Stimulating the patient’s immune system represents a promising therapeutic strategy to fight cancer. However, low immunogenicity of the tumor cells within an immune suppressive milieu often leads to weak anti-tumor immune responses. Additionally, the immune system may be impaired by accompanying aggressive chemotherapies. We show that mitoxantrone, bound to superparamagnetic iron oxide nanoparticles (SPIONs) as the transport system, can be magnetically accumulated in adherent HT-29 colon carcinoma cells, thereby inducing the same cell death phenotype as its soluble counterpart, a chemotherapeutic agent and prototypic inductor of immunogenic cell death. The nanoparticle-loaded drug induces cell cycle stop, apoptosis and secondary necrosis in a dose- and time-dependent manner comparable to the free drug. Cell death was accompanied by the release of interleukin-8 and damage-associated molecular patterns (DAMPs) such as HSP70 and ATP, which fostered chemotactic migration of monocytes and maturation of dendritic cells. We furthermore ensured absence of endotoxin contaminations and compatibility with erythrocytes and platelets and investigated the influence on plasma coagulation in vitro. Summarizing, with magnetic enrichment, mitoxantrone can be accumulated at the desired place, sparing healthy peripheral cells and tissues, such as immune cells. Conserving immune competence in cancer patients in the future might allow combined therapeutic approaches with immune therapies . In recent years, immune therapies have constituted substantial improvements in tumor treatment. However, these therapies often fail due to the low immunogenicity of tumor cells and the surrounding immunosuppressive milieu, both resulting in a weak anti-tumor immune response and enabling immune therapies only in a subgroup of patients . Moreove®). All cells were cultured in a cell culture incubator  at 37 °C, 5% CO2 and 95% humidified air. Cells were regularly checked for mycoplasma contamination using PCR kit Venor®GeM . Before every experiment, count and viability was analyzed using MUSE® Count & Viability assay kit in MUSE cell Analyzer . A cell viability above 95% was required to start an experiment.The adherent colon carcinoma cell line HT-29  was cultivated in McCoy’s 5A medium  with either 10% fetal calf serum (FCS)  for magnetic accumulation or cell death kinetics, or 10% Panexin NTA  whenever analyses of DAMPs or cytokines were planned, to minimize interactions with the detection. In preceding experiments, we confirmed that HT-29 cells showed similar behavior, no matter if cultivated in medium containing FCS or Panexin. The monocytic cell line THP-1  was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2 mM glutamine, 10% FCS (Biochrom AG), 100 U/mL penicillin, and 100 μg/mL streptomycin , representing an endpoint fluorescent microplate assay intended for in vitro quantitative determination of endotoxins in pharmaceuticals and biological substances. Importantly, false positive reactions by β-glucan reactions  are thereby eliminated. SPIONs of three different batches were taken up in endotoxin-free water in concentrations of 25 µg/mL and 50 µg/mL and tested in duplicates. Samples spiked with 5 EU/mL endotoxin served as inhibition/enhancement controls (IEC). 100 µL of mixture of assay reagent liquid  was added to every sample and standard. The reaction was monitored for 90 min at 37 °C in the Microplate Reader Filter Max F5 . The endotoxin level in EU/mL per sample was calculated from the standard curve (non-linear 4-parameter logistic regression). The regression coefficient Rg, room temperature) of freshly drawn sodium citrate-anticoagulated blood from healthy donors with normal coagulation times. Ex vivo use of human blood for nanoparticle testing was approved by the ethics committee of the FAU Erlangen-Nürnberg . 450 µL plasma was incubated for 30 min at 37 °C with 50 µL of SPION dilutions, resulting in final iron concentrations of 20, 100, or 500 µg/mL. Addition of 50 µL H2O served as negative control. After that, plasma was pipetted into pre-warmed cuvettes, a metal ball was added, and coagulation was started by addition of the respective coagulation reagent to each cuvette. The samples were analyzed regarding activated partial thromboplastin time (aPTT), prothrombin time (PT) and thrombin time (TT), using DiaSys coagulation kits  in a blood coagulometer  according to the manufacturer’s instructions. Standard plasma served as the control for normal and abnormal coagulation times.To analyze whether SPIONs influence plasma coagulation, platelet-poor plasma (PPP) was obtained by centrifugation  and at 2500× g for 15 min to receive platelet-poor plasma as background control (blank). To ensure normal platelet function, all aggregation assays were performed within 4 h of blood collection. To analyze whether SPIONs induce platelet aggregation, 90 µL PRP was incubated with 10 µL SPION dilutions, resulting in final iron concentrations of 20, 100 and 500 µg/mL. Phosphate-buffered saline  served as negative control (NC), H2O as vehicle control (VC) and 100 µg/mL collagen as positive control (PC) . Samples were incubated with continuous shaking for 15 min at 37 °C. Then, 50 µL of each sample were diluted in PBS and analyzed by flow cytometry. Data analysis was performed with KaluzaTM software version 1.2.Sodium-heparin anticoagulated blood was collected from healthy volunteers and centrifuged at 200× g for 15 min at room temperature (RT). The hemoglobin content of the whole blood samples was determined and adjusted to 5 mg/mL in PBS. SPIONs were incubated with diluted blood in final iron concentrations of 20, 100 µg/mL for 3 h at 37 °C and carefully mixed every 30 min. 1% Triton X-100 , PBS and H2O served as positive, negative and vehicle controls, respectively. To detect interference of SPIONs with the assay, the positive control (PC) was spiked with SPIONs. SPIONs diluted in H2O in the respective concentrations served as background controls. After incubation, the tubes were centrifuged for 15 min at 800× g at RT to achieve sedimentation of erythrocytes. The supernatant was transferred into new tubes and centrifuged for 1 h at 18.000× g at RT to sediment the SPIONs. To determine the content of free hemoglobin, 100 µL supernatant was transferred into the wells of a 96-well plate and incubated with 100 µL Drabkin’s solution  for 3–5 min at 56 °C on a heating plate until the content of the wells became clear. Drabkin’s reagent converts unstable released hemoglobin and its derivatives to methemoglobin and then to stable cyanmethemoglobin, which was measured at 590 nm on Microplate Reader Filter Max F5. The absorption values measured from the released hemoglobin of the positive control were set to 100%.Lithium heparin anti-coagulated blood was taken from healthy donors. Hemoglobin-free plasma was prepared as a control by centrifugation of the blood at 800× 5 HT-29 cells were seeded into 12-well plates and cultured overnight. The next day, a 96-well plate containing magnets was positioned directly under the 12-well plates, so that each cell-containing well possessed one central magnet. The cells were incubated with SPIONs, free MTO, or SPIONMTO for 5 h in FCS-containing HT-29 medium. Then, the medium was removed, cells were washed with PBS, fixed with 3% paraformaldehyde  in PBS and stained with 10 µg/mL Hoechst 33342 . Fluorescence microscopy pictures were prepared using Zeiss Axio Observer.Z1 fluorescence microscope  in tile modus and single tiles were stitched to a complete overview picture. Analysis of MTO distribution was performed with ZEN 2012 software (Blue Edition) (Carl Zeiss AG). 1 × 104 cells/well. The plates were incubated for adherence for either 72 h or 96 h. After that, the medium was replaced with 1 mL Panexin- or FCS-containing medium and the cells were treated with SPIONs, free MTO or SPIONMTO. To analyze cell proliferation, an IncuCyte life cell imaging system  was used. All samples were run in triplicates. From the microscopic pictures the amount of area covered by cells (confluence) was calculated. The plates were observed until 100% confluence of the untreated control group was achieved. Confluence was evaluated with Microsoft Excel. In FCS- and Panexin-containing media, cell proliferation was comparable (data not shown).HT-29 cells were seeded into 24-well plates with 1 mL FCS-containing HT-29 medium in a concentration of 1 × 1024 h, 48 h or 72 h after treatment, supernatants of the cells were collected and frozen at −80 °C for further use. Then, cells were harvested using 0.05% Trypsin . 25 µL aliquots of the cells were either fixated in ethanol (70%) and frozen at −20 °C for cell cycle analysis or stained for cell viability measurement in flow cytometry. For that purpose, 10 µL of cells were stained with 10 µg Hoechst 33342 , 1 µL AnnexinA5-fluorescein isothiocyanate (FITC) or 1 µL AnnexinA5-allophycocyanin  and 66.6 ng propidium iodide  per 1 mL Ringer’s solution  for 30 min at 4 °C.2HPO4, 0.8 mL 0.1% Triton X-100 , pH 7.8) following Riccardi et al. [For cell cycle characterization, the ethanol-fixated cells were washed with PBS and incubated for 5 min with 500 µL DNA extraction buffer . FITC and PI were excited at 488 nm and fluorescence was detected on fluorescence (FL) 1 sensor, 525/38 nm, bandpass (BP) and FL 3 sensor, 620/30 nm BP, respectively. APC and MTO were excited at 638 nm and fluorescence was detected at FL6, 675/20 nm BP or FL7 sensor, 725/20 nm BP, respectively. Electronic compensation was used to eliminate any fluorescence bleed-through. The data were analyzed in KaluzaTM software version 1.2  and processed in Microsoft Excel. d-luciferin and 2.5 µL firefly luciferase 5 mg/mL) was added. The ATP-mediated chemiluminescence was measured after incubating for 40 min at 28 °C employing Microplate Reader Filter Max F5. The ATP content of the samples was quantified using a standard curve ranging from 0.1 pM to 1 µM ATP. The data were analyzed with Microsoft Excel.To analyze the amount of ATP in cell supernatants, the ATP Determination kit  was used according to the manufacturer’s instructions. In brief, supernatants frozen at −80 °C were thawed gradually and mixed. 10 µL were placed in each well of a white 96-well plate . Then, 90 µL reaction mix  according to the manufacturer’s instructions. In detail, wells of a flat-bottom high-binding 96-well microplate  were coated with 100 µL capture antibody (2.0 µg/mL in PBS) overnight at room temperature. After blocking the plate with 300 µL blocking buffer composed of 1% BSA  and 0.05% NaN3  in PBS for 90 min at room temperature, 100 µL of the samples or standards was pipetted into the wells and incubated for two hours. Subsequently to placing 100 µl of detection antibody (3.6 µg/mL in 1% BSA in PBS) into the wells and incubating for two hours, 100 µL of Streptavidin-HRP (50 µL in 10 mL in 1% BSA in PBS) was pipetted into the wells and incubated at room temperature for 20 min. Between each step the plate was washed with 200 µL 0.05% Tween20  in PBS. Finally, the plates were developed by adding 100 µL freshly prepared substrate solution  consisting of 1 mL of 0.01 g 3,3‘,5,5‘-tetramethylbenzidine  dissolved in 10 mL dimethyl sulfoxide, 9 mL of 0.1 M Na2HPO4, 0.05 M citric acid, pH 4.5-5.5 and 2 µL H2O2). The plate was incubated for 20 min and reaction was stopped with 100 µL of 2N H2SO4 . 170 µL of the samples were then transferred into a clear 96-well plate  and measured in Microplate Reader Filter Max F5 at 450 nm. The HSP70 content in the samples was quantified using a standard curve ranging from 312.5 µg/mL to 10 mg/mL HSP70. The data were analyzed with Microsoft Excel.Heat Shock Protein 70 (HSP70) in the cell supernatant was determined using the Total HSP70 DuoSetMTO was assessed in a 96-well Chemo-Tx plate . In brief, the microplate wells were filled with 30 µL of supernatant. MCP-1  was used as a positive control. After placing the filter frame, the filter top sites with 5 µm pores were filled with 25 µL of THP-1 monocytes at a concentration of 1 × 106 cells/mL. Following incubation for 1 h at 37 °C, migrated non-adherent cells from the lower wells were fixed and counted using flow cytometry. All samples were run in quadruple and averaged. The mean number of migrated cells in the negative control (MCP-1 unstimulated) samples was set as 1.The chemotactic capacity of supernatants from HT-29 cells treated with SPIONg for 25 min at room temperature without brakes. The PBMCs were washed three times in PBS containing 2% EDTA  and resuspended in DC medium composed of RPMI medium  supplemented with 1% heat inactivated 0.22 µm filtered human AB-plasma , 1% l-Glutamine, and 0.4 µg/mL Gentamycin . After seeding the cells in cell culture dishes  and incubating them for 1.5 h at 37 °C for adherence, non-adherent cells were removed and 10 mL DC medium was added to the remaining adherent monocytes. Further incubation for 6 days with recurring fresh DC medium substitution , containing 275 U/mL IL-4 and 800 U/mL GM-CSF  generated the iDCs.After giving informed consent, healthy volunteers provided leukocytes in human leukoreduction system chambers . As described by Schaft et al. , monocyt5 HT-29 cells cultivated with 6 mL Panexin-medium in T 25 cell culture flasks  were treated with 1 µM or 5 µM SPION, MTO and SPIONMTO as described above. After 72 h, the supernatants were separated from the cells and frozen at −80°C for further experiments. On day 6, 100 µL DC medium containing 1 × 106 iDCs, which were harvested from the dishes, centrifuged at 200× g for 12 min and resuspended in 10 mL DC medium, were added to 2 mL HT29 cell supernatant per well of a 6-well plate. For every sample at least one duplicate was prepared. To generate mature DCs (mDCs), a cytokine cocktail containing IL-1β (200 U/mL), IL-6 (1000 U/mL), TNF-α , and PGE2  in DC medium served as a positive control. Untreated DCs served as negative immature DC controls (iDCs). After 24 h of co-incubation, supernatants of DCs were frozen for further cytokine analysis at −80 °C. DCs were harvested, washed and adjusted to a density of 5 × 104 cells per 50 µL in ice-cold PBS containing 2% FCS. Staining for DC activation markers was performed with fluorochrome-conjugated antibodies and their appropriate isotype controls for 30 min at 4 °C, using CD197-PE, CD25-FITC, CD83-PE/cy7, CD40-PerCP/Cy5.5, CD209-APC, CD80-APC, CD86-PerCP/Cy5.5, CD70-PE  and HLA-DR-eFluor450 . Stained DCs were analyzed in flow cytometry . We identified DCs using the forward and side scatter properties and excluded CD3, CD14, CD16, CD19, CD20, CD56 staining typical for T cells, monocytes, granulocytes, B cells and NK cells, respectively. The gated cell population was positive for CD11c and CD1c, both known markers for DCs. Data were analyzed using KaluzaTM software version 1.2. 4 × 10g for 5 min and the supernatant was removed. The pellet was washed with 300 µL Ringer’s solution and finally resuspended in 150 µL Ringer’s solution. The samples were measured in flow cytometry with excitation at 488 nm and 638 nm and detection in FL2 (575/30 BP) and FL6 (675/20 BP). LEGENDplexTM data analysis software was used to analyze the data.Cytokines released from DCs were analyzed using the LEGENDplex human IL-8 detection kit or the LEGENDplex human inflammatory panel  according to the manufacturer’s instructions. In short, 10 µL of assay buffer, 10 µL of supernatant, 10 µL mixed beats and 10 µL detection antibody were combined in small tubes  and incubated at room temperature, in the dark, on a plate shaker for two hours. Then, 10 µL Streptavidin-phycoerythrin was added and incubated for further 30 min. Next, the tubes were centrifuged at 1000× t-test in the samples passing the normality test and non-parametric Mann–Whitney U test in those failing.Data were processed in Microsoft Excel . Graphs and statistics were prepared in GraphPad Prism software, version 8 . Group differences were calculated after performing a normality test (Shapiro-Wilk), using Student’s MTO are intended for use in magnetic drug targeting, implying that after intraarterial application, they will come into close contact with components of the blood. Therefore, it is mandatory that the particles are free of endotoxins to prevent immune stimulation. To exclude assay interference by the nanoparticles, the samples were spiked with endotoxin and spiking recovery was determined. Dilution of the SPIONs to 25 µg/mL Fe content resulted in an acceptable endotoxin spiking recovery of 64%. For 25 µg/mL <0.05 EU/mL endotoxin was detected, showing that the nanoparticles are eligible for further investigations  with magnets (0.5 cm diameter) positioned under the cell-containing wells  as seen in MTO- and SPIONMTO-treated cells in a dose-dependent manner. After 72 h, cells treated with MTO in free or nanoparticle-loaded form are characterized by degraded DNA (Effects on cell viability were assessed using Annexin A5-FITC (Ax) and propidium iodide (PI) staining. Ax binds phosphatidylserine, which switches from the inner to the outer leaflet of the plasma membrane when cells undergo apoptosis . The plarocesses B. With tdetected . Althougdetected . Becauseaded DNA . So far,MTO-treated cells, we detected ATP and HSP70 after 72 h incubation in a dose-dependent manner  or only a small amount of ATP or HSP70 was released . When analyzing supernatants of SPION-, MTO- or SPIONt manner A,B. At treleased A,B. In creleased C. For SP counted D. SupernMTO-treated cells were able to induce maturation of dendritic cells. For this purpose, we co-incubated the supernatants with immature dendritic cells (iDC) and analyzed cell morphology and surface molecules. iDCs in the presence of SPION-treated supernatants showed the same behavior as the control group, with typical dendritic cell morphology and no induction of activation markers on the cell surface  principle for animal experimentation. Immunogenically dying CT26 colon carcinoma cells killed by MTO in vitro and adorned with CpG nanoparticles (adjuvant TLR9-agonist) have been already shown to foster maturation of DCs, T cell responses and antitumor responses in mouse models [We are aware that the gold standard, however, should be animal experiments because investigations of the development of adaptive immune responses need a complex in vivo immune environment. As requested by the consensus guidelines, two sets of experiments should be performed in vivo: first, vaccination assays, where cells are killed in vitro and injected subcutaneously into immune-competent mice to investigate their potential in inducing a protective anticancer immune response against a challenge with living tumor cells one week later. Second, therapeutic assays, where immune-competent and immune-deficient tumor-bearing mice are treated with the putative ICD inducer, followed by monitoring of the tumor size . MTO itse models . With oue models .MTO in tumor-bearing rabbits previously, we observed that the tumor shrank over several weeks, which is more indicative of an immune-mediated effect than immediate tumor lysis [MTO and magnetic accumulation in tumor tissue, an immunogenic tumor response can be finally achieved by cytotoxic T cells.Our in vivo therapeutic setting using an electromagnet with a maximum magnetic field of 1.01 T at the very tip of the pole show at a current of 72 A to magnetize the SPIONs was constructed for the use of larger organisms than mice, which makes the procedure more time-consuming, more expensive, enables only smaller group sizes and must be planned carefully. When we performed MDT with SPIONor lysis . We alsoor lysis . AdvanceNanoparticles which can induce localized cell death with immunogenic properties might thus be used to turn immunologically classified “cold” tumors with low intratumoral lymphocyte infiltration rates into “hot” immune-infiltrated tumors ,52 to en


LSD1 forms complexes with the CoREST family of transcriptional corepressors, which are molecular bridges that bring HDAC1/2 and LSD1 enzymes to deacetylate and demethylate the tail of nucleosomal histone H3. To test the hypothesis that LSD1-complexes are involved in initial modifications associated with pilocarpine-induced epilepsy, we studied the expression of main components of LSD1-complexes and the associated epigenetic marks on isolated neurons and the hippocampus of pilocarpine-treated mice. Using a single injection of 300 mg/kg of pilocarpine and after 24 h, we found that protein levels of LSD1, CoREST2, and HDAC1/2 increased, while CoREST1 decreased in the hippocampus. In addition, we observed increased histone H3 lysine 9 di- and trimethylation (H3K9me2/3) and decreased histone H3 lysine 4 di and trimethylation (H3K4me2/3). Similar findings were observed in cultured hippocampal neurons and HT-22 hippocampal cell line treated with pilocarpine. In conclusion, our data show that muscarinic receptor activation by pilocarpine induces a global repressive state of chromatin and prevalence of LSD1-CoREST2 epigenetic complexes, modifications that could underlie the pathophysiological processes leading to epilepsy.Epilepsy is a neurological disorder of genetic or environmental origin characterized by recurrent spontaneous seizures. A rodent model of temporal lobe epilepsy is induced by a single administration of pilocarpine, a non-selective cholinergic muscarinic receptor agonist. The molecular changes associated with pilocarpine-induced seizures are still poorly described. Epigenetic multiprotein complexes that regulate gene expression by changing the structure of chromatin impose transcriptional memories. Among the epigenetic enzymes relevant to the epileptogenic process is lysine-specific demethylase 1   •Pilocarpine modifies LSD1-CoREST-HDAC1/2 complexes in HT-22 cell and hippocampal neurons.•Activation of cholinergic muscarinic receptors induces a global repressive state of chromatin in hippocampus.•Pilocarpine-induced seizures associate to LSD1-CoREST-HDAC1/2 modifications and repressive chromatin. One central feature of epilepsy is the recurrence of epileptic seizures and the greater probability of having a new seizure after having suffered one ,2. Tempoepilepsy . AlthougSeveral lines of evidence focused on revealing the molecular mechanisms of TLE have shown that histone modifications are implicated in the pathophysiology of epilepsy, highlighting the eventual role of proteins that regulate gene expression such as Methyl-CpG Binding Protein 2 (MeCP2), Repressor Element-1 Silencing Transcription Factor (REST) and others . In thisLSD1 is a ubiquitous epigenetic enzyme that demethylates mono and dimethylated lysines 4 and 9 in the histone H3 favoring transcriptional repression or activation, respectively ,9. LSD1 Considering the role of the LCH complex in regulating the expression of neuronal excitability genes, we hypothesized that changes in the expression of its components could account for the epigenetic events associated with the chromatin status in the early phase of epileptogenesis. We aimed to expand the knowledge related to the epigenetic nature of epileptogenesis by focusing on the description of chromatin changes occurring after an epileptic seizure and the enzymes involved in this process. To this end, we studied the expression of LCH components and epigenetic marks in the hippocampus of mice scoring between 2 and 5 on the Racine scale  after a 22.1Fifty-six male C57BL/6 mice, six weeks old (21–25 g) and three pregnant adult Sprague–Dawley rats (250–300 g) were obtained from the animal facility of the Faculty of Biological Sciences of the Pontificia Universidad Católica de Chile.ad libitum.All experimental procedures (protocol ID 03082015) were approved by the Bioethical Committee of the Faculty of Biological Sciences of the Pontificia Universidad Católica de Chile. All procedures were conducted to reduce the number of mice used when possible and to reduce their pain level and discomfort as much as possible. Animals were grouped in a climate-controlled vivarium on a 12 h light/dark cycle with food and water provided 2.2Animals were injected with the muscarinic receptor antagonist scopolamine methyl nitrate  to minimize peripheral pilocarpine-induced side-effects. Pilocarpine  was injected 30 min after SMN. We selected this dose of pilocarpine because it induced seizures in all mice and did not require the administration of anticonvulsants. The mortality rate was 9.09%. Control animals were injected with SMN, and 30 min later, an equivalent volume of the pilocarpine solution vehicle (0.9% NaCl) was injected. All animals were videotaped for 2 h to register their behavior and the severity of seizure was evaluated using the Racine scale .2.3These experiments were carried out in three mice anesthetized with urethane , using the protocol of Meza et al., 2018 .2.4HT-22 mouse hippocampal neuronal cell line (SCC129 Sigma-Aldrich) were cultured in Dulbecco's modified Eagle's high-glucose medium  supplemented with 2 mM of glutamine (Gibco), 10% fetal bovine serum , 1% penicillin/streptomycin and maintained at 37 °C and 5% CO2.l-lysine-coated covers and maintained in Neurobasal medium supplemented with B27, 1% penicillin/streptomycin, and 2 mM of glutamine. On the second day of culture, 2 μM Cytosine-Arabinoside (AraC) was added 24 h to inhibit glial proliferation. The cells were maintained at 37 °C and 5% CO2 during 7 days in vitro (DIV) before starting the experiments to allow the expression of the LCH complex components [Rat embryos of E18 pregnancy day were used for primary culture studies. Pregnant rats were decapitated, and embryos were dissected in Hank's medium. Embryos were decapitated with scissors and the hippocampus dissected out for cell culture, essentially as we have described . Primarymponents . At DIV 2.5.Twenty-four hours after pilocarpine injection, mice were deeply anesthetized with chloral hydrate , and brain tissue was fixed by transcardial perfusion of physiological saline followed by 4% paraformaldehyde in phosphate buffer saline (PBS 1X), pH 7.5. Later, brains were removed and postfixed in 4% paraformaldehyde for 2 h and maintained in 20% sucrose in PBS 1X for 48 h. Brains were sectioned in 40 μm coronal slices using a cryostat  from Bregma −1.7 mm to −2.46 mm, according to the atlas of Franklin and Paxinos .For immunodetection, the slices were permeabilized in 0.3% of Triton x-100 in PBS1x for 2 h and incubated in a blocking solution  in PBS1x) for 2 h. The primary antibody solution was 0.05% of Triton x-100/5% NDS in PBS1x overnight at 4 °C. After washing with 0.3% of Triton x-100 in PBS1x, the slices were incubated for 2 h with the secondary antibodies in 0,05% of Triton x-100/1% NDS in PBS1x at RT in the dark. Finally, the slices were stained with 1 μg/mL Hoechst 33,342 (Abcam) and mounted on glass slides with 0.1% gelatin. After drying were mounted with Dako Fluorescence Mounting Medium (Agilent Technologies) .2.6Cells were fixed with 4% paraformaldehyde in PBS 1X for 15 min and permeabilized with 0.25% Triton-X100 in PBS 1X for 5 min. Then, the cells were incubated with 3% BSA in PBS 1X for 1 h, followed by primary and secondary antibodies in the same solution for 1 h each, at RT in a humid chamber. Finally, the cells were stained with 1 μg/mL Hoechst 33,342 (Abcam). Coverslips were mounted on DAKO Fluorescence Mounting Medium (Agilent Technologies).2.7Images of 2560 × 1920 pixels from tissue and cells were acquired with an Olympus DS-Fi2 epifluorescence microscope equipped with a Nikon DS-Fi2 camera with standard QC capture software (Q-Imaging). The tissue images were acquired with appropriate 10× objectives and cells were acquired with appropriate 40× and 100X (using oil immersion). The time of exposition for each used laser was the same for vehicle and pilocarpine samples. Immunofluorescence quantification was performed using ImageJ software . Fluorescence intensity was calculated as the Mean Gray Value/Area (IntDen/Area) corrected by the background. The area selected to calculate the fluorescence intensity on dentate gyrus was from 998,520 to 1,151,360 pixels, using the same area for vehicle and pilocarpine for each antibody evaluated. Three to nine hippocampal slices were analyzed per animal per experiment. Six to eight fields of cultured neurons were analyzed by experiment, and for HT-22 cells, between thirty to hundred cells were quantified.2.8Extracted proteins were fractionated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were transferred to PVDF membrane (Millipore) blocked with 5% dry fat milk in TBS, 0.1% Tween 20 (TBS-T). Primary antibodies were incubated in 3% BSA in TBS-T overnight at 4 °C, and secondary antibodies were incubated in TBS-T for 45 min at room temperature and were revealed with SuperSignal West Pico Chemiluminescent Substrate .2.9g 3 min and then nuclei were purified by hypotonic lysis as previously described [g and then clarified supernatants containing solubilized histones were transferred to a new tube. Histones were precipitated in 25% trichloroacetic acid, centrifuged 10 min at 12,000×g, and extensively washed with acetone. Finally, histones were resuspended in RIPA buffer, quantified, and then subjected to Western blot analyses.Dissected hippocampal regions from control and pilocarpine-treated animals were trypsinized 15 min at 37 °C in PBS1X supplemented with protease inhibitors. Tissues were then mechanically homogenized by 30x syringe pressing movements. Cells were centrifuged at 1400×escribed . Nuclei 2.10g and then loaded into a 5–20% Sucrose gradient prepared in a buffer containing 50 mM HEPES-KOH (pH 7.5), 1 mM (CH3COO)2 Mg, 80 mM CH3COOK and 0.5% (v/v) Triton X-100. Ultracentrifugation was performed for 16 h at 300,000×g at 4 °C. Fractions enriched in LSD1 were pooled and subjected to immunoprecipitation.Purified nuclei from mice hippocampus were subjected to extraction in a buffer containing 420 mM KCl. Salt-extracted material was clarified by 30 min centrifugation at 15,000×2.11Nuclear extracts or LSD1-enriched fractions from sucrose gradient experiments were dialyzed against immunoprecipitation buffer . Immunoprecipitation was performed using 45 μL Agarose-conjugated Protein A (Pierce #20333) and 1 g of anti-LSD1 antibody (ab17721) every 700 g protein in the input. Immunocomplexes were separated by centrifugation after 12 h of incubation at 4 °C. Beads were extensively washed against the CoIP buffer, and immunocomplexes were eluted by boiling the beads in 1X Laemmli Sample Buffer before Western blot analysis.2.12Mouse anti-CoREST1 ; rabbit anti-CoREST2 ; rabbit anti-LSD1/KDM1 ; rabbit anti-HDAC1 ; mouse anti-HDAC2 ; rabbit anti-H3K4me2 ; rabbit anti-H3K9me2 ; H3K4me3 ; rabbit anti-H3K9me3 ; rabbit anti-H3Ac ; goat anti HP1α ; rabbit anti-H3 (Abcam ab1791); mouse anti-GAPDH ; donkey anti-rabbit AlexaFluor 488, donkey anti-mouse AlexaFluor 594, donkey anti-goat AlexaFluor 594 ; Peroxidase-conjugated secondary antibodies .2.13Total RNA was isolated from mice hippocampus 7- or 24- hours after pilocarpine injection using Trizol reagent (Invitrogen Life Technologies) and reverse transcribed using MMulV (Thermo Scientific). Primers were based on the coding frame of the mouse rcor1 gene: F: 5′TCAGCAGACCACATCGTCAC3'; R:5′CATGAGGCTACAGTGCCCAA3'; rcor2 gene:F:5′AGGACAGAAGGGACTAGGGC3′; R:5′GTCACAGCCAGGAAGCAAGA3′; rcor3 gene: F:5′CACGGGGATGTTGGGATGAG3′ R:5′CGTGAAGTTAGGGAGGTCCG3'. Primers were manufactured by IDT™. All PCRs were performed with 5 x HOT FIREPol® EvaGreen® qPCR Mix Plus (Solis BioDyne) using a LightCycler 2 (Roche).2.14Total RNA was isolated from mice hippocampus 7 h after pilocarpine injection using Trizol reagent (Invitrogen Life Technologies), and reverse transcribed using MMulV (Thermo Scientific). Rqf-PCR was performed as previously described . PCR pri2.15U test or unpaired Student's t-test, and One-way ANOVA, multiple comparisons, or Two-way ANOVA followed by Bonferroni post hoc test were used to determine the statistical significance of the differences, using Prism 7.0 software. The data they were expressed as mean ± SEM, and statistical significance was established at P < 0.05. The number of independent experiments and p values can be found in figure legends.Non-parametric Mann-Whitney 33.1The LCH complex regulates the expression of genes involved in neuronal excitability. Therefore, we wondered whether the LCH complex's components and chromatin's functional status in the hippocampus are modified in the model of epileptogenesis induced by pilocarpine. For this purpose, we first characterized the pilocarpine-induced epilepsy model in our laboratory. The protocol used consisted of an injection of pilocarpine (300 mg/kg) 30 min after injecting scopolamine (SMN) to minimize peripheral pilocarpine-induced side effects A. LatencTwenty-four hours after pilocarpine injection, the hippocampi were removed for Western blot analysis A. We obsTo investigate whether the observed changes in protein levels are due to a change in transcript levels, we performed a qPCR study at short (7 h) and long times (20 h) after pilocarpine injection. Previously, it was shown that pilocarpine-induced seizures modify transiently the expression of the LSD1 neuronal variant, nLSD1 in the hippocampus . We also3.2To assess the integrity of the LCH complex against pilocarpine-induced seizure, we tested whether immunocomplexes formed between endogenous CoREST1 and LSD1 proteins in the hippocampus were modified after pilocarpine injection. To this end, we biochemically enriched hippocampal LSD1 after separating hippocampal nuclear proteins on a 5–20% sucrose gradient and pooling LSD1-enriched fractions A. Next, 3.3Changes in the protein levels of LCH-complex components prompted us to study whether the histone substrates of LCH complexes are also affected by the pilocarpine treatment. The evidence accumulated so far shows that LSD1 can demethylate H3K4me1/me2 and H3K9me1/me2, inducing gene repression and activation, respectively ,9,25. He3.4To test the idea that the LCH complex is a target of the cholinergic system, we evaluated the effect of pilocarpine and SMN, a specific antagonist of M1/M2 receptors, in isolated hippocampal neurons. To achieve this goal, rat primary cultures at DIV7 of E18 embryos were treated during 24 h with pilocarpine (200 μM) alone or in the presence of SMN (10 μM), and then subjected to immunofluorescent analysis. Consistent with in vivo data, pilocarpine increased LSD1 protein levels in cultured hippocampal neurons, effect that was reversed by SMN. Similarly, increased H3K9me2 induced by pilocarpine was reversed by SMN . On the To further inquire in the role of muscarinic receptors regulating LCH complex components and epigenetic status of chromatin, we tested the effect of pilocarpine in HT-22, a hippocampal cell line that expresses M1 and M2 muscarinic receptors . Similar4This study provides evidence that the cholinergic system regulates components of the LSD1-CoREST-HDAC1/2 complex through M1/M2 muscarinic receptors. We detected changes in main components of the LCH complex produced by pilocarpine in vivo, in cultured neurons, and HT-22 cells, and global changes in chromatin structure revealed by increased levels of repressive histone modifications and decreased activating marks.LSD1, when bound to CoREST proteins, is a transcriptional corepressor that removes methyl groups from lysine residues in chromatin histones whose methylation is linked to active transcription . PilocarHDACs have a crucial role in suppressing gene transcription by condensing the chromatin structure, removing the acetyl groups of the amino-terminal lysines from the histone tails, which gives histones a positive net charge, making it more compact, characteristic of gene repression ,30. ThisA deficiency of HDAC2 is known to cause an increase in the number of synapses and the facilitation of memory . After pInterestingly, genome-wide profiling studies of hippocampal gene expression changes on animals subjected to pilocarpine treatments have shown that a large subset of genes change their pattern of expression at different time points after presenting a status epilepticus, suggesting that a global and dynamic reprogramming of gene expression is occurring . SimilarThe state of the chromatin and the components of the LCH repressor complex change as a result of the neuronal hyperexcitability produced by pilocarpine. Interestingly, these changes principally occurred at the dentate gyrus of the hippocampus, a region that has high expression levels of M1 muscarinic receptors compared to others . Our in National Agency for Research and Development (ANID), Chile .We gratefully acknowledge funding by the V·N and C.R conceived and designed the study, participated in the acquisition, analysis, interpretation of data, and the manuscript's writing. M.P.G., G.M and MOC participated in data acquisition and the editing of the manuscript. M.E.A. supervised the development of the project, contributed to the writing, reviewed, and approved the manuscript's final submission.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The device was applied for detection of sarcosine, a potential prostate cancer biomarker, in urine for the first time. The morphology and structures of MXene have been characterised by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Electrochemical measurements, SEM and AFM analysis revealed that MXene interfaced with chitosan is an excellent support for enzyme immobilisation to fabricate a sensitive biosensor exhibiting a low detection limit of 18 nM and a linear range up to 7.8 µM. The proposed biosensing method also provides a short response time of 2 s and high recovery index of 102.6% for detection of sarcosine spiked into urine sample in a clinically relevant range.Two-dimensional layered nanomaterial Ti PSA is a glycoprotein released into the blood stream by the prostate and the protein is responsible for seminal fluid liquefaction. Since PSA is organ-specific rather than cancer-specific, the protein is released into the blood stream not only as a result of PCa development and progression, but under other conditions such as prostate inflammation. Hence, PSA cannot be used as a reliable early stage diagnostic PCa biomarker because of the high false-positive/negative results provided , 2, withTwo-dimensional (2D) nanomaterials, namely graphene , find nu3C2 MXene and haemoglobin-based device was developed for determination of H2O2 [2 [So far, only a limited number of MXenes-based biosensors have been developed . A prist of H2O2  or NaNO2 H2O2 [2 . MXene,  H2O2 [2 , acetylc H2O2 [2  and tyro H2O2 [2  for ultr3C2TX  MXene was used as an enzyme immobilisation support to construct a biosensor for sensitive detection of a small molecule—sarcosine , sarcosine (98%), glutaraldehyde solution (50 wt. % in H2O), chitosan (medium molecular weight), acetic acid, phosphate buffer (PB) components (KH2PO4 and K2HPO4) and Surine™ (a negative urine control for toxicology) were purchased from Sigma-Aldrich . Micropolish alumina powder (1.0 and 0.3 µm) and polishing pads for disc electrodes were purchased from Buehler .Sarcosine oxidase  from 2.22O2 and some other small molecules as well.MXene was prepared by in situ-formed hydrofluoric acid (HF) (reaction of lithium fluoride (LiF) with hydrochloric acid (HCl)) using a previously described protocol . This MX2.3d = 3 mm, Bioanalytical systems, West Lafayette, IN, USA) used as a working electrode. An Ag/AgCl/3 M KCl reference electrode and a counter platinum electrode  were applied in a three-electrode cell system. Chronoamperometric detection of H2O2 (at different rpm) was performed on a rotating disc electrode employed as a working electrode . Cyclic voltammetry and chronoamperometry were performed in 0.1 M phosphate buffer (PB) pH 7.4 purged with nitrogen for 15 min prior to use to eliminate the interference from an oxygen reduction reaction. Cyclic voltammetry measurements were run in the potential window from 0.1 V (volt) to -1.0 V at a scan rate of 100 mV s-1. Chronoamperometry measurements were run at a fixed potential value of -0.7 V. All electrochemical measurements were run under Nova Software 1.10 , and data acquired were evaluated using OriginPro 9.1 .All electrochemical procedures were carried out on a laboratory potentiostat/galvanostat Autolab PGSTAT 302N with an impedimetric module  with a GCE (2.4-1 in distilled water) were mixed and shaken overnight at a temperature of 20 °C and at a rotation speed of 1500 rpm. After preparation, 20 µL of the above-prepared mixture was cast onto the surface of polished GCEs. After drying, 10 µL of 5% or 1% glutaraldehyde solution was applied to the surface for 10 min. Construction and behaviour of the biosensor without using glutaraldehyde solution was also studied. Finally, 20 µL of the enzyme solution was cast onto the surface and allowed to dry at room temperature in a laminar box. Before electrochemical measurements, all the as-prepared electrodes were immersed in PB pH 7.4 to remove residual components. For the purpose of biosensor preparation and its performance, different configurations and nanocomposite preparation protocols were used  and 40 µL MXene dispersion  to obtain wetting angle and free surface energy for MXene-based interfaces. The droplet volume was 1 μL and the testing liquid was distilled water and diiodomethane. Free surface energy was determined using the two-liquid Owens–Wendt method, where the total surface energy 2.6The morphology and structure of MXene and the as-prepared MXene-chitosan mixture were characterised by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM images of MXene and the MXene-chitosan composite were recorded using a Nova NanoSEM 450  microscope, applying accelerating voltage. A peak force tapping mode atomic force microscopy  in air was carried out on a Bioscope Catalyst instrument and Olympus IX71 microscope in conjunction with NanoScope 8.15 software .3Five different configurations were tested during biosensor preparation . Among a3.1Initial scanning electron microscopy-based characterisation of MXene showed the presence of MXene flakes with the size from a few μm to tens of μm . Atomic Wetting angle and surface energy calculation using the Owens–Wendt method for two liquids (water and diiodomethane) were applied to further investigate the surface modifications during individual steps of biosensor preparation. The hydrophilicity of bare rotating glassy carbon electrode (GCE) (90.0 ± 2.3)° increased upon modification of the electrode by MXene (71.9 ±5.0)° and further by the MXene-chitosan composite (69.5 ± 3.6)°. After SOx adsorption, the biosensor surface becomes quite hydrophobic again (87.4 ± 4.9)° .3.22-purged PB pH 7.4. At this pH value, SOx has a maximum stability [2O2 on unmodified GCE and MXene-modified GCE is shown in 2O2 reduction. The effect of concentration of SOx applied for patterning of MXene-modified GCE was optimised in the range from 0.19 mg/mL to 0.77 mg/mL, with an optimal SOx concentration of 0.38 mg/mL identified.Since MXene undergoes irreversible anodic oxidation at +0.43 V , 16, cyctability . A direc2O2 produced by catalytic decomposition of sarcosine by SOx present on the surface of the electrode started at -0.4 V, with the peak maximum present at -0.7 V. The cathodic peak for H2O2 reduction increased with increasing biomarker concentration. Calibration curves were constructed by plotting current response versus sarcosine concentration in the first CV scan with subtraction of a background CV.To evaluate the bioelectrochemical activity of the biosensor and to construct the corresponding calibration curves, the final sarcosine concentration was in the range of 2.5–50 µM , left. EmaxI): (2.61 ± 0.15) μA (0% GA), (1.96 ± 0.26) μA (1% GA), and (1.42 ± 0.10) μA (5% GA). Thus, glutaraldehyde has a negative effect on the SOx activity, which can be explained by the formation of a multi-point attachment of SOx with the carrier, leading to the enzyme inactivation [Immobilisation of SOx on the modified electrode was optimised using various glutaraldehyde concentrations  and LOD=18.0 nM (S/N=3). Moreover, the biosensor exhibits a very short response time of 2 s  spiked into 10x diluted artificial urine . The value of the obtained recovery index was 102.6%. This means that under such conditions, there were no interferents affecting the biosensor’s performance. The results prove that the as-fabricated biosensor is reliable tool for sarcosine detection in urine samples.4In this paper, a MXene-based biosensing platform was successfully fabricated for indirect sensitive detection of sarcosine. The sarcosine biosensor displayed low detection limit of 18 nM and a linear range up to 7.8 µM for sarcosine, with correlation coefficient of 0.998 and a response time of 2 s. The SOx/MXene-Chi/GCE device was further used for sarcosine determination in artificial urine with a recovery index  of 102.6%. These excellent operational parameters make MXene a potential electrochemical biosensing platform for protein-based biosensors with application in biomedical detection and diagnostics. In future work, we would like to test the device for analysis of human urine samples from healthy individuals and patients with prostate cancer.Figs. S1-S5Supplementary Materials


Nilaparvata lugens. NlPPP1α and NlPPP1β are highly conserved whereas NlPPP1-Y, NlPPP1-Y1 and NlPPP1-Y2 are lowly conserved among insects. NlPPP1α and NlPPP1β exhibited a ubiquitous expression, while NlPPP1-Y, NlPPP1-Y1, and NlPPP1-Y2 were obviously detected from the 4th instar nymph to imago developmental stages in males, especially detected in internal reproductive organ and fat bodies of the male. Injection nymphs with dsRNA of NlPPP1α or NlPPP1β was able to reduce the target gene expression in a range of 71.5–91.0%, inducing a maximum mortality rate of 95.2% or 97.2% at 10th day after injection and eclosion ratio down by 65.5–100.0%. Injection with dsNlPPP1Ys targeted to NlPPP1-Y, NlPPP1-Y1and NlPPP1-Y2 was able to induce a maximum mortality rate of 95.5% at 10th day after injection, eclosion ratio down by 86.4%. Knock-down one of the male-biased NlPPP1 genes has no effect on survival and eclosion ratio. Injection of 4th instar nymph with dsNlPPP1Ys led to reduced oviposition amount and hatchability, down by 44.7% and 19.6% respectively. Knock-down of NlPPP1-Y1 or NlPPP1-Y2 gene did not significantly affect oviposition amount but significantly affected hatchability. The results indicate that the male-biased NlPPP1 genes have overlapping functions in N. lugens development, and NlPPP1-Y1 and NlPPP1-Y2 may play important roles in spermatogenesis and fertilization. The dsNlPPP1β and dsNlPPP1Ys in this study could be the preferred sequence in RNAi and low-conserved male-biased NlPPP1 genes could be potential target for N. lugens control.Ten distinct cDNAs encoding five different protein phosphatases 1 (PPP1) were cloned from  Nilaparvata lugens, Hemiptera: Delphacidae) is one of the most destructive rice pest, which causes serious damage to rice crop through phloem sap sucking and nutrient depletion1. In the last decades, the main means of pest control for N. lugens is to apply pesticides and resistant rice varieties. However, N. lugens has developed resistance against most of the insecticides and adaptation to resistant rice varieties3. With the development of molecular biology of pests, cloning and identifying the genes related to the growth and development of pests can help us to understand the virulent mechanism of pests, and also bring chances for finding new molecular targets and designing alternative control strategies.The Brown planthopper  can be classified into three groups: serine/threonine phosphatases, the protein Tyr phosphatase superfamily, and Asp-based protein phosphatases. In eukaryotic cells, eight types of serine/threonine phosphatases have been identified6. Among these, PPP1 is a ubiquitously expressed, highly conserved and abundant eukaryotic protein serine/threonine phosphatase that regulates diverse cellular processes such as cell-cycle progression, protein synthesis, muscle contraction, glycogen metabolism, transcription and neuronal signalling9. Each functional PPP1 enzyme consists of a catalytic subunit and a regulatory subunit. Catalytic subunit has three subunits-PPP1α, PPP1β and PPP1γ10, and can form as many as 650 distinct complexes with PPP1-interacting proteins11. It is estimated that around one third of all eukaryotic proteins are dephosphorylated by PPP1. The catalytic subunit of PPP1 is highly conserved among all eukaryotes, with approximately 70% or greater protein sequence identity in any pairwise alignment12. In Drosophila Melanogaster, PP1α isozyme encoded by three genes named after their respective chromosomal location: PP1α13C (FlyBase: Pp1-13C), PP1α87B (Pp1-87B), and PP1α96A (Pp1α-96A). Only one gene, PP1β9C , encodes the PP1β type14, and PpY-55A, PpN-56A, PpD5, PpD6, Pp1-Y1 and Pp1-Y2, which are Drosophila specific intron-less phosphatases with male biased expression15. In Drosophila, PPP1α is essential for mitosis17. Loss of PPP1β leads to increased levels of actin disorganization, crumpled or blistered wings19 and disrupt oocyte polarization20. PPP1 was suggested to be involved in the regulation of glycolysis which plays important roles in the internal metabolism of Spodoptera litura during metamorphosis21. PPP1 in tick play a role in modulating tick salivary secretion22. In fungal pathogen Candida albicans, PPP1 may contribute to pathogenicity23. For male biased PPP1, deletion of the testis-specific PPP1γ2 gene in mice results in defective sperm development and motility26. Also in nematode Caenorhabditis elegans, sperm specific PPP1 phosphatases are required for chromosome segregation during sperm meiosis and necessary for the ability of sperm to fertilize27. Armstrong et al. reported that a male specific protein phosphatase PPY in Drosophila may be required to prevent cyst cell division, increase transcription for provision of nutrients to the germ cells and/or provide a signal for spermatocyte differentiation28.Protein phosphorylation is a common means to the regulation of most cellular processesN. lugens has not been revealed, and whether they can be used as targets for controlling N. lugens has not been explored. In this study, we report the isolation of ten cDNA clones encoding five distinct catalytic subunits of type 1 protein phosphatases  from N. lugens. To explore a potential role for PPP1 in N. lugens, the present study examined the mRNA expression levels of the NlPPP1 during the nymph development and in diverse tissue. The effects of knockdown the expression of NlPPP1 by RNAi method were also examined. These results demonstrated that NlPPP1α, NlPPP1β and NlPPP1-Y play important roles in N. lugens development. Male biased NlPPP1-Y1 and NlPPP1-Y2 play important roles in spermatogenesis and fertilization ability. Our data also reveals NlPPP1Ys can be the preferred targets for N. lugens control by means of RNAi.In summary, PPP1 is an important functional gene for eukaryotic growth and development and metabolic regulation. However, the PPP1 family genes in Nilaparvata lugens were collected in field of China National Rice Research Institute, Fuyang, Zhejiang, China and reared on the susceptible rice variety Taichung Native1 in wire mesh cages at 27 ± 2 °C with 80 ± 5% relative humidity under a 16 h light/8 h darkness photoperiod.Adult females and males, 2 days after eclosion, were immobilized by placing them in a freezer for 15 min, and their midguts (50), salivary glands (100), fat bodies (50) and internal reproductive organs (50) were dissected with tweezers. Eggs (200) and the individuals from the day 1 of the 1st instar (100), 2nd (100), 3rd (50), 4th (20) to day 3 of the 5th instar nymphs (10) and newly emerged female (10) and male adults (10) were randomly selected respectively. The number of insects in each sample is given in parentheses above. All samples were collected in triplicate. The samples were frozen in liquid nitrogen and stored at − 80 °C prior to RNA extraction.N. lugens at different developmental stages and from different tissues using an RNeasy Mini Kit  according to the manufacturer’s instructions. The RNA was quantified and the quality verified by NanoDrop 2000 spectrophotometer . A total of 500 ng RNA was used for reverse transcription in a 10 μL reaction with the ReverTra Ace qPCR RT Master Mix with gDNA Remover Kit . Synthesized cDNA was diluted tenfold and used as template for quantitative PCR.Total RNA was isolated from N. lugens and designed using National Center for Biotechnology Information (NCBI) primer design tool (www.ncbi.nlm. nih.gov/tools/primer-blast). All the primer used in this study were synthesized by Invitrogen Co., Ltd Shanghai China and listed in Table 2.1Topo vector  and then the plasmids from positive colonies were sequenced with the M13 primer pair on ABI Prism 3100 DNA sequencer .The cDNA sequences were analysis with BLAST against N. lugens genome (Nilaparvata lugens (taxid: 108931)).The open reading frame (ORF) was determined using ORF Finder (https://www.ncbi.nlm.nih.gov/gorf/gorf.html). The translated amino acid sequence was used as a query to identify homologous proteins and compared with other PPP1 deposited in GenBank using the BLASTp tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The molecular weight (Mw) and isoelectric point (pI) of NlPPP1 were calculated by the Compute pI/Mw tool (https://web.expasy.org/compute_pi/). The phylogenetic tree of PPP1 was constructed using the maximum likelihood method with MEGA 5.0(https://megasoftware.net/). All the PPP1 sequences from N. lugens were aligned in a multiple sequences alignment using CLUSTAL X and edited with GeneDoc software. The phosphorylation sites were predicted using Netphos3.0 server (https://www.cbs.dtu.dk).The PPP1 gene was amplified with primer pairs which based on our transcriptome database from whole bodies of NlPPP1 transcript levels were quantified in tissues and development stages with specific primers for NlPPP1α, NlPPP1β, and NlPPP1-Ys which were designed based on the cDNA sequence obtained. The RT-qPCR (20 μL per reaction) used 3.0 μL cDNA template, 0.4 μL of each primer (10 mM) and 10 μL SYBR Premix . RT-qPCRs were carried out using an ABI 7,500 Real-time PCR system  in a two-step reaction  followed by a melt curve analysis at the end of the run. Each experiment consisted of 3 separate biological replicates, each of which was comprises 3 technical replicates. Relative expression levels were calculated using the 2−∆∆CT method29. The N. lugens housekeeping genes for β-actin (FJ948574) and 18S rRNA (JN662398) were used as the reference genes30. Fold induction values of target genes were calculated with the ΔΔCt equation and normalized to the mRNA level of target genes in control which were defined as 1.0.31. A PCR method using the plasmid with NlPPP1 insert as template was used to generate the templates for the dsRNA synthesis. The GFP gene (ACY56286) was used as a control. Approximately 70 ng of dsRNA was injected into each newly moulted third-instar nymphs or fourth-instar nymphs (3-day old) N. lugens. The survived nymphs in each treatment were selected and reared on 60- to70 day-old rice variety TN1 in one cage. A total of 175 nymphs  for each treatment were used for each dsRNA injection. RNAi efficiency for dsNlPPP1α and dsNlPPP1β was assessed at 4 days post injection by RT-qPCR. Total RNA was extracted from 5 nymphs sampled from each treatment and each replicate. RNAi efficiency for male-biased gene was assessed at 4 days after emergency by RT-qPCR, internal reproductive organs dissected from approximately 5 males were used in each sample.The dsRNA synthesis, microinjection experiment and bioassay was conducted based on our previously described method32. Duncan’s tests were used to determine differences between the treatment and control. Values of P < 0.05 were considered significantly, all values were expressed as mean ± SEM.The survival rates of the injected 3rd-instar nymphs were observed at 24 h intervals with duration of 10 days. Once the 4th-instar nymphs after injection emerged, each female was matched with one male and each pair was allowed to reproduce separately. For the male-biased gene, the injected male was matched with untreated female. In total, 15 single pairs per gene were successfully mated. The number of newly hatched nymphs was recorded every other day until no more nymphs were observed for two successive days. The number of unhatched eggs was also recoded under a light microscope. Eggs were scraped from the leaf sheaths and blades using a pin. All analyses were performed with the data procession system (DPS) of Tang and Fengg for 15 min at 4 °C, and the supernatants were used for total protein (TP) determination with the Insect TP Elisa Kit . The optical density was read at 450 nm on a Sunrise ELISA reader .Two days after emergence, a total of 40 male internal reproductive organs  were randomly selected and dissected from the treatment group injected with dsNlPPP1Ys and the control group injected with dsGFP. A total of 20 female internal reproductive organs  were randomly selected and dissected from females mated with injected with dsNlPPP1Ys and females mated with injected with dsGFP. The male and female internal reproductive organs were homogenized with a glass tissue grinder in 200 μL 0.8% NaCl buffer. Homogenates were centrifuged at 10,000×N. lugens. Totally 10 cDNA clones were isolated. Based on the comparative study of their nucleotide and deduced amino acid sequences with those reported, these cDNA clones were named NlPPP1α, NlPPP1β, NlPPP1-Y1, NlPPP1-Y2 and NlPPP1-Y. There are 6 transcript variants were identified for NlPPP1-Y named as NlPPP1-Y-X1-6  for NlPPP1-Y transcript variants were retained, which share common 5-UTR (582 bp in length) and ORF  and three different lengths 3-UTR . The 5-UTR is the same with NlPPP1-Y-X6.Based on the assembled transcriptome which constructed in our laboratory, five different pair of PCR primers were designed and used to clone the PPP1 gene from NlPPP1α, NlPPP1β, NlPPP1-Y, NlPPP1-Y1 and NlPPP1-Y2 contains 7, 3 and 4 exon and is located at scaffold 2,314, 31,341, 1,294, 10 and 1,537 in the N. lugens genome respectively. No intron was found in cDNA sequences of NlPPP1-Y1 and NlPPP1-Y2. The blast information, deduced amino acid lengths, molecular weights and isoelectric points of the NlPPP1 cDNA clones are illustrated in Table Analysis of the genomic position and structure showed that NlPPP1α and NlPPP1β is 66.9%, NlPPP1-Y1 and NlPPP1-Y2 is 89.5%, while NlPPP1-Y is divergent from other PPP1s which showed about 54% identity with NlPPP1-Y1 and NlPPP1-Y2  of PPP family within the catalytic domain were found. The conservative catalytic domain starting from the second α-helix (α1) and ending with the last β-strand (β14) as reported by Goldberg32 were showed in Fig. 35. In Drosophila and N. lugens, this TPPR segment is retained in DmPP1α (PP1α-96A), NlPPP1α, and NlPPP1β, but absent in DmPP1α1 (DmPP1α-87B), DmPP1α2 (DmPP1α-13C), and male-biased NlPPP1-Ys. As a characteristic of most Ser / Thr / Tyr protein kinases, a large number of potential phosphorylation site were also identified in NlPPP1, among them, there are 9 to 17 Ser sites, 5 to 9 Thr sites, and 4 to 6 Tyr sites   Fig. .Figure 3NlPPP1 in N. lugens was determined using RT-qPCR. NlPPP1α and NlPPP1β were expressed in all developmental stages and both sexes , fat bodies(FB), guts(GT), legs(LG), male internal reproductive organ(MIRO), and female internal reproductive organ (FIRO). The RT-qPCR results demonstrated that ues Fig. A, B. To ely Fig. C–E. TheiNlPPP1 genes or different region were synthesized. The dsRNA name, length, targeted position and the largest length of 100% similarity stretch between the dsRNA and NlPPP1s were listed in Table NlPPP1β, 5′-UTR of NlPPP1-Y and 5′-UTR of NlPPP1-Y1 respectively. The sequence of dsNlPPP1α showed 100% identity with 34 bp stretch in NlPPP1β. dsNlPPP1-Y2 with 78 bp stretch in NlPPP1-Y1, dsNlPPP1Ys showed over 20 bp stretch in NlPPP1-Y1 and NlPPP1-Y2. Moreover, dsNlPPP1Ys also showed over 50 bp stretch with more than 95% and 85% identity in NlPPP1-Y1 and NlPPP1-Y2 respectively.Six dsRNAs targeted to different N. lugens. The survival rate at 3rd day after injection was significantly lower in nymphs injected with dsNlPPP1α (83.1 ± 1.8%) and dsNlPPP1β (55.2 ± 3.5%) than with the dsGFP (100.0%). Ten days after injection, the survival rate of nymphs decreased to 4.8 ± 1.6% (dsNlPPP1α) and 2.8 ± 1.2% (dsNlPPP1β) .NlPPP1α and NlPPP1β transcript levels in nymphs after the injection of dsNlPPP1α at 4th day were significantly down-regulated  and by 31.9% , and the offspring significantly decreased by 55.4% (from 289 to 129) and 44.3% (from 289 to 161), relative to dsGFP-treated males mated with the control females respectively  were predicted to exhibit off-target cross-reaction44. The question of over how much length and how much similarity is necessary to observe off-target cross-reaction remains open in N. lugens. In our study, all target NlPPP1 genes were successfully silenced by using dsRNAs containing over 25 bp with 100% similarity. The efficient RNAi effect was also observed between NlPPP1-Y1 with dsNlPPP1Ys and NlPPP1-Y2 with dsNlPPP1Ys. NlPPP1-Y1 has 21 bp with 100% similarity or 54 bp with 95% similarity with the dsNlPPP1Ys. NlPPP1-Y2 has 20 bp with 100% similarity or 59 bp with 85% similarity with the dsNlPPP1Ys. RT-qPCR showed the expression of NlPPP1-Y1 and NlPPP1-Y2 reduced by 44.1% and 54.8% respectively in males injected with dsNlPPP1Ys when compared with males injected with dsGFP.RNAi silences gene expression through the production of small interfering RNAs (siRNAs). In 2. RNAi-based pest control strategies are emerging as environment friendly and species-specific alternatives for the use of conventional pesticides45. As the critical molecular switch in cell, protein phosphatase is a preferably considered target when designing RNAi-based pest control strategy as it affects numerous proteins dephosphorylation. NlPPP1-Y, NlPPP1-Y1, and NlPPP1-Y2 have low homology to known PPP1. The dsNlPPP1Ys sequence presents 100% similarity stretch with other organism is short than 10 bp. This means that the sequence of dsNlPPP1Ys varies greatly among insect species and the possibility of off-target effects is tiny. The dsNlPPP1Ys also showed ability to inducing high mortality rate, low eclosion rate and fecundity by interfering three male-biased NlPPP1 genes. In the application of RNAi to conserved genes at the cDNA level, the 3′-UTR is a good candidate sequence46. The dsNlPPP1β designed based on 3′-UTR also showed ability to inducing high mortality rate, low eclosion ratio and fecundity by silencing NlPPP1β genes. Therefore, NlPPP1-Ys would be a high efficient potential target gene used for N. lugens control. The selected dsNlPPP1β and dsNlPPP1Ys can be the preferred target sequence used for N. lugens control by means of RNAi.The overuse of conventional synthetic insecticides caused not only the serious detrimental effect on the environment but also the emergence of pest insect resistance to insecticidesSupplementary Table 1


PurposeTo evaluate the intensity modulated radiotherapy (IMRT) quality assurance (QA) results of the multichannel film dosimetry analysis with single scan method by using Gafchromic™ EBT3  film under 0.35 T magnetic field.Methods®  linear accelerator (Linac). Film dosimetry QA plans were generated for all IMRT treatments. Multichannel film dosimetry for red, green and blue (RGB) channels were compared with treatment planning system (TPS) dose maps by gamma evaluation analysis.Between September 2018 and June 2019, 70 patients were treated with ViewRay MRIdianResultsThe mean gamma passing rates of RGB channels are 97.3% ± 2.26%, 96.0% ± 3.27% and 96.2% ± 3.14% for gamma evaluation with 2% DD/2 mm distance to agreement (DTA), respectively. Moreover, the mean gamma passing rates of RGB channels are 99.7% ± 0.41%, 99.6% ± 0.59% and 99.5% ± 0.67% for gamma evaluation with 3% DD/3 mm DTA, respectively.ConclusionThe patient specific QA using Gafchromic™ EBT3 film with multichannel film dosimetry seems to be a suitable tool to implement for MR-guided IMRT treatments under 0.35 T magnetic field. Multichannel film dosimetry with Gafchromic™ EBT3 is a consistent QA tool for gamma evaluation of the treatment plans even with 2% DD/2 mm DTA under 0.35 T magnetic field presence. Onliny (SBRT) , 2, 4, 5y (SBRT) .There are different IMRT QA verification solutions which work properly under magnetic field such as Sun Nuclear ArcCHECK-MR™ with helical diode array , PTW Octavius™ 1500 with plane parallel ion chambers two-dimensional (2D) array (PTW GmbH Freiburg Germany), ScandiDos DELTA4 TM  with 2.5D diode array and Ashland Gafchromic™ EBT3  film with 28 active layer. These systems can be compared in terms of dose response under magnetic field, detector type, number of detectors, measurement resolution, detector dimension, physical design, weight and cost.eff EBT3 = 7.46; Zeff Water = 7.30) hence EBT3 does not have over-response with low-energy photons [Gafchromic™ EBT/EBT2 and EBT3 films were widely used for patient-specific dose verifications for long time -16. EBT3 photons , 12, 13. photons ,12. On t photons -16.As a consequence, physical characteristics, