Mutations in leucine-rich repeat kinase (LRRK2) are the most common cause of heritable Parkinson’s disease (PD), and the most common mutations in LRRK2 lead to elevated kinase activity. For these reasons, inhibitors targeting LRRK2 have been the subject of intense research and development. However, it has been difficult to develop preclinical models that recapitulate PD-relevant LRRK2 phenotypes. The primary pathology in PD is the Lewy body (LB), which is a cytoplasmic aggregate of α-synuclein. The recent demonstration that LB-like aggregates of α-synuclein can be induced in primary neurons has provided a robust model for testing genetic modifiers of PD-relevant aggregation and neurodegeneration. In this study, we test the modulation of α-synuclein pathology by LRRK2 in primary neuron cultures using biochemistry and immunocytochemistry. We find that expression of familial mutant G2019S LRRK2 does not dramatically elevate the pathological burden of α-synuclein or neurodegeneration in neurons. We further test three LRRK2 inhibitors in two strains of wildtype neurons and find that even robust LRRK2 inhibition is insufficient to reduce α-synuclein pathology. LRRK2 inhibitors similarly had no effect in neurons with α-synuclein pathology seeded by human brain-derived pathological α-synuclein. Finally, we find that this lack of pathological modulation by LRRK2 was not confined to hippocampal neurons, but was also absent in midbrain dopaminergic neuron cultures. These data demonstrate that LRRK2 activity does not have more than minor effects on α-synuclein pathology in primary neurons, and more complex models may be needed to evaluate the ability of LRRK2 inhibitors to treat PD. Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Patients with this disease experience rigidity, resting tremors, and slowness of movement; 80% of patients will develop dementia throughout the disease course . The cliLRRK2 is a widely expressed protein with unclear functions. It has kinase, GTPase and scaffolding domains and has been implicated in intracellular trafficking and cytoskeletal modeling . LRRK2 mTo understand the relationship between LRRK2 and α-synuclein and develop a model of LRRK2-dependent PD phenotypes, early studies crossed mice expressing wildtype or G2019S LRRK2 mice with mice overexpressing α-synuclein with the familial A53T mutation. These studies all found little or no effect of LRRK2 expression on α-synuclein pathology , 14, 20.All housing, breeding, and procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and approved by the University of Pennsylvania Institutional Animal Care and Use Committee. C57BL/6 J  and B6.Cg-Tg(Lrrk2*G2019S)2Yue/J  mice were obtained from Andrew West . The G2019S mice have multiple inserts of the gene, but were heterozygous at these loci. Subsequently, mice were bred to homozygosity at loci as determined by quantitative PCR and outbreeding. The expression level of G2019S LRRK2 was thereby stabilized in this line of mice. All experiments shown use homozygous G2019S mice. CD1  mice were obtained from Charles River, Wilmington, MA.Purification of recombinant α-synuclein and generation of α-synuclein PFFs was conducted as described elsewhere , 33, 34.Primary hippocampal neuron cultures were prepared from postnatal day (P) 1 NTG and G2019S mice and embryonic day (E) 16–18 CD1 embryos. Dissociated hippocampal neurons were plated at 17,000 cells/well (96-well plate) or 1,000,000 cells/well (6-well plate) in neuron media   for postnatal cultures) supplemented with B27 , 2 mM GlutaMax , and 100 U/mL penicillin/streptomycin . LRRK2 inhibitors PF-475 and PF-360 were synthesized at Pfizer, Inc. MLi-2 was obtained from Tocris Bioscience (5756). All LRRK2 inhibitors were reconstituted at 10 mM in DMSO and stored at − 20 °C. There were further diluted to the final concentration indicated in neuron media with DMSO as a vehicle control. Neurons were either fixed with 4% paraformaldehyde, 4% sucrose in phosphate-buffered saline for immunocytochemistry or scraped for biochemistry.The ventral mesencephalon and striatum were dissected from P2 NTG and G2019S mice in Hibernate A medium (ThermoFisher A1247501) with B27  and 0.5 mM GlutaMax . The tissue was then digested in papain  for ~ 20 min at 37 °C. Cells were mixed at a ratio of 1:1 and plated at a density of 34,000 cells/well in 96-well black-walled plates (Perkin Elmer 50–905-1605) in Neurobasal A medium  supplemented with B27 and 0.4 mM GlutaMax, 50 ng/mL BDNF (PeproTech 450–02) and 30 ng/mL GDNF (Millipore Sigma GF030).g for 30 min. The pellet was sonicated and again spun at 100,000 x g for 30 min in 1 volume 1% TX-100 solution to remove remaining TX-100-soluble proteins. This pellet was suspended in 0.5 volumes 2% SDS solution, sonicated and spun once more at 100,000 x g for 30 min. The first and final supernatant were kept for immunoblot analysis. Western Blot analysis was performed with primary antibodies targeting α-synuclein , pS129 α-synuclein , LRRK2 , pS935 LRRK2 , p62  or GAPDH . Primary antibodies were detected using IRDye 800  or IRDye 680  secondary antibodies, scanned on Li-cor Odyssey Imaging System and analyzed using Image Studio software. Values obtained from this program were normalized to average PFF alone values.Proteins from primary neuronal cultures treated with PBS or α-synuclein PFFs were sequentially extracted as described previously , 33, 34.w/v) high salt (HS) buffer (50 mM Tris-HCL (pH 7.4), 750 mM NaCl, 10 mM NaF, 5 mM EDTA, protease and phosphatase inhibitors), followed by homogenization with a dounce homogenizer and centrifugation at 100,000 x g for 30 min. The HS wash was repeated and the resulting pellet was then homogenized with 9 volumes HS buffer with 1% TX-100 and centrifuged at 100,000 x g for 30 min. The pellet fraction was then homogenized with 9 volumes HS buffer with 1% TX-100 and 30% sucrose and centrifuged at 100,000 x g for 30 min to float away the myelin, which was discarded. The pellet was then homogenized with 9 volumes HS buffer with 1% Sarkosyl, rotated for 1 h at room temperature and centrifuged at 100,000 x g for 30 min. The resulting pellets were washed once with Dulbecco’s PBS and re-suspended in Dulbecco’s PBS by brief sonication . This suspension was termed the “sarkosyl insoluble fraction” containing pathological α-synuclein and used for the cellular assays described here. The amounts of α-synuclein in the sarkosyl insoluble fractions were determined by sandwich ELISA as described previously [Frozen postmortem human cingulate gyrus or frontal cortex brain tissue containing abundant α-synuclein-positive inclusions was selected for sequential extraction based on IHC examination of these samples as described  using prImmunostaining of neuronal cultures was carried out as described previously . BrieflyAll statistical analyses were done in GraphPad Prism. The analysis used for each data set is described in the accompanying figure legend. More specifically, for experiments directly comparing only NTG and G2019S neurons Fig.  or KruskTo establish a model in which the effect of LRRK2 mutations on α-synuclein could be observed, we used a transgenic BAC mouse which expresses mouse LRRK2 with the familial G2019S mutation under the endogenous LRRK2 promoter (B6.Cg-Tg(Lrrk2*G2019S)2Yue/J, . Upon inHowever, it has recently been demonstrated that G2019S neurons may show time-dependent changes in α-synuclein pathology that don’t become apparent until 18 DPT . TherefoThe lack of an effect of LRRK2 inhibitors on α-synuclein pathology in NTG neurons was surprising given previous publications showing a reduction of α-synuclein pathology in response to both LRRK2 inhibition  and knocWe sought to further validate this finding with a larger family of LRRK2 inhibitors at increased concentrations that would leave no residual LRRK2 activity. We chose an additional, validated LRRK2 inhibitor (MLi-2) for further investigation and added these inhibitors at concentrations ranging from 30 to 300 nM to CD1 neurons. Use of these inhibitors resulted in near complete depletion of pS935 LRRK2 Fig. . The neuWe then tested whether inhibition of LRRK2 activity can alter α-synuclein pathology induced by a means other than PFFs. We have recently demonstrated the ability of LB α-synuclein purified from human brain to induce pS129 α-synuclein pathology in WT neurons . CorticaAll the experiments to this point were performed in primary hippocampal neuron cultures. These cultures develop robust α-synuclein pathology and can be obtained in an abundance suitable for both immunocytochemistry and biochemistry. However, they are not the neurons most affected in PD patients. In order to address whether we were missing a phenotype that is dependent on expression of mutant LRRK2 in dopaminergic neurons, we developed ventral midbrain and striatum primary neuron co-cultures. When treated with α-synuclein PFFs at 7 DIV and allowed to age 14 DPT, dopaminergic neurons positive for TH develop cell body and neuritic pS129 α-synuclein pathology Fig. . We treaIn this study, we utilized primary neuron cultures to assess the status of α-synuclein pathology in relation to LRRK2 activity. We performed biochemical and immunocytochemical analysis of primary hippocampal and midbrain neurons cultured from two strains of wildtype mice and a mouse expressing LRRK2 with the familial G2019S mutation. To further manipulate LRRK2 activity, we used three validated LRRK2 inhibitors. In none of these assays did we observe more than a mild LRRK2-dependent alteration in α-synuclein pathology.The LRRK2*G019S neurons we describe have ~ 25-fold elevated LRRK2 levels and ~ 50-fold elevated kinase activity Fig. . DespiteWe induced pathological α-synuclein inclusion formation in NTG and G2019S neurons through addition of recombinant α-synuclein PFFs or human brain-derived LB α-synuclein. We designed our initial experiments to test the relationship between LRRK activity and α-synuclein at 14 DPT, before the onset of neurodegeneration. We found that there was no alteration of α-synuclein pathology in G2019S neurons. A recent publication using a similar model showed a mild elevation in α-synuclein in G2019S hippocampal neurons at 18 DPT, while detecting no difference at 7 DPT . We founTo further explore the role of endogenous LRRK2 in α-synuclein pathogenesis, we cultured wildtype hippocampal and midbrain neurons, and showed using biochemistry and immunocytochemistry that LRRK2 kinase inhibition is unable to alter induced α-synuclein pathology Figs. , and 6. LRRK2 is the most common cause of inherited PD. Even so, only around 30% of those with G2019S mutation in LRRK2 will go on to develop PD . It is tThere is substantial genetic and pathological data indicating that mutations in LRRK2 lead to an increased susceptibility to PD, and LRRK2 inhibitors are currently in clinical trials for the treatment of PD. Yet, how mutations in LRRK2 predispose patients to PD is still unclear. In this study, we show that in primary neurons, only after the onset of neurodegeneration does G2019S LRRK2 elevate α-synuclein pathology. Further, while LRRK2 inhibitors can rescue this elevated pathology, they show no benefit to wildtype neurons. Future studies of the link between LRRK2 mutations and the pathophysiology of PD will be critical to inform clinical studies of LRRK2 inhibitors.


