphPad Prism 6.0 software. Data were expressed as mean ± S.D., and analyzed by one-way ANOVA with multiple comparisons test (three or more datasets in a group). Differences were considered to be statistically significant when miR-375, we used bioinformatics software of MirTarget algorithm and miRDB to predict downstream target gene of miR-375. The data showed that miR-375 could bind to 3′-UTR region of Ctgf gene (Ctgf mRNA is bona fide target of miR-375. The result demonstrated that the relative luciferase activity of the wild-type Ctgf 3′-UTR reporter was significantly suppressed in the HEK-293T cells with the treatment of miR-375 compared with the cells treated with miR-NC. In contrast, the luciferase activity of the binding site mutant Ctgf 3′-UTR reporter was unaffected by co-transfection of miR-375 mimic compared with NC co-transfected (miR-375 might provide the protection in the injury of cerebral I/R through inhibiting Ctgf expression.To determine the target gene of tgf gene A, which nsfected B. These miR-375 against brain I/R injury, we first established the model of I/R rat brain and determined the effect of miR-375 mimic on infarction volumes by TTC staining, with Sham, I/R, I/R + NC, I/R + FNS as control. As shown in miR-375 mimic or FNS (miR-375 mimic and FNS compared with the I/R group with NC treatment or without treatment (miR-375-treated I/R group was linked to the up-regulation of miR-375 (miR-375 (miR-375 does protect rat from injury of cerebral I/R.To demonstrate the protective role of c or FNS B. In thereatment D and 2E. miR-375 C. Of not(miR-375 C,D. ThismiR-375/CTGF-mediated protection from injury of cerebral I/R, we observed histological integrity and tissue damage in rat brains after subjecting to I/R. As indicated in miR-375 mimic clearly attenuated the degeneration and cytoplasmic vacuolization induced by I/R. Furthermore, a large number of TUNEL-positive cells was observed in the brain sections of rats subjected to I/R injury, whereas TUNEL-positive cells were significantly reduced in the miR-375 mimic group compared with the NC group (miR-375 mimic treated group (To clarify the downstream mechanism of NC group B, which NC group D. Consised group C.miR-375 mimic-treated group compared with the NC-treated group (miR-375 mimic treatment (miR-375 could exert protective role against cerebral I/R injury through suppressing apoptosis and peroxidation.The ROS and MDA levels, known as the indication of intracellular oxidative stress, were significantly much higher in the I/R + NC group than in the Sham group. Of note, there was significant reduction in ROS and MDA level in the ed group E,F. In preatment G. CollecmiR-375 underlying its protection from cellular H/R injury, H/R P12 cell model was established through 6 h of hypoxia and 48 h of reoxygenation. In line with the in vivo results, the expression of miR-375 was decreased and mRNA and protein expression of Ctgf was elevated in H/R P12 cells (To further prove the mechanism of 12 cells A,B. Afte12 cells C.miR-375 levels on H/R P12 cell growth, cell viability was assessed through CCK-8 assay. H/R PC12 cells treated with miR-375 mimic showed increased cell viability compared with H/R P12 cells with NC both 48 and 72 h after H/R (miR-375 mimic-treated H/R PC12 cells than in the NC-treated group (To determine the effects of fter H/R D. In added group E.. For example, progenitor cells from periventricular region proliferate and migrate into the hippocampus to regenerate new neurones after ischemia and reduce neurological deficits in vivo. Herein, we thus explored effects of miR-375 on PC12 cell migration. The results showed that miR-375 mimic significantly promoted the migration of H/R PC12 cells, as indicated by transwell assay (miR-375 mimic (Considering that cell migration plays an important role in the injury-induced neurogenesis and tissue regeneration ,14. For ll assay G,H. In t75 mimic F.miR-375 against H/R injury was associated with PI3K/AKT and p21-dependent signaling pathway. As expected, miR-375 mimic enhanced the protein expression of PI3K, p-AKT as well as p21 in H/R PC12 cells (It has been known that the PI3K/AKT signaling and p21 are closely related to cell survival, growth, and migration ,16. Thus12 cells I.miR-375 inhibited the expression of Ctgf in PC12 cells and protected the cells from H/R-induced injury through promoting cell proliferation and migration by p21/PI3K/AKT signaling pathways.Taken together, these results indicate that miR-375. We thus hypothesized that miR-375 attenuated the H/R injury through inhibiting the expression of Ctgf. To confirm this, we co-transfected miR-375 mimic and pcDNA3.0-Ctgf or pcDNA3.0 vector to verify the effect of Ctgf on H/R PC12 cell proliferation and migration. As indicated in Ctgf mRNA in miR-375 mimic-transfected group was obviously restored by the addition of pcDNA3.0-Ctgf. Consistent with restored Ctgf expression in the presence of pcDNA3.0-Ctgf, H/R PC12 cell proliferation reflected by EdU assay was decreased in the group treated with pcDNA3.0-Ctgf compared with the group without pcDNA3.0-Ctgf (As shown in the above studies, Ctgf is a direct target of 3.0-Ctgf B. Simila3.0-Ctgf C. Moreov3.0-Ctgf D. These miR-375 expression is significantly down-regulated in the rat brain with I/R. With the in vivo and in vitro I/R stroke models, we found that miR-375 mimic provides significant protection from injury of cerebral I/R, which is reflected by reduced infarct volumes and cell apoptosis, increased proliferation and migration of PC12 cells. Mechanistically, our findings showed that miR-375 binds to 3′-UTR region of Ctgf mRNA, subsequently leading to the decreased expression of Ctgf in the I/R brain. Furthermore, we showed that miR-375/Ctgf-mediated protective effects are associated with p21/PI3K/Akt signaling pathways.Previous studies have demonstrated that expressions of miRNAs would be changed in the brain in response to I/R . ElucidamiR-375 was originally identified from murine β-cells as a regulator of insulin secretion [miR-375 has been shown to be found in multiple tissues or organs including brain [miR-375 expression was down-regulated in several neural injury models including cerebral I/R injury [miR-375 level in the brain was associated with the increased injury induced by I/R. In agreement with previous study [miR-375 expression was significantly decreased in the I/R rat brain in our study. Importantly, we for the first time confirmed that miR-375 does play a positive role in the protection from cerebral I/R injury, which is shown by overexpression of miR-375 in the in vivo and in vitro I/R models.ecretion . At presng brain –20. AccuR injury ,10. Howeus study , miR-375miR-375 have been reported to include 3′-phosphoinositide-dependent protein kinase-1, hipposignaling effector YAP, the p53 gene and SP1, most of which are involved in the development and progression of tumor [miR-375 using bioinformatics software of MirTarget algorithm and miRDB and a Dual-luciferase reporter assay. Ctgf, also known as CCN2, was first identified in 1991 to describe a novel polypeptide growth factor secreted by human endothelial cells [miR-375 leads to Ctgf down-regulation, which subsequently markedly reduced the injury induced by I/R, which was reflected by increased proliferation, migration, and decreased apoptosis of PC12 cells. Meanwhile, overexpression of Ctgf suppressed miR-375-mediated proliferation and migration in the H/R PC12 cells. Combined with other results, our findings suggest that Ctgf is one of the pivotal factors contributing to the injury of cerebral I/R. Also, miR-375-mediated protection from cerebral I/R injury was at least in part linked to down-regulation of Ctgf expression.Target genes of of tumor –24. Hereal cells . Ctgf haal cells . Recentlal cells . Ctgf haal cells . Similaral cells . In linemiR-375 as a key factor which provides the protection from cerebral I/R injury. Inhibition of Ctgf, a target gene of miR-375, is an important reason of miR-375-mediated protective effects. Thus, our study suggests that Ctgf should be a potential therapeutic target against the injury of cerebral I/R.In summary, we here identified


Staphylococcus haemolyticus isolates has been reported in several European countries. Here, we report the first two complete genome sequences of S. haemolyticus sequence type 25 (ST25) isolates 83131A and 83131B. Both isolates were isolated from the same clinical sample and were first identified through shotgun metagenomics.The emergence of nosocomial infections by multidrug-resistant  Staphylococcus haemolyticus isolates causing hospital infections has been associated with an unusual genome plasticity generated by a large number of insertion sequences (A recent worldwide increase in the number of multidrug-resistant (MDR) equences . In partequences , are knoequences .S. haemolyticus isolates from the same clinical sample , originally identified through shotgun metagenomics .Here, we report the complete genome sequences of two coexisting ST25 DNA extraction was performed with the DNeasy UltraClean microbial kit . DNA concentration was measured with a Qubit 2.0 fluorometer . Both strains were sequenced using the MiSeq platform , generating 2 × 250-bp reads, and the MinION instrument  in order to close the first genomes of this lineage. The Illumina and MinION reads were assembled using Unicycler v0.4.1 , 60 tRNAs, and 19 rRNAs. The genome of the ST25 S. haemolyticus 83131B isolate has a circular chromosome with 2,593,418 bp and a GC content of 32.8%, 2,437 CDS, 60 tRNAs, and 19 rRNAs. The 83131A isolate harbors one circular plasmid of 16,882 bp (p83131A-A), and the 83131B strain harbors two circular plasmids of 16,882 bp (p83131A-B) and 2,366 bp (p83131B).The genome of the ST25 S. haemolyticus available. The most closely related genome was from an S. haemolyticus ST42 strain (GenBank accession number CP011116) isolated during a nosocomial outbreak in Brazil , CP024810 (p83131A-A), CP025397 (p83131A-B), and CP025398 (p83131B).The GenBank genome accession numbers for the sequences described in this study are