Furthermore, improvements in the rota-rod performance and clasping score were observed in R6/2 HD mice after oral administration of βL compared to that of vehicle control-treated mice. Taken together, our data show that βL is a potential therapeutic candidate for the treatment of HD-associated phenotypes, and increases in Sirt1 level, CREB phosphorylation and PGC-103B1 deacetylation can be the possible underlying mechanism of the effects of βL.Huntington’s disease (HD) is one of the most devastating genetic neurodegenerative disorders with no effective medical therapy. β-Lapachone (βL) is a natural compound obtained from the bark of the Lapacho tree and has been reported to have beneficial effects on various diseases. Sirt1 is a deacetylase of the sirtuin family and deacetylates proteins including the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) which is associated with mitochondrial respiration and biogenesis. To examine the effectiveness of βL on HD, βL was orally applied to R6/2 HD mice and behavioral phenotypes associated with HD, such as impairment of rota-rod performance and increase of clasping behavior, as well as changes of Sirt1 expression, CREB phosphorylation and PGC-1α deacetylation were examined. Western blot results showed that Sirt1 and p-CREB levels were significantly increased in the brains of βL-treated R6/2 mice. An increase in deacetylation of PGC-1α, which is thought to increase its activity, was observed by oral administration of βL. In an  Huntington’s disease (HD) is a progressive brain disorder caused by the expansion of a CAG repeat in the huntingtin gene. This mutation results in the production of the polyglutamine expanded huntingtin protein (mHtt), leading to involuntary choreiform movements, cognitive impairment, and neuropsychiatric symptoms , 2. The Tabebuia avellanedae) [The quinone-containing compound, β-Lapachone  is a natural product derived from the roots of the south American Lapacho tree (lanedae) . βL has lanedae) –15. Sinclanedae) , 17. βL lanedae) , 19. In lanedae) –22, whillanedae) . AlthougSirt1 is the most important and best characterized sirtuin involved in gene expression, cell cycle regulation, DNA repair, stress response, apoptosis, neurogenesis, metabolism, and aging –28. Sirtin vitro HD model. This HD model was used to show the change of mitochondrial superoxide level by βL treatment.In the present study, we investigated whether βL affects behavior phenotypes associated with HD, including the rota-rod performance impairment and clasping behavior. Furthermore, its effects on brain-derived Sirt1, PGC-1α acetylation status, and phosphorylated CREB level were examined. Primary neuronal cells from R6/2 mice shows mitochondrial dysfunction along with mHtt aggregation and can be used as an All animal experiments were performed in accordance with the National Institutes of Health guide for the care and use of laboratory animals . All animal studies were approved by the Institutional Animal Care and Use Committee at Seoul National University Hospital.We use transgenic mice of the R6/2 line (B6CBA-Tg(HDexon1)62Gpb/3J, 111 CAGs) and their WT littermates . The R6/2 transgenic mouse model is the most widely used animal model of HD and expresses exon 1 of a human mHtt. These mice were obtained by crossing ovarian transplant hemizygote females with B6CBAF1/J males. The mice were housed in groups with ad libitum access to food and water and a 12 hours light/ 12 hours dark cycle. The genotype was assessed using a PCR assay, and CAG repeat length was measured by DNA sequencing  The average number of CAG repeat was 166 ± 1.1 n = 4, .Considering protective effect of βL, oral-injection was started at 5 weeks old. Dosage was set at 70 mg per body weight (kg) and calculated by the body weight measured just before the experiment. βL was dissolved in 0.1% sodium lauryl sulfate (SLS) solution at a concentration of 7 mg/ml for oral gavage, and oral administration was performed by sonde for once a day during 6 weeks. Same volume of 0.1% SLS was used for vehicle control group.The Rota-rod test was conducted using a rota-rod apparatus . Mice were placed on the rod with an accelerating rotating speed from 4 to 40 rpm over a period of 3 minutes with a 15 minutes rest between trials. Mice were trained on three consecutive days for three trials per day at 4 weeks of age. Three trials were performed and the mean latencies to fall were used to analyze data. Rota-rod evaluation was performed every week from 5 to 11 weeks. Clasping test is the behavior test to evaluate motor impairment of HD transgenic mice and the motor functions of the mice were tested using clasping test. In each session, the mouse was suspended by the tail for 30 sec and the time during which the hindlimbs are entirely retracted and touching the abdomen was measured. Clasping test was performed at 11 weeks of age.Brains of R6/2 mice were isolated at 11 weeks of age, immediately frozen on liquid nitrogen, and stored at -80°C until protein extraction. Protein was extracted using RIPA buffer  containing freshly added protease inhibitor and phosphatase inhibitor . The protein content was determined using a BCA protein assay kit . Thirty micrograms of protein samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membrane . Membranes were blocked with 5% non-fat dried milk dissolved in 1× TBST  for 1 h at room temperature. Blots were then incubated at 4°C overnight with primary antibodies diluted as recommended in the manufacturer's instructions. The following primary antibodies were used: anti-Sirt1 , p-CREB , CREB  and anti-β-actin . Blots were then incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies , and developed using ECL solution . Between each incubation step, the membrane was washed three times with 1× TBST. ImageJ software was used for obtaining intensity values of the blots.in vitro HD model by isolating neuronal stem cells (NSCs) from R6/2 HD mice [5 cells/ml in 25 cm2 flask. NSCs were incubated in culture medium consisting of DMEM/F12, 1% PSA , 2% B27 Supplement , 10 ng/mL EGF  and 10 ng/mL bFGF  at 37°C in a 95% O2, 5% CO2 humidified atmosphere. For MitoSOX red analysis, NSCs were differentiated in the differentiation medium, which was composed of DMEM/F12, 1% PSA, 2% B27, and 5% FBS. Cultured neurospheres reaching 150 μm diameter were transferred to sterile tissue culture tubes and spun at 1,000 rpm for 5 min. Neurosphere pellets were resuspended with differentiation medium. Approximately ten neurospheres were then transferred into individual 24-well tissue culture plates containing poly-L-lysine-coated cover glass.We developed an  HD mice , 48. To PGC-1α lysine acetylation was analyzed by immunoprecipitation of PGC-1α followed by western blot using acetylated-lysine antibody . Tissue protein extracts and protein content were prepared using RIPA buffer and BCA protein assay kit. For immunoprecipitation, 500 μg of protein lysates samples were incubated at 4°C rotation overnight with anti-PGC1α  diluted as recommended in the manufacturer's instructions. Purified proteins were separated by 8% SDS-PAGE and immunoblotting using anti-PGC1α  and acetylated-lysine . The supernatant and precipitated protein was immunoblotted with PGC-1α and actin antibody to confirm the immunoprecipitation process .in vitro HD model NSCs were differentiated in a 24-well plate for 3 days. For MitoSOX Red  staining, cells were washed once with PBS  and stained with MitoSOX Red for 15 min at 37°C. Cells were washed again with PBS and analyzed by flow cytometry  and image with fluorescence microscopy . The FACS and fluorescence was analyzed by with Winmdi 2.9, or captured by fluorescence microscopy according to the manufacturer’s instructions.For t-test. Rota-rod test was analyzed using one-way ANOVA. The data were analyzed using Prism software , and a 2-tailed probability value below 0.05 was considered statistically significant.All values indicated in the figures are presented as mean ± standard error mean (SEM), and results of western blot, flow cytometry and clasping test were analyzed using Student's To examine the effects of βL on Sirt1 and CREB phosphorylation and PGC-1α acetylation, βL was orally administered to R6/2 mice for 6 weeks from 5 to 11 weeks of age. Brains of R6/2 mice administered with βL or the vehicle were isolated and protein levels were analyzed using western blot. Treatment with βL promoted the expression of Sirt1 and p-CREB in the brain of R6/2 mice . To confin vitro HD model with βL and mitochondrial superoxide level was measured. For the in vitro HD model, NSCs were isolated from the brains of R6/2 HD mice and culture by the neurosphere method. HD NSCs could be differentiated in a growth factor-deprived medium and showed mitochondrial dysfunction and mHtt aggregation in vitro [in vitro HD model.To investigate whether βL has a mitochondrial protective role in a HD cell model, we treated an in vitro , 48. OurOur data show that R6/2 mice exhibit impairments in the rota-rod performance from 5 to 11 weeks of age, whereas the motor performance was maintained in WT littermates. Next, we administered βL to female R6/2 mice from 5 to 11 weeks of age and rota-rod performance was examined. R6/2 mice injected with βL showed overall longer mean latencies to fall with a statistically significant difference at 10 weeks of age compared to control R6/2 mice administered with vehicle . We alsoin vitro HD neuronal cells upon βL treatment. We also found that behavioral deficits in performance on rota-rod and clasping test were improved in βL-administered R6/2 mice.Collectively, our results demonstrate that βL could increase Sirt1, p-CREB, and PGC-1α deacetylation in the brains of R6/2 HD mice. In addition to these molecular changes, we observed decreased mitochondrial superoxide levels in +-dependent deacetylase activity and it plays a role in cellular metabolism [Sirt1 is an evolutionarily conserved protein with NADtabolism . It is atabolism . Activattabolism , 34. Thitabolism . Given tThe transcription factor CREB is known as a major regulator of PGC-1α transcription. CREB expression and phosphorylation is a potent survival signal for neuronal cells . Mice wiin vitro primary NSC model, βL showed a decrease in superoxide level but no difference in JC-1 assay, which is an experiment to confirm mitochondrial membrane potential Click here for additional data file.S2 FigTotal brain lysates was immunoprecipitated with anti-PGC-1α antibody. The supernatant and precipitated protein was immunoblotted with PGC-1α and actin antibody.(TIF)Click here for additional data file.S3 FigHD neuronal cells were treated with or without βL at 24h after seeding. Cells were subjected to JC-1 staining at 48h after treatment, and data showed no significant change by βL treatment (n = 3 each).(TIF)Click here for additional data file.S4 FigβL was orally administered to male R6/2 mice at 5 to 11 weeks of age and rota-rod performances were measured every week. No significant change was observed between vehicle and βL group.(TIF)Click here for additional data file.S5 FigThe brains of R6/2 mice administered vehicle or βL were isolated and mHtt aggregation was examined using immunohistochemistry of EM48 staining. βL group showed tendency of mHtt aggregate size to be small in cortex and striatum. Size bar = 100 μm.(TIF)Click here for additional data file.


The potential inflammatory signaling associated with the toxicity of HA/β-Ga2O3:Cr3+ NPs in primary cortical neurons was examined. We observed a dose-dependent decrease in cell viability and an increase in apoptosis in neurons following various concentrations  of HA/β-Ga2O3:Cr3+ NPs treatment. Consistently, constitutively active forms of calcineurin (48 kDa) were significantly elevated in cultured primary cortical neurons, which was consistent with calpain activation indicated by the breakdown products of spectrin. Moreover, HA/β-Ga2O3:Cr3+ NPs result in the elevation of LC3-II formation, SQSTM/p62, and Cathepsin B, whereas phosphorylation of CaMKII (Thr286) and Synapsin I (Ser603) were downregulated in the same context. Taken together, these results demonstrate for the first time that calpain activation and a disturbance of autophagy signaling are evoked by exposure to HA/β-Ga2O3:Cr3+ NPs, which may contribute to neuronal injury in vitro.The toxicity of engineered nanoparticles remains a concern. The knowledge of biohazards associated with particular nanoparticles is crucial to make this cutting-edge technology more beneficial and safe. Here, we evaluated the toxicity of Ga Nanotechnologies have been steadily growing and are used for the delivery of therapeutic material, leading to new important applications in health and life-science . Nanosciin vitro models of neural cells have been scarce  and inducible nitric oxide synthases (iNOS) in the cortex exposed to air with high concentrated metal NPs. BBB injury has also been observed  is a defense mechanism against potentially neurotoxic molecules and structures . The appn scarce . Additiognostics . Anothergnostics . The neugnostics . Exampleobserved . For theobserved .2O3) is a wide band gap semiconductor that is applied in optical components and optical catalysis, for example, catalytic reactions involving CO, H2 and methanol  when preparing Ga2O3 (β-Ga2O3: Cr3+) NPs enables the NPs to emit fluorescence and near-infrared persistent luminescence  could effectively absorb antineoplastic drugs, and they were shown to bind with CD44, which is highly expressed in tumor cells and is primarily distributed at the tumor site , were purchased from Aladdin .All chemicals were purchased from Sigma-Aldrich Chemical Co.  unless otherwise noted. Sodium hyaluronate  and Gallium Oxide . The zeta potential was determined using a Zetaplus/90plusZeta potential and laser particle size analyzer  after 100-fold dilutions of the prepared dispersion were made with distilled water  were purchased from American Type Culture Collection  and cultured in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum . Cells were grown in a 5% CO2 incubator at 37 °C. Then, the cells were seeded in 6-well plates at a density of 1 × 106 cells/mL and treated with HA/β-Ga2O3:Cr3+ NPs at various concentrations from 1 to 100 µg/mL for 12 h before harvesting.Primary cortical neurons were obtained from 17-day-old embryonic mice and cultured in neurobasal medium  with 2% B27, 1% antibiotics and 0.25% GlutaMAX™ Supplement  for 10 days . The cel2O3:Cr3+ NPs exposure (2O3:Cr3+ NPs for 12 h. The medium was replaced with 100 µL of MTT (5 mg/mL), and incubated at 37 °C for 4 h. Then, added 200 µL DMSO to each well and incubated at room temperature for 15 min. The absorbance was detected with an automatic microplate reader .The cell viability were monitored by a 3--2,5-diphenyltetrazolium bromide (MTT) assay with or without HA/β-Gaexposure . Cells wHarvested primary cortical neurons were used to detect the expression level of proteins related to neuron synapse structures and functions. Western blotting was carried out by SDS polyacrylamide gel electrophoresis (SDS-PAGE) as described previously . The proImmunofluorescence changes of Cathepsin B were examined by confocal microscopy . BrieflyIn Situ Cell Death Detection Fluorescein  following the manufacturer’s instructions , was used to examine the ROS levels in SH-SY5Y cells after exposure to NPs. Cells were cultured on glass coverslips overnight and then treated with various doses of NPs for 12 h. Before being fixed in 4% formaldehyde, cells were incubated with 2′, 7′-dichlorofluorescein diacetate for one hour . Nucleust-test. These differences were considered to be significant at P < 0.05. All data are expressed as the mean ± S.E.M.The significance of the differences between more than two groups was determined using a one-way ANOVA. The significance of the differences between two groups was determined using a 2O3:Cr3+ NPs in different kinds of culture media was detected. As shown in 2O3:Cr3+ NPs for 12 h. The results showed that cell morphology of neurons was normal at concentrations lower than 5 µg/mL, but cell deformation was observed at concentrations higher than 25 µg/mL . The results of NPs toxicity were further confirmed using the TUNEL assay  was significantly decreased after treated with 25 and 50 µg/mL HA/β-Ga2O3:Cr3+ NPs  was increased after cells were incubated with HA/β-Ga2O3:Cr3+ NPs (P < 0.01). It has been established that calcium-sensitive proteases such as calpain can lead to increased spectrin cleavage. Here, HA/β-Ga2O3:Cr3+ NPs treatment resulted in calpain activation, as shown by the elevated levels of spectrin breakdown products (120 kDa) (P < 0.01).Cacineurin . We inve2O3:Cr3+ NPs, compared to the control group (P < 0.01). As shown in P < 0.05). Taken together, these results indicated that the fusion of autophagosomes and lysosomes in the autophagy flux may be blocked in primary cortical neurons after treatment with HA/β-Ga2O3:Cr3+ NPs for 12 h, thereby resulting in the aberrant accumulation of LC3-II and p62. Consistently, the fluorescence intensity of Cathepsin B (green) was significantly enhanced in a concentration-dependent manner following treatment with HA/ β-Ga2O3:Cr3+ NPs . The final concentration of NPs we used as a drug carrier was 40 µg/mL  for 12 h, whereas the expression level of total synapsin I did not change. Interestingly, we also observed that phospho-synapsin I (Ser603) levels were transiently elevated following exposure to HA/β-Ga2O3:Cr3+ NPs at 5 µg/mL, and the same phenomenon was also observed for the phosphorylation of CaMKII. This turnover may be interpreted as a compensatory mechanism of the neurons. The activation of CaMKII is closely related to changes in phosphorylation of its post-synaptic and pre-synaptic substrates, such as synapsin I and GluR1  are poorly understood and their potential toxicity vary depending on the exposure conditions (2O3:Cr3+ NPs, an important limitation of the present study was that lack of extensive characterization of the solubility and release of the Ga3+ or Cr3+ over the time course of the experiment. Therefore, a good safety margin should be further studied in an animal model to investigate the neuronal toxicity of HA/β-Ga2O3:Cr3+ NPs in the context of an intact brain with or without risk factors.The homeostasis of autophagy-lysosome signaling plays a critical role in protecting against neurovascular injury . Based ofunction . During function . Here, tagy flux , we alsofunction . Indeed,nditions . Besides2O3:Cr3+ NPs could induce calpain activation and the disturbance of autophagy signaling, which may result in neuronal damage.In summary, our results suggested that the exposure of neurons to HA/β-Ga10.7717/peerj.4365/supp-1Figure S1There was no significant difference in the size distribution of NPs in the media with or without 10% FBS.Click here for additional data file.10.7717/peerj.4365/supp-2Figure S22O3:Cr3+ NPs was increased, scale bar = 20 µm.The increased fluorescence means that the ROS level was significantly increased as the doses of HA/β-GaClick here for additional data file.10.7717/peerj.4365/supp-3Data S1Click here for additional data file.


Silybum marianum (milk thistle) is a medicinal plant used for the treatment of various liver disorders. This study examined whether the main flavonolignans from S. marianum  and their 2,3-dehydro derivatives  activate the Nrf2 pathway, which regulates the expression of genes encoding many cytoprotective enzymes, including NAD(P)H:quinone oxidoreductase 1 (NQO1). After 48 h of exposure, 2,3-dehydrosilydianin at concentrations of 25 μM and higher significantly elevated the activity of NQO1 in murine hepatoma Hepa1c1c7 cells. In contrast, other tested compounds at non-cytotoxic concentrations had a mild or negligible effect on the NQO1 activity. Using a luciferase reporter assay, 2,3-dehydrosilydianin was found to significantly activate transcription via the antioxidant response element in stably transfected human AREc32 reporter cells. Moreover, 2,3-dehydrosilydianin caused the accumulation of Nrf2 and significantly induced the expression of the Nqo1 gene at both the mRNA and protein levels in Hepa1c1c7 cells. We found that 2,3-dehydrosilydianin also increased to some extent the expression of other Nrf2 target genes, namely of the heme oxygenase-1 gene (Hmox1) and the glutamate-cysteine ligase modifier subunit gene (Gclm). We conclude that 2,3-dehydrosilydianin activates Nrf2 and induces Nrf2-mediated gene expression in Hepa1c1c7 cells.  These flavonolignans originate biosynthetically from the flavanonol taxifolin  and coniferyl alcohol. Their oxidation at the flavonoid moiety yields the corresponding 2,3-dehydroflavonolignans  moiety. They are found in some species of the families Asteraceae, Fabaceae, Poaceae, and others olignans , formalli) direct scavenging of reactive oxygen species (ROS), ii) chelation of transition metal ions, iii) inhibition of ROS-generating enzymes, and iv) upregulation of antioxidant enzymes HMOX1 gene in human hepatoma Huh-7 cells Silymarin is clinically used for its hepatoprotective effects in the complementary therapy of liver disorders caused by various hepatotoxic compounds and viral infections. In addition, anticancer, cardioprotective, neuroprotective, UV-protective, hypocholesterolemic and some other effects have been reported for silymarin in animal models 22.1Silybin (SB) was isolated from silymarin  as described previously 2.23 and 10% heat- and charcoal-treated fetal bovine serum (FBS). The stable human mammary AREc32 reporter cell line 2. For experiments, cells were seeded into multiwell plates and the experiments were performed after 24 h of stabilization in fresh complete culture medium. Cells were treated with the tested compounds (in 0.1% (v/v) DMSO) and negative controls were treated with 0.1% (v/v) DMSO alone.The murine hepatoma Hepa1c1c7 cell line  was cultured in Minimum essential medium α  supplemented with 2.2 g/L NaHCO2.34 cells/well in 96-well plates) were treated for 48 h with 0.1% (v/v) DMSO (control), 1.5% (v/v) Triton X-100 (positive control) or with the tested flavonolignans. After treatment, the cell viability was determined by MTT reduction assay. Cells were washed with phosphate-buffered saline (PBS) and incubated for 2 h at 37 °C in serum-free medium containing 0.5 mg/mL 3--2,5-diphenyltetrazolium bromide (MTT). After incubation, the medium was removed and formazan produced by active mitochondria was dissolved in DMSO containing 1% (v/v) ammonia. The absorbance at 540 nm was measured on a spectrophotometric plate reader and used to calculate relative cell viability, where cells treated with DMSO alone represented 100% viability.Hepa1c1c7 cells (1 × 102.44 cells/well in 96-well plates), the activity of NQO1 was determined spectrophotometrically as described previously w/v) bovine serum albumin, 1.5% (v/v) Tween-20, 7.5 mM FAD, 150 mM glucose-6-phosphate, 2 U/mL glucose-6-phosphate dehydrogenase , 50 mM NADP+, 25 mM menadione and 0.7 mM MTT. The mixture was incubated for 5 min at room temperature and the reaction was stopped with 50 μL of dicumarol suspension . The absorbance of the reduced MTT corresponding to the activity of NQO1 was measured at 610 nm on a spectrophotometric plate reader. The absorbance values were normalized to the protein content and used for the calculation of fold changes versus the control.After the treatment of Hepa1c1c7 cells (1 × 102.54 cells/well in 96-well plates) were treated for 24 h with the tested compounds. After treatment, the plate was frozen and kept at −20 °C for 24 h and then the luciferase activity was measured on a GloMax-Multi+ microplate luminometer  using the Bright-Glo Luciferase Assay System (Promega). The luminescence values were normalized to the protein content of the cells and used for the calculation of fold changes versus the control.AREc32 cells (1 × 102.65 cells/well in 6-well plates), total RNA was extracted using TRI Reagent Solution . RNA samples (2 μg) were reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and real-time PCR was performed in a LightCycler 480 II system (Roche) using TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays, consisting of specific primers and FAM dye-labeled TaqMan minor groove binder probes (Applied Biosystems). The assay ID was Mm00477784_m1 for Nfe2l2 (Nrf2), Mm01253561_m1 for Nqo1, Mm00516005_m1 for Hmox1, Mm00802655_m1 for Gclc, Mm00514996_m1 for Gclm and Mm99999915_g1 for Gapdh. Amplification conditions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Crossing point values, equivalent to CT, were determined using second derivative maximum analysis. Relative changes in gene expression were calculated by the comparative CT method using the 2ΔΔCT− equation with results normalized to Gapdh mRNA levels.After the treatment of Hepa1c1c7 cells (4 × 102.75 cells/well in 6-well plates), total cellular extracts were prepared as described previously After the treatment of Hepa1c1c7 cells  silybin, silychristin and silydianin, isolated from commercially available extract from the fruits of S. marianumii) 2,3-dehydrosilybin, 2,3-dehydrosilychristin and 2,3-dehydrosilydianin, which were prepared by oxidation, by molecular oxygen under basic conditions, of the compounds listed in the first item This study was designed to investigate the ability of six flavonolignans to activate the Nrf2 pathway in cells. The study included 3.2The effect of 2,3-dehydrosilydianin on the transcriptional activity of Nrf2 was examined using stably transfected human AREc32 reporter cells, which contain a luciferase reporter gene controlled by eight copies of the ARE 3.3The activity of Nrf2 depends on the interaction between Nrf2 and its negative regulator, Kelch-like ECH-associated protein 1 (Keap1). Under homeostatic conditions, Keap1 binds to Nrf2 and targets it for ubiquitination and proteasomal degradation. In contrast, stress conditions associated with the oxidation or covalent modification of cysteine residues in Keap1 and/or with the phosphorylation of Nrf2 inactivate Keap1 and thus stabilize Nrf2, which in turn accumulates in the cell and increases the expression of Nrf2 target genes 3.4Nqo1, Hmox1, Gclc and Gclm. As expected, 5 μM sulforaphane (a positive control) Nqo1 gene. At concentrations of 25 and 50 μM, 2,3-dehydrosilydianin elevated Nqo1 mRNA levels to 1.6-fold and 2.3-fold, respectively, compared to the control. The expression of the other tested genes was also affected to some extent by 2,3-dehydrosilydianin, but only at a concentration of 50 μM, where the increase in mRNA levels of Hmox1, Gclc and Gclm were 2.2-fold, 1.3-fold and 1.5-fold, respectively (Nqo1 gene To further investigate the effect of 2,3-dehydrosilydianin on Hepa1c1c7 cells, we analyzed the expression of selected Nrf2 target genes, including ectively . Westernectively . These r4tert-butyl hydroperoxide-induced lipid peroxidation with a higher potency than silydianin Our study examined the effect of six flavonolignans, namely of silybin, silychristin, silydianin and of their 2,3-dehydro derivatives, on the Nrf2 pathway. We have demonstrated that 2,3-dehydrosilydianin, in contrast to the other tested compounds, activates the Nrf2-dependent gene expression in vitro. This is mainly documented by the activation of the antioxidant response element in AREc32 reporter cells, and by the accumulation of Nrf2 and induction of NQO1 at the mRNA, protein and activity levels in Hepa1c1c7 cells. The difference in the effect of 2,3-dehydrosilydianin and silydianin confirms the importance of the 2,3-double bond in the structure of flavonolignans for certain biological activities. For instance, 2,3-dehydrosilydianin was shown to inhibit Since the main silymarin components silybin, silychristin and silydianin did not activate the Nrf2 pathway, our results cannot explain the effects of silymarin and silybin on the expression and/or activity of Nrf2-regulated proteins observed in vivo The authors declare that there are no conflicts of interest.


However, the high conductivity inherent in BiFeO3-based ceramics complicates measurement of the ferroelectric properties. In the present work, doping with niobium (Nb) is carried out to reduce the conductivity of (Bi0.5K0.5)TiO3-BiFeO3. Powders of composition 0.4(K0.5Bi0.5)Tix1−NbxO3-0.6BiFex1−NbxO3  are prepared by the mixed oxide method and sintered at 1050 °C for 1 h. The effect of Nb doping on the structure is examined by X-ray diffraction. The microstructure is examined by scanning electron microscopy. The variation in relative permittivity with temperature is measured using an impedance analyzer. Ferroelectric properties are measured at room temperature using a Sawyer Tower circuit. Piezoelectric properties are measured using a d33 meter and a contact type displacement sensor. All the samples have high density, a rhombohedral unit cell and equiaxed, micron-sized grains. All the samples show relaxor-like behavior. Nb doping causes a reduction in conductivity by one to two orders of magnitude at 200 °C. The samples have narrow P-E loops reminiscent of a linear dielectric. The samples all possess bipolar butterfly S-E loops characteristic of a classic ferroelectric material. Nb doping causes a decrease in d33 and Smax/Emax.Ceramics in the system (Bi This is due to the high Curie temperature (~810–820 °C) and large remnant polarization  of BiFeO3 [3 is itself piezoelectrically active, with a direct piezoelectric coefficient d33 ≈ 30 pC/N and a large peak-to-peak strain of up to 0.36% measured in S vs. E curves at low frequency [3, manifesting in strongly nonlinear (frequency and stress amplitude-dependent) piezoelectric properties. Domain walls are also strongly pinned by charged defects, probably acceptor-oxygen vacancy defect pairs, making the material properties dependent on the sintering and poling conditions [3 are often inferior to those of single crystal or thin film BiFeO3 [Solid solutions containing perovskite BiFeOrature ~80–820 °C f BiFeO3 . BiFeO3 requency ,6. Non-1nditions ,8. This nditions , means tm BiFeO3 ,8.3 with other perovskites such as BaTiO3 and PbTiO3 have been studied [xBaTiO3-(1−x)BiFeO3 system has an MPB at x = 0.33 between rhombohedral and pseudocubic phases [d33 > 100 pC/N, bipolar strain of ~0.15% at an electric field of 6 kV/mm) and good temperature stability [et al. studied the system x(K0.5Bi0.5)TiO3-(1−x)BiFeO3 (KBT-BFO) and found it to have an MPB between rhombohedral and pseudocubic phases at x ≈ 0.4 [Smax/Emax ≈ 225 pm/V, 33d* ≈ 130 pm/V) and good temperature stability (k33 ≈ 0.36 at 300 °C). Mozorov et al. also found ceramics in this system to have good inverse piezoelectric properties, making them potentially suitable for actuator applications [In order to improve the electrical properties, solid solutions of BiFeO studied ,11,12. T studied . The xBac phases . Compositability ,15. Mats x ≈ 0.4 . Composiications ,18.3-based ceramics tend to have high leakage currents, which makes measuring the ferroelectric properties and poling the ceramic difficult. Doping with Mn was found to reduce the leakage current density of BiFeO3 thin films under high electric fields [3-BiFeO3 ceramics [0.5Bi0.5)TiO3-0.6BiFeO3 ceramics by three orders of magnitude [vs. electric field and the strain vs. electric field behavior of the ceramics. Further work should be carried out to optimize the dopant level.BiFeOc fields . It was ceramics ,20. Our agnitude . The rho2+, Mn3+ or Mn4+ form [2+ or Mn3+ states [3+ or Ti4+ [x(K0.5Bi0.5)TiO3-(1−x)BiFeO3. Doping with Nb was found to reduce the conductivity of BiFeO3 and BaTiO3-BiFeO3 ceramics by six orders of magnitude [0.5Bi0.5)TiO3-0.6BiFeO3 ceramics with Nb in order to reduce their conductivity. The effect of Nb doping on the structure, microstructure, and electrical properties of 0.4(K0.5Bi0.5)TiO3-0.6BiFeO3 ceramics will be described.Mn can be present in perovskite ceramics in Mnn4+ form , but is + states . Mn acts or Ti4+ . In addiagnitude ,25. In t3 (space group R3m). Second phases of Bi25FeO40 (ICDD card #46-0416) and Bi2O3 (ICDD card #76-2478) are also present. The amount of second phase increases as the amount of Nb substitution in the powders increases. XRD traces of the samples sintered at 1050 °C for 1 h are shown in 3. The KBT-BFO 0 and 1Nb samples are single-phase, but the KBT-BFO 3Nb samples have small amounts of Bi2O3 (ICDD card #71-0467) and BiFe2O4 (ICDD card #74-1098) secondary phases. The unit cell parameters of the sintered samples are given in 3 for 0.4(K0.5Bi0.5)TiO3-0.6BiFeO3 [X-ray diffraction (XRD) traces of the powders after calcination at 900 °C for 5 h are shown in .6BiFeO3 . The samSEM micrographs of the samples sintered at 1050 °C for 1 h are shown in 3 powders and sintered BiFeO3 ceramics [3 was found to be thermodynamically unstable, decomposing to Bi25FeO39 and Bi2Fe4O9 upon heat treatment between 447–767 °C [et al. also showed that the incorporation of even a small amount of a third component that formed a solid solution with sillenite (Bi25FeO39) could induce the formation of secondary phases of Bi2Fe4O9 and Bi25FeO39 by shifting the composition into a three phase region [0.5Bi0.5)TiO3-0.6BiFeO3 behaves in a similar manner to BiFeO3 regarding the effect of dopant addition on the formation of secondary phases. The secondary phases all melt at temperatures below the sintering temperature [Secondary phases often appear in both calcined BiFeOceramics . BiFeO3 7–767 °C  and at 87–767 °C . Valant perature  and so mvs. temperature for the samples sintered at 1050 °C for 1 h are shown in maxT increasing with the measurement frequency and the maximum value of the relative permittivity εrmax decreasing with the measurement frequency. Note that the rapid decrease in permittivity at temperatures above the peak temperature maxT is, in fact, an artifact. The decrease in permittivity to negative values is determined by the lead wire inductance at high temperatures when the sample resistance is small. Further increases in permittivity at high temperature for the lower measurement frequencies (1 and 3.16 kHz) are due to dc polarization. The incorporation of Nb causes a decrease in the value of εrmax, particularly for the KBT-BFO 3Nb sample. Nb incorporation causes an increase in maxT at 10 kHz  > maxT (10 kHz). The εr vs. temperature peaks also become substantially broader for the KBT-BFO 3Nb sample, suggesting two peaks. This is indicated by the lower maxT at 1 MHz than at 10 kHz in Plots of relative permittivity t 10 kHz . Tmax slvs. temperature for the samples sintered at 1050 °C for 1 h are shown in DC. The component of loss tangent due to dc conductivity is proportional to σDC/ωε. A steeper increase with temperature is indicated for the lower frequency values. The curves show a minimum point or the point of the largest slope change at low temperature, which is related to the ferroelectric transition. The temperature of this characteristic point increases with frequency and with Nb doping. Below the characteristic points, the frequency dispersion of the ferroelectric origin is indicated by the loss tangent increasing with frequency, and this dispersion becomes stronger for larger Nb doping. This is also indicated by the difference between 1 MHz and 10 kHz values of loss tangent at 40 °C in Plots of loss tangent vs. inverse temperature for the samples are shown in The dc conductivity responsible for the large apparent loss tangent at high temperatures and apparent relaxor-like dielectric response can be obtained from the resistance magnitude of the skewed arc responses, as indicated in the previous reports ,26. Plot3 and the solid solutions of BiFeO3 such as KBT-BFO. Many workers have proposed that BiFeO3 is an n-type semiconductor and that conductivity is governed by the presence of Fe2+ and oxygen vacancies [3 is a p-type semiconductor [et al. found that BiFeO3 thin films prepared by RF magnetron sputtering displayed p-type semiconducting behavior [3 samples that had been heat-treated in O2 at ~125 bar were p-type semiconducting [et al. found that BiFeO3 was a p-type semiconductor up to ~100 °C, after which it became n-type semiconducting [4+ ions. On the basis of conductivity measurements carried out in oxidizing and reducing atmospheres, it was suggested that the conductivity in KBT-BFO ceramics with high BiFeO3 content (≥40 mol %) is p-type [Several causes have been proposed for the high conductivity of BiFeOacancies ,31,32,33acancies . Recent onductor ,36. Undebehavior . Masó annducting . Makhdoonducting . Both grcontent ≥ mol % is5+ r6 = 0.064 nm, Fe3+ r6 = 0.065 nm, Ti4+ r6 = 0.061 nm, K+ r6 = 0.138 nm, Bi3+ r6 = 0.103 nm) [2O5 can enter the KBT-BFO crystal lattice as a donor dopant according to the following defect equations:A comparison of the different ionic radii of the metal cations in 6-fold co-ordination shows that Nb will likely substitute for Fe or Ti (Nb.103 nm) . The sliTherefore, p-type conductivity in KBT-BFO is expected to decrease. The activation energy values of dc conductivity may be related to the electronic structure modified by Nb doping as well as by the ferroelectric transition.vs. electric field are shown in vs. electric field curves are given in Smax/Emax taken from the unipolar loops are given in Smax/Emax. The unipolar loop for the KBT-BFO 3Nb sample is noticeably narrower than those of the KBT-BFO 0 and 1Nb samples. Values of 33d measured by 33/d31d meter are also given in 33d also decreases with increasing Nb content. Due to the low values of d33, it was not possible to measure tk and pk by impedance spectroscopy.Curves of polarization 33d are smaller than those measured by other workers [d33, Smax/Emax and the strain-electric field hysteresis caused by Nb incorporation may be due to a reduction in the domain wall mobility, and hence extrinsic contributions to the strain [Values of low-field  workers ,46. The e strain . The smae strain ,45.r and tanδ. The present work as well as the previous work suggests a correlation between the loss tangent (at low field) and the piezoelectric response in KBT-BFO. The values of relative permittivity near room temperature do not exhibit significant differences with doping (d33) and d33 was also found for BiFeO3 at low frequencies (<1 Hz) [et al. found a strong extrinsic contribution to the piezoelectric properties in BiFeO3 [i.e., electrical conduction. The reduction in conductivity caused by Nb doping of KBT-BFO may therefore lead to the reduction in extrinsic contribution to the piezoelectric properties  ,8. Rojacn BiFeO3 ,8. They operties .0.5Bi0.5)Tix)(1−NbxO3-0.6BiFex) were prepared by ball milling appropriate amounts of K2CO3 , Bi2O3 , TiO2 , Fe2O3  and Nb2O5  for 24 h in high purity ethanol (99.9%) in a polypropylene jar with ZrO2 milling media. Before milling, all starting materials were dried at 250 °C for 5 h to remove any adsorbed moisture. After milling, the ethanol was evaporated using a hotplate/magnetic stirrer. The dried powder was crushed in an agate mortar and pestle and sieved to pass 180 μm mesh. Powders were calcined in a high purity alumina crucible with lid at 900 °C for 5 h with heating and cooling rates of 5 °C·min−1. Calcined powders were examined by X-ray diffraction  using Cu Kα radiation, a scan range of 10°–90° in 2θ, a step size of 0.026° and a scan speed of 3°·min−1.Powders of composition 0.4(K2 (~147 MPa). Samples were buried in 0.4(K0.5Bi0.5)TiO3-0.6BiFeO3 packing powder in double high-purity alumina crucibles with lids and sintered at 1050 °C for 1 h. Heating and cooling rates were 5 °C·min−1.To make samples for sintering, 0.5 g of powder was pressed by hand in a 10 mm diameter steel die into pellets. The pressed pellets were then cold isostatically pressed at 1500 kg/cm−1. Unit cell parameters were calculated by the least-squares method using the program Jade 6.5 . To observe the microstructure, samples were vertically sectioned using a low speed diamond saw and polished to a 1 μm finish. Samples were thermally etched and the microstructure observed in a scanning electron microscope  equipped with an energy dispersive X-ray spectrometer . The mean and standard deviation of the grain size was measured from the micrographs using an image analysis program (ImageJ v1.46r). For each sample, at least 250 grains were measured.The density of the sintered samples was measured using the Archimedes method in deionized water. XRD was used to analyze the phase composition of sintered samples as before. For calculation of the unit cell parameters, Si (99.9% Alfa Aesar) was added as an internal standard and the scans repeated with a scan range of 20°–80° in 2θ, a step size of 0.013° and speed of 1°·min−1 in oxygen in the temperature range 800–30 °C. P-E loops were measured at room temperature at a frequency of 10 Hz using a Sawyer Tower circuit . For the measurement of piezoelectric properties, samples were poled in silicone oil at room temperature under an electric field of 5 kV/mm. The piezoelectric charge coefficient 33d was measured using a Piezo d33/d31 meter . Strain vs. electric field curves were measured using a contact type displacement sensor  at 50 mHz.For the measurement of dielectric properties and polarization-electric field hysteresis loops, the samples were parallel polished on both sides and then silver paste was applied and fired onto both sides of the samples. Variation in relative permittivity with temperature was measured using an impedance analyzer  during the cooling of the samples at 1 °C·min0.5Bi0.5)Tix1−NbxO3-0.6 BiFex1−NbxO3  lead-free piezoelectric ceramics has been studied. The incorporation of Nb causes an increase in the second phase content of both calcined powders and sintered ceramics. Nb doping causes a small increase in the unit cell parameter and a small decrease in the rhombohedral distortion of the unit cell. The samples show relaxor-like ferroelectric behavior. However, this is attributed to the high p-type conductivity originating from BiFeO3. Nb incorporation causes an increase in activation energy from 0.72 to 0.93 and 1.13 eV below 300 °C and a reduction in conductivity by one to two orders of magnitude at 200 °C. Maximum εr is reduced by half. While the frequency dispersion in εr is substantially reduced with Nb doping, a strong dispersion in tanδ characteristic of ferroelectricity can be observed. Nb incorporation causes a reduction in the piezoelectric properties and in the strain-electric field hysteresis.The effect of Nb content on the structure, microstructure, and electrical properties of 0.4(K


Fulminant meningococcal sepsis, characterized by overwhelming innate immune activation, mostly affects young people and causes high mortality. This study aimed to investigate the effect of targeting two key molecules of innate immunity, complement component C5, and co-receptor CD14 in the Toll-like receptor system, on the inflammatory response in meningococcal sepsis.Neisseria meningitidis administered over 3 h. The piglets were randomized, blinded to the investigators, to a positive control group (n = 12) receiving saline and to an interventional group (n = 12) receiving a recombinant anti-CD14 monoclonal antibody together with the C5 inhibitor coversin.Meningococcal sepsis was simulated by continuous intravenous infusion of an escalating dose of heat-inactivated p = 0.006). The following inflammatory mediators were substantially reduced in plasma in the treatment group: Interferon-γ by 75% (p = 0.0001), tumor necrosis factor by 50% (p = 0.01), Interleukin (IL)-8 by 50% (p = 0.03), IL-10 by 40% (p = 0.04), IL-12p40 by 50% (p = 0.03), and granulocyte CD11b (CR3) expression by 20% (p = 0.01).A substantial increase in plasma complement activation in the untreated group was completely abolished in the treatment group (Inhibition of C5 and CD14 may be beneficial in attenuating the detrimental effects of complement activation and modulating the cytokine storm in patients with fulminant meningococcal sepsis. Neisseria meningitidis, characterized by whole-body inflammation and severe disturbances in homeostasis leading to high mortality despite optimal antimicrobial and intensive care treatment [8/mL in the plasma [Fulminant meningococcal sepsis is a rapid and devastating infection caused by reatment –3. Withie plasma –3. Treate plasma –3. Active plasma , 5. Howee plasma –9.Neisseria species. TCC may also be formed in the fluid phase as a soluble form (sC5b-9) and then serves as a valuable marker of complement activation.Molecular structures of meningococci other than LPS activate different parts of the innate immune system, including the complement system and additional TLRs beside TLR4-MD2 . CD14 seE. coli induced sepsis [We have hypothesized that a combined inhibition of complement and CD14 will efficiently attenuate the inflammatory response in various clinical situations including sepsis . Previoud sepsis  and mousd sepsis , 20.The aim of the present study was to investigate the effect of blocking both C5 and CD14 on the inflammatory response induced in vivo in a porcine model of meningococcal sepsis. The model has been developed by our group as the first large animal model simulating meningococcal sepsis and has been proven to be a valuable tool to study inflammatory mechanisms of the disease and potential interventions , 22.For inhibition of porcine CD14, a recombinant variant of a well-established anti-porcine CD14 mouse monoclonal IgG2b antibody (Mil2) was used. This novel anti-porcine CD14 antibody (rMIL2) was recently constructed by our group as a mouse-human chimeric, IgG2/IgG4 hybrid antibody keeping the antigen binding site the same as for Mil2 . Thus, rN. meningitidis 44/76 (H44/76) was obtained from the National Institute of Public Health, Oslo, Norway. It is characterized as B:15:P1.7,16:L3,7,9 and was originally isolated from a female patient with lethal meningococcal septic shock [The international reference strain ic shock . MeningoSus scrofa domesticus, outbred stock) of either sex, weighing 6 kg (range 5.5–6.5 kg) were used. All piglets were clinically healthy and thus none were excluded from the study. The piglets were randomized into two groups, each consisting of 12 animals: an intervention and a positive control group. In addition, two sham control piglets that received saline instead of bacteria were included as reference. The groups were blinded for the investigators. The number of piglets in each group was decided according to previous analysis and experience showing that at least 10 animals in each group were needed to demonstrate differences [Piglets  before oral intubation using a 4.9-mm inner diameter tube with cuff. After intubation, an i.v. infusion of propofol (10 mg/kg/h) and fentanyl (50 μg/kg/h) was started and mechanical ventilation was established. A 5 F pulmonary artery catheter  was inserted via the left external jugular vein and guided into a distal pulmonary artery by pressure wave-form analysis. A cannula was inserted in the left carotid artery for continuous recording of arterial pressure and intermittent blood sampling. A urinary catheter was inserted into the urinary bladder via a cystotomy. After the completion of surgery and insertion of catheters, the animals were placed on their right side, remaining in this position for the rest of the experiment.N. meningitidis. Septic shock was obtained according to the Third International Consensus Definitions for Sepsis and Septic shock, as vasopressors were needed to keep the blood pressure above 65 mmHg [After induction of anesthesia and surgery, the intervention group received first a bolus of coversin (1 mg/kg) and thereafter a bolus of rMIL2 (5 mg/kg), administered about 10 min before induction of sepsis. Excess coversin from the bolus not bound to C5 is rapidly removed from circulation . Therefo 65 mmHg . Due to To limit the need for inhibitors, the size of the pigs used in the present study was reduced from about 30-kg to about 6-kg piglets compared to previously published studies employing the porcine model of meningococcal sepsis , 21. How9 bacteria/kg. The positive control group received the same volume of saline as the intervention group. The sham animals also received the same volume of saline. Blood samples were drawn before i.v. infusion of intervention agents (Tbasis), before start of bacterial infusion (T0), and 60, 120, and 180 min after induction of sepsis . All animals received the same background infusion of Ringer’s acetate, i.e., 10 mL/kg/h during surgery and until 60 min after induction of sepsis, and thereafter 20 mL/kg/h to 120 min and 30 mL/kg/h to 180 min after induction of sepsis. In addition, 10% glucose was given i.v. at 5 mL/kg/h throughout the experimental course to avoid hypoglycemia. Inotrop and vasopressor therapy were given when needed as a therapy for severe and lethal hypotension.Each piglet received a total of 8.4 × 10A commercially available enzyme immune assay  was used to test functional activity of the classical complement pathway. The kit detects human complement activity, but has been shown to cross-react efficiently with pig . Samplesg at 4 °C for 20 min, immediately aliquoted, and stored at −70 °C.Soluble TCC (sC5b-9) was measured using multiplex xMAP technology  as previously described , 31. SamThe cytokines TNF, IL-1β, IL-4, IL-6, IL-8, IL-10, and IL-12p40 and Interferons (INF)-α and INF-γ were analyzed in EDTA-plasma obtained as described for soluble TCC above, using multiplex technology .4Cl/10 mM NaHCO3-/0.12 mM EDTA (Tritiplex III) for 8–10 min in the dark at ambient temperature. Samples were centrifuged at 300 g for 5 min at 4 °C, and the cells were washed with PBS, 0.1% BSA . The samples were then resuspended in PBS, 0.1% BSA before flow cytometry . Values are given in mean fluorescence intensity (MFI).Leukocyte activation was measured by flow cytometry. In pig whole blood, neutrophils are clearly discriminated from mononuclear cells, but lymphocytes and monocytes cannot be separated by forward/side scatter dot plots. For measurement of wCD11R3 on neutrophils (the pig ortholog to human CD11b), blood was collected at Tbasis and T180, anticoagulated with EDTA, and stained with mouse anti-porcine wCD11R3 FITC clone 2 F4/11 or the isotype-matched mouse IgG1-FITC negative control Ab . The samples were incubated for 15 min in the dark, and red cells were lysed with 0.16 M NHStatistical analyses were performed with GraphPad Prism version 6 . The positive control group was compared with the treatment group using two-way ANOVA. Area under the curve (AUC) was used as a single measure of the total difference between groups in the amount of each cytokine secreted.t test. For all statistical analyses, p < 0.05 was considered statistically significant. Two sham animals were included as references.The difference in wCD11R3 upregulation on granulocytes in the intervention group and the positive control group was measured at only one time-point (T180) in addition to baseline and was analyzed by Student’s p = 0.01)  complement arbitrary units (CAU)/L at T0 to mean 1233 (±871) CAU/L at T180 (p = 0.0001), and the area under the curve (AUC) was 75% lower in the treatment group than in the positive control group. No increase was seen in sham animals.In the positive control group, IFN-γ increased from <lower detection limit (LDL) at T0 to mean 16 (±7.5) pg/mL at T120 and 14 (±6.4) pg/mL at T180 , and the AUC was 50% lower in the treatment group than in the positive control group. No increase was seen in sham animals.In the positive control group, TNF increased from <LDL at T0 to mean 44 (±15.1) ng/mL at T120 . No increase was seen in sham animals.In the positive control group, IL-1β increased from <LDL at T0 to mean 0.1 (±0.05) ng/mL at T120 and 0.9 (±0.5) ng/mL at T180 . No increase was seen in sham animals.In the positive control group, IL-6 increased from <LDL at T0 to mean 0.8 (±0.3) ng/mL at T120 and 2.6 (±0.9) ng/mL at T180 , and the AUC was 50% lower in the treatment group than the positive control group. No increase was seen in sham animals. One animal of the treatment group showed excessively increased IL-8 level at T180 right before it died (83 ng/mL). This value was 33 SD higher than the mean value of the rest of the treatment group. Thus, it was regarded as an outlier and excluded from statistical analysis.In the positive control group, IL-8 increased from <LDL at T0 to mean 1.8 (±1) ng/mL at T120 and 4.5 (±1.6) ng/mL at T180 , and the AUC was 40% lower in the treatment group than the positive control group. No increase was seen in sham animals.In the positive control group, IL-10 increased from mean 0.05 (±0.01) ng/mL at T0 to 4.6 (±0.8) ng/mL at T120 and 6.6 (±2.3) ng/mL at T180 , and the AUC was 50% lower in the treatment group than the positive control group. No increase was seen in sham animals.In the positive control group, IL-12p40 increased from mean 0.5 (±0.01) ng/mL at T0 to 1.8 (±0.3) ng/mL at T120 and 10.0 (±2.5) ng/mL at T180 .p = 0.01).In the positive control group, wCD11R3 increased from mean MFI 51 (±9) at T0 to MFI 700 (±150) at T180, whereas in the treatment group wCD11R3 increased from mean MFI 46 (±14) at T0 to MFI 568 (±68) at T180  in the treatment group implies that combined treatment influenced white blood cell function not only by decreasing cytokine release but also by decreasing expression of surface molecules. In complex with CD18, wCD11R3 constitutes complement receptor 3 (CR3), which is active in phagocytosis of bacteria. Phagocytosis per se is obviously beneficial in meningococcal disease in order to clear bacteria from the circulation. However, it does not need to be as efficient in the presence of antibiotics, and a limited decrease of phagocytosis may possibly be beneficial due to less induced oxidative burst, which otherwise may be harmful to surrounding tissue .We suggest that the course of meningococcal sepsis observed in piglets in the present study represents a realistic model of the situation in patients with fulminant meningococcal sepsis. We demonstrate that efficient complement inhibition can be obtained in this model. Furthermore, we find that combined inhibition of the two key innate immune systems, complement with the central component C5 and the TLRs with the co-receptor CD14, can modulate the inflammatory response in fulminant meningococcal sepsis, provided the C5 and CD14 inhibition is initiated early. Modulation of this inflammatory response may be of potential benefit in patients with meningococcal sepsis.


The phase, shape, and size of the resulting core–shell NPs are confirmed by transmission electron microscopy and X-ray diffraction. Coated with a NaGdF4:10%Er3+ active shell around the NaGdF4:12%Er3+ core NPs, a maximum luminescent enhancement of about 336 times higher than the NaGdF4:12%Er3+ core-only NPs was observed under the 1540 nm excitation. The intensity ratio of green to red was adjusted through the construction of the core–shell structure and the change of Er3+ concentration in the shell. By analyzing the lifetimes of emission bands and exploring the energy transition mechanism, the giant luminescence enhancement is mainly attributed to the significant increase in the near-infrared absorption at 1540 nm and efficient energy migration from the shell to core.NaGdF Upconversion (UC) refers to nonlinear optical processes characterized by the successive absorption of two or more pump photons via intermediate long-lived energy states, followed by the emission of the output radiation at a shorter wavelength than the pump one . With th3+-doped active core to increase the intensity of UC.Moreover, the major challenge for UCNPs is still how to gain stronger upconversion luminescence with more optimized structures and synthesis methods. To overcome this challenge, many efforts were made, including changing the composition, tuning the morphology and size, and surface modification –17. Amon3+ ions as luminescent centers [3+, Tm3+, and Ho3+ are thought to be desired candidates. Moreover, Er3+ turns out to be the most effective one during the UC process, owing to its similar energy gaps. But, if simply increasing the concentration of Er3+ in the NPs, the luminescence shows a nonlinear relationship with the concentration of Er3+ and sometimes the luminescence intensity even decrease with the augment of Er3+, due to the influence of energy migration and concentration-quenching effects at higher concentrations.Commonly, an emissive UCNP consists of an inert host matrix and active Ln centers . As an i3+ ion-doped NaGdF4 was developed in this work. Compared to the inert shell, the UC luminescence was largely enhanced by adopting the method of the active shell and optimizing the Er3+ concentration. The luminescence properties of novel NaGdF4:12%Er3+@NaGdF4: 10%Er3+ active-core/active-shell NPs (Er3+ doping in mol% relative to Gd) were also discussed in detail under the 1540-nm excitation.Except successful construction of the active-core/active-shell structure, a strategy to improve the intensity of upconversion luminescence by only Er3COONa; reagent grade, 99%), trifluoroacetic acid , gallium oxide , and europium oxide . Cyclohexane , oleic acid , oleylamine , and absolute ethanol were used. Deionized water was used throughout. All chemical materials were used as received without further purification.The starting materials were sodium trifluoroacetate (CF3COO)3; RE = Gd, Er) precursor was prepared as follows: rare earth oxides were dissolved in trifluoroacetic acid (CF3COOH) with certain amount of deionized water until the solution was transparent. The solution was then filtered, followed by drying at 140 °C for dozens of hours. The synthesis basically followed the routes previously reported in literature [3COONa (1 mmol), Gd(CF3COO)3 (0.88 mmol), and Er(CF3COO)3 (0.12 mmol) powder were introduced to a three-necked flask (100 ml) containing 8 ml of OM and 16 ml of OA at room temperature. After vigorous stirring for about 15 min, the mixture was then heated to 120 °C under the protection of nitrogen or argon atmosphere and maintained at this temperature for another 30 min under magnetic stirring to remove the oxygen and residual water. At this end, the mixture was totally clear forming a slight yellow color. The mixture was then heated slowly to 275 °C in the presence of argon atmosphere and maintained at the temperature for 30 min. After then, the solution was cooled down naturally to room temperature. Finally, NPs were then precipitated using excess ethanol and collected via centrifugation at 7000 rpm for 5 min. After washed with ethanol for several times, the as-prepared nanocrystals were dried in air at 70 °C overnight.At the beginning, rare earth (RE) trifluoroacetate , core NPs of NaGdF4 doped with 12% Er3+ ion concentration were firstly prepared following the procedure as described above. The procedure for the growth of shell on core is similar to the case of core except that 0.5-mmol core NPs re-dispersed in 4 ml cyclohexane were added simultaneously with rare earth trifluoroacetates to the mixture of OA and OM solution and the heating temperature was enhanced to 300 °C.To prepare the NaGdFStructures of the samples were investigated by X-ray diffraction (XRD) using X’TRA (Switzerland ARL) equipment provided with a Cu tube with Ka radiation at 1.54056 Å. The size and shape of the samples were observed by a JEM-2100 transmission electron microscope . Luminescence spectra were obtained by the Acton SpectraPro Sp-2300 spectrophotometer with a photomultiplier tube equipped with 1540 nm as the excitation source. The fluorescence decay curves in visible region were recorded on a FLSP920 fluorescence spectrophotometer and using a Shimadzu R9287 photomultiplier (200–900 nm) as the detectors. All measurements were performed at room temperature.4:12%Er3+ core NPs, NaGdF4:12%Er3+@NaGdF4 active-core/ inert-shell NPs, and NaGdF4:12%Er3+@NaGdF4:10%Er3+ active-core/active-shell NPs, respectively. Obviously, all the as-prepared NPs are uniform in size and morphology. The NaGdF4:12%Er3+ core-only NPs consist of monodisperse NPs with an average diameter of ~12.5 nm. After coated with a shell, the diameter of the NPs tends to increase to about 175 nm  structure. Coating with a shell, the NaGdF4:12%Er3+@NaGdF4 active-core/inert-shell NPs and the NaGdF4:12%Er3+@NaGdF4:10%Er3+ active-core/active-shell NPs show hexagonal phase, and the identified diffraction peaks are also in well agreement with the XRD pattern of β-NaGdF4 (JCPDS card no. 27−0699). No obvious peak from other phases or impurities is observed [Figure  nm Fig. , c. As d nm Fig.  confirm observed .Fig. 1a3+, originating mainly from the following four transitions: 2H11/2→4I15/2 (528 nm), 4S3/2→4I15/2 (540 nm), 4F9/2→4I15/2 (660 nm), and 4I9/2→4I15/2 (810 nm). Compared with the NaGdF4:12%Er3+ core-only NPs and the NaGdF4:12%Er3+@NaGdF4 active-core /inert-shell NPs, great enhancement of upconversion luminescence was obviously observed from the NaGdF4:12%Er3+@NaGdF4:10%Er3+ NPs. As seen in Fig. 4:12%Er3+@NaGdF4:10%Er3+ NPs shows the strongest luminescence with shorter concentration step. Specifically, Fig. 4:12%Er3+ core-only NPs, NaGdF4:12%Er3+@NaGdF4 active-core/ inert-shell NPs, and NaGdF4:12%Er3+@NaGdF4:x%Er3+ active-core/active-shell NPs. Compared with the NaGdF4:12%Er3+ core-only NPs, the enhancement factor of the NaGdF4:12%Er3+@NaGdF4 active-core/inert-shell NPs is about 30 times larger. While when it comes to the NaGdF4:12%Er3+ @NaGdF4:10%Er3+ active-core/active-shell NPs, a largest enhancement factor up to approximately 336 is observed. Comparing the as-obtained NPs with same size, the upconversion luminescence tends to decrease firstly and then gets the largest enhancement at 10 mol% to ~336 times, so the energy transfer reaction plays the main role in the huge enhancement of luminescence other than the effect of size [Figure  of size . Apparen4:12%Er3+ NPs capped with NaGdF4 gain an enhancement factor about 30. However, the luminescence of the NPs tends to decrease first when doped the Er3+ ions with 6 mol%. As reported before, the decrease is mainly caused by the less efficiency of sensitizers in shell than that in the core [3+ concentration strengthened the function of the active shell, leading to more efficient energy migration from the shell to the core. When the concentration of the shell comes to 10 mol%, the interaction of the Er3+ ions in the core and the shell may reach the peak as observed, bringing the largest enhancement of luminescence to 336 times. Other values of doping concentration are not beneficial for the higher emission intensity enhancement, suggesting that the higher or lower doping concentration may lead to the cross relaxations (CR) or the reduction of the luminescent activators [As is known, the emission intensity of NPs will be enhanced by coating an inert shell over them, due to the confine of surface passivation . The inethe core . The lumtivators , 32.G/IR) is studied in Fig. G/IR decreases to ~1.09. The reduction of non-radiative decay (caused by suppression of surface quenching) perhaps increased the probability of the CR process of Er3+ ions, resulting in the enhanced population on 4F9/2 level [G/IR ratio is tuned by constructing the core–shell structure and changing the Er3+concentration in the shell. Essentially, the green and red emissions are mainly originated from the transitions of 2H11/2, 4S3/2→4I15/2, and 4F9/2→4I15/2 [3+ in the shell directly changes the distance between the neighboring emitters, causing the cross relaxation to different extent [4:12%Er3+ core NPs, NaGdF4:12%Er3+@NaGdF4 active-core/inert-shell NPs, and NaGdF4:12%Er3+ @NaGdF4: x%Er3+ active-core/active-shell NPs are presented. Coated with an inert or active shell, the bandwidth values with the full width at half maximum (FWHM) are all adjusted to lower values. The luminescent properties were well improved by tuning the FWHM values with core–shell structure.The effect of the shell structure on the intensity ratio of the green to red emission  active-core/active-shell NPs under 1540-nm excitation are proposed. The corresponding decay lifetimes can be calculated by the second order exponential curve fitting:I is the luminescence intensity, A1 and A2 are constants, t is the time, and τ1 and τ2 are the short and long lifetimes for exponential components, respectively. The average decay lifetime τ is determined by the formula:As can be seen in Fig. 2H11/2 (528 nm), 4S3/2 (540 nm), 4F9/2 (660 nm), and 4I9/2 (810 nm) are populated again through the energy transfer process between Er3+ ions. Comparing the values of the decay lifetime about the as-obtained NPs, one can find that while continuing to increase the Er3+ concentration higher than 6 mol%, the lifetime begin to decrease. The phenomenon can be explained by the fact that the probability of CR process is largely related to the ions concentration [2H11/2, 4S3/2, and 4I9/2 appear no obvious signal rising edge, indicating that they are populated by non-radiative relaxation [4:12%Er3+@NaGdF4:10%Er3+ NPs at 660 nm presents a typical slow rising edge. This phenomenon is a direct evidence of an energy transfer upconversion process of state 4F9/2 [4:12%Er3+@NaGdF4 active-core/inert-shell NPs and the NaGdF4:12%Er3+@NaGdF4:10%Er3+ active-core/active-shell NPs under the 810 nm emission are smoother than other profiles, indicating that the population ions on the 4I9/2 state have less difference between different core–shell compositions.The results of the calculation are all shown in Fig. ntration . The prolaxation . Contrar), 4F9/2 60 nm, ante 4F9/2 . As depi3+ ions is exhibited in Fig. 4I13/2. With the excitation of 4I13/2 state, the excited state absorption (ESA) process arises immediately, resulting in more high-lying levels to be excited. After coating with an inert shell, the surface quenching is largely confined and the depletion of low-lying levels is reduced to enhance the population of high-lying ones, bringing intensive luminescence . From the decay profile of 810 nm, the lifetime of the as-obtained NPs decreases firstly while doping the Er3+ ions into the shell, which is another direct evidence of the ETU process, populating the 4S3/2 and 2H11/2 state from the 4I9/2 state.As analyzed above, the energy transfer mechanism between the states of Er4:12%Er3+@NaGdF4 active-core/inert-shell NPs and the NaGdF4:12%Er3+@NaGdF4:10%Er3+ active-core/active-shell NPs exhibit a typical slow rising edge. As other work reported before, the rising edge is proved to be another evidence of CR as theoretical transients [4F3/2 + 4I15/2→4F9/2 + 4I13/2 makes large contribution to the population of the 4F9/2 state due to the higher and higher concentration of Er3+ in the shell, which also can be obtained from the calculated results of the lifetimes (Fig. 4F3/2 is found, proving the existence of CR. Consequently, under the co-effect of core/shell structure and optimized Er3+ concentration in shell, the energy transfer reaction is extremely improved, leading to the great luminescent enhancement of active-core/active-shell NPs.By further studying the temporal behavior of the red emission, one can find that the decay curves of 660 nm of the NaGdFansients , 41. Commes Fig. . Besides3+ ions single-doped NaGdF4 without any other lanthanide-doped ions. Hexagonal NaGdF4:12%Er3+@NaGdF4:10%Er3+ active-core/active-shell NPs have been synthesized and present a significant intensive emission of the upconversion luminescence compared to NaGdF4:12%Er3+@NaGdF4 active-core/inert-shell and NaGdF4:12%Er3+ core-only NPs. Acting both as the emitter and the sensitizer, the luminescence of single Er3+-doped NaGdF4 UCNPs can be greatly enhanced by forming the structure of an active shell over the core. By the optimization of the concentration of Er3+ ions in the shell, the upconversion luminescent properties are greatly promoted and the enhancement of UC luminescence intensity is up to ~336 times greater than the NaGdF4:12%Er3+ core-only NPs, which is much larger than simply increasing the Er3+ concentration in NPs.In conclusion, a strategy was put forward to enhance the upconversion luminescence by forming active-core/active-shell structure with Er


The avian origin influenza A virus (IAV) H7N9 has caused a considerable number of human infections associated with high rates of death since its emergence in 2013. As a vital component of the host innate immune system, the nucleotide-binding domain leucine-rich repeat containing receptor, pyrin domain containing 3 (NLRP3) inflammasome plays a critical role against H1N1 viral infection. However, the function of NLRP3 inflammasome in host immunological responses to the lethal H7N9 virus is still obscure. Here, we demonstrated that mice deficient for NLRP3 inflammasome components, including NLRP3, caspase-1, and Apoptosis-associated speck-like protein containing a CARD (ASC), were less susceptible to H7N9 viral challenge than wild type (WT) controls. Inflammasome deficiency in these animals led to significantly milder mortality and less pulmonary inflammation compared with WT mice. Furthermore, IL-1 receptor deficient mice also exhibited a higher survival rate than WT controls. Thus, our study reveals that the NLRP3 inflammasome is deleterious for the host during H7N9 infection in mice, which is due to an overwhelming inflammatory response via caspase-1 activation and associated IL-1 signal. Therefore, fine-tuning the activity of NLRP3 inflammasome or IL-1 signaling may be beneficial for the host to control H7N9 associated lethal pathogenesis. The infected patients often develop severe symptoms, including pneumonia and acute respiratory distress syndrome (ARDS)4. The H7N9 infection had caused 1230 confirmed human cases, with 428 deaths by 22 February 2017 [data from FAO (Food and Agriculture Organization of the United Nations)]. The severe symptoms and high mortality associated with H7N9 infection pose a big threat to the public health. Although recent studies of H7N9 have achieved great progress, clear definition of the host immunological responses to H7N9 is still lacking6.In March 2013, the novel avian-origin influenza A virus (IAV) H7N9 was identified in China. H7N9 exhibits low pathogenicity in birds, but has an ability to bind certain mammalian cell receptors, and replicates at temperature close to the normal body temperature of mammals7. A rapid antiviral response, i.e. type I interferon production, to facilitate intracellular control of viral replication; and a proinflammatory response characterized by secretion of cytokines and chemokines, which promotes recruitment of various immune cells for viral clearance8. As a key mediator of inflammation, the proinflammatory cytokine IL-1β plays a crucial role in the pathogenesis of various diseases9. The maturation and secretion of IL-1β require a protein complex called inflammasome, which activates caspase-1 to cleave proIL-1β to its mature form10. More importantly, caspase-1 and the NLRP3 inflammasome have been found essential for protecting mice against H1N1 IAV infection13.Influenza virus infection mainly induces two types of innate immune responses15. Employing this murine model, in the current study we demonstrate a detrimental role for the NLRP3-caspase-1 inflammasome and associated IL-1 signal in H7N9 infection of mice. Our data suggest that dampening the NLRP3 inflammasome activity or inhibiting IL-1 signaling should be beneficial for the host during a lethal H7N9 challenge.In a previous study, we have demonstrated that H7N9 influenza virus isolated from human patients could establish successful infection of mice11. To assess whether the NLRP3 inflammasome could be activated during H7N9 challenge, we transfected LPS-primed BMDCs (bone marrow derived dendritic cells) with H7N9 RNA and measured the production of IL-1β. As expected, robust IL-1β secretion was detected in the H7N9 RNA-transfected samples, while the production of an inflammasome-independent cytokine TNF-α was unaltered  infected with H7N9 virus as described15. We found that the Nlrp3−/− mice survived significantly better than the WT controls     Fig. . In term.i) Fig. . Moreovets) Fig. . Howevernot Fig. . Notablyice Fig. .Figure 2Nlrp3−/−, Casp1/11−/− and WT mice, lung tissues were collected and H&E stained samples were examined by microscopy. On 3 d.p.i, infiltration of inflammatory cells was observed in the infected lungs of all strains of mice. There was an obvious thickening of alveolar septum, destruction of partial alveolar structure, as well as pulmonary septal rupture  on 3 d.p.i. Meanwhile, the mean fluorescence intensities (MFIs) of CD11b , F4/80  and CD11c  in WT BALF cells were significantly increased compared with that of Casp1/11−/− mice  on 3 d.p.i  of H7N9 infected WT or ned Fig. . The perp.i Fig. . For Ly6p.i Fig. . For lymp.i Fig. . Thus, d16. To investigate the role of caspase-1 in H7N9 infection related inflammation, cytokines and chemokines in the supernatants of lung homogenates were analyzed  and IL-18 (1.168 ± 0.3761 ng/g tissue) were significantly lower than those in WT tissues  on 7 d.p.i (P < 0.01) . The levels of other chemokines, including MIP-1β, IP-10, CXCL1, CCL2 and GM-CSF were just slightly lower in the lungs of Casp1/11−/− mice than that in WT counterparts on either 3 d.p.i or 7 d.p.i. Therefore, caspase-1 was involved for an optimal production of IL-1β, IL-18 as well as MIP-1α in the lung in response to H7N9 challenge. To note, the production of above mentioned cytokines and chemokines from Nlrp3−/− mice showed a similar pattern as that from Casp1/11−/− mice  Fig. . Other cant Fig. . The levses Fig. . Other cice Fig. . As for ice Fig. , indicatAsc−/− and Il1r1−/− mice with H7N9 virus and monitored their weight change and mortality daily. Our results demonstrated that the mortality of Asc−/− and Il1r1−/− mice was lower compared with that of the WT mice  on 8 and 9 d.p.i.  or CD11c+ (high-expressed on dendritic cells) cells in the BALF of WT mice were significantly more than those in Casp1/11−/− mice at 3 d.p.i. There were also Ly6c+ (expressed on monocytes), Ly6g+ (high-expressed on neutrophils) and CD3+ (expressed on T cells) in the BALF of WT mice exhibited higher level, though it was not significant differences  cells were increased, the percentage of the most infiltrated cells were decreased in the BALF at 7 d.p.i. , the local inflammation was limited Fig. , and mac~72 hoursCasp1/11−/− mice could not returned to the initial level (0 d.p.i) . Thus, WT mice suffered more due to the lung damage and inflammation in the early phase (before 7 d.p.i) of H7N9 infection and either died of organ failure, or recovered at the later phase. In contrast, Casp1/11−/−mice did not develop as strong inflammation and lung damage as seen in WT counterparts in the early phase, consequently fewer of them died. However, Casp1/11−/− mice failed to recover completely at the later phase. These results might suggest a potentially dual role for caspase-1 in H7N9 infection, detrimental role to the host upon H7N9 infection before 7 d.p.i. and possibly necessary role for recovery from the lung inflammation after 7 d.p.i. phase, likely due to a contribution to tissue regeneration. But it should be kept in mind that the comparison between 20% surviving WT and 60% surviving Casp1/11−/− is not justified, and more stringent experiments are needed for further explorations on the role of caspase-1 to host recovery.From 7 d.p.i. to 14 d.p.i, the body weight of survived WT mice recovered completely, whereas .i) Fig. . Microsc12. Inflammation is essential to protect host from infection, but can also cause tissue damage or dysfunction of important organs and lead to death21. Dysregulation of the proinflammatory response may result in a “cytokine storm” that contributes to a severe viral pneumonia and serious complications in the lung21. The highly pathogenic avian influenza virus H5N1 causes hypercytokinemia and severe tissue damage22. It is reported that production of proinflammatory cytokines and chemokines, including IL-6 and IP-10, is remarkably increased in H7N9 infected patients23, and H7N9 infection associated inflammation in the lungs resulted in rapidly progressive pneumonia and development of ARDS in the majority of hospitalized patients25. In addition, the H5N1 IAV infected patients who progressed severe lung injury have elevated levels of CCL2, IP-10, CXCL9 and IL-8 compared with the seasonal H1N1 infected patients26. Therefore, for the highly pathogenic influenza virus the hyperactivated proinflammatory responses may be detrimental for the host. Moreover, a recent study demonstrates that NLRP3 inflammasome plays both a protective and detrimental role during H1N1 and H3N2 infection of mice depending on the disease phase13. In our study, we found the inflammasome activation was deleterious in the case of H7N9 infection, possibly due to overwhelming and fatal acute inflammation in the lungs following H7N9 infection. Pathology and infiltrating cellular analyses as well as cytokine and chemokine analyses all supported this possibility  can activate granulocytes and lead to acute neutrophilic inflammation. It also induces the synthesis and release of IL-1β, IL-6 and TNF-α from fibroblasts and macrophagesNlrp3−/− and Casp1/11−/− mice compared with WT on 7 d.p.i  assay25. All experiments related to H7N9 virus were performed in biosafety level 3 laboratories in Shanghai Public Health Clinical Center (SPHCC), and followed the standard operating protocols approved by the Institutional Biosafety Committee at SPHCC, Fudan University.A/Shanghai/4664 T/2013(H7N9) (GenBank No. KC853225-KC853232) strain was used in this study. Isolation of H7N9 virus was described before and the viral titer was measured via tissue culture infective dose (TCIDNlrp3 deficient mice (a gift from Dr. Warren Strober) and Asc deficient mice (a gift from Dr. Vishva M. Dixit) had been described36. Caspase-1/11 and IL-1 receptor 1 deficient mice were purchased from the Jackson Laboratory . Herein, these mice were described as Nlrp3−/−, Asc−/−, Casp1/11−/− and Il1r1−/−. All mice were housed under specific pathogen-free (SPF) condition at the animal facility at Institut Pasteur of Shanghai. Mice were transferred to the Animal Biosafety Level 3 (ABSL3) Laboratory at SPHCC three days before infection. Mice care and experimental procedures complied with national guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) at SPHCC, Fudan University.All mice used in our experiments are on a C57BL/6 genetic background and all experiments were carried out with age and gender matched mice . C57BL/6 mice were purchased from the B&K Universal Group Limited . 37. Briefly, bone marrow cells were harvest and lysed with RBC lysis buffer, and seeded in 10 cm dish with 10 ml RPMI1640 with GM-CSF (20 ng/ml). The media were refreshed 3 days later and the cells were harvested for experiments on day 6.Bone marrow derived dendritic cells (BMDCs) were generated as describedAntibodies for immunoblotting include: rabbit anti-mouse ASC , mouse anti-mouse caspase-1 , rabbit anti-mouse mature and pro–IL-1β  and mouse anti-mouse β-actin .H7N9 RNA was extracted with QIAamp Viral RNA Mini Kit according to the manufacture’s protocol. And we measured the concentration of viral RNA with Nanodrop 2000 . BMDCs were primed with 500 ng/ml LPS  for 4 hours, then the different doses of H7N9 viral RNA were transfected with Lipofectamine 2000  according to the manufacture’s protocol. Briefly, Lipofectamine 2000 (2 ul/ml) and viral RNA were diluted in opti-MEM , then mixed together for 20 minutes and the mixture was added into cells. 12 hours later, the supernatant was harvested for ELISA or inflammasome activation assay, and the cells were collected for Western Blotting and ASC oligomerization detection.36. Brifely, H7N9 RNA transfected BMDCs were lysed with lysis buffer , pelleted and washed with PBS, and cross-linked with disuccinimidylsuberate (DSS) before subjected to immunobloting.ASC oligomer was prepared as described4 TCID50 of H7N9 virus in a volume of 50 µl intranasally (i.n.) after sevoflurane  inhalation anaesthesia, and monitored for clinical signs, body weight changes and physical survival during the 14 days observation period. The infected mice were sacrificed on 3, 7 or 14 days post infection for collection of samples for analysis as reported15. To monitor the body weight changes and survival rate, 14 to 22 mice were used in each group (n = 14~22). For the rest analysis of murine samples, more than 3 mice were used in each group.Mice were infected with 5 × 10Lung homogenate supernatants and serum were collected and stored at −80 °C before analysis. The levels of cytokines and chemokines in the collected samples were determined quantitatively using a ProcartaPlex Multiplex Immunoassay kit  according to the manufacturer’s protocol.Bronchoalveolar lavage fluid (BALF) was obtained via flushing the lungs three times with 0.5 mL/time of PBS. The single-cell suspensions from BALF of H7N9 infected mice were first treated with anti-mouse CD16/CD32 antibody  to block Fc receptor, then surface stained with antibodies against B220, CD3, CD11b, CD11c, Ly6c, Ly6g and F4/80, and fixed with 4% paraformaldehyde before analysis with FACS Array . The final graphical output was generated through FlowJo software .15. Calculation was carried out by comparing H7N9 Hemagglutinin with GAPDH, which served as an internal control GAPDH (ΔΔCT method).Total RNA was extracted from lung homogenates using a kit  according to the manufacturer’s protocol. Relative Quantification of H7N9 viral load via RT-PCR was conducted as reported previouslyExcised mouse lung tissues were fixed in 4% paraformaldehyde followed by paraffin embedding and sectioning, then subjected to hematoxylin and eosin (H&E) staining. Images were collected by Leica DMI3000 microscopy with indicated magnifications.38 and modified by the pathologist, Dr. Y Feng, in author list of this manuscript.Four indexes were analyzed for scoring on each slide: 1) Blood vessel region inflammation, 2) Pulmonary alveoli inflammation, 3) Lung tissue inflammation, 4) Bronchial inflammation. The detailed criteria were listed in Table All statistical analysis was conducted using GraphPad Prism Software via parametric unpaired t-test if not noted otherwise . P < 0.05 was defined as statistically significant.supplementary figures 1, 2 and 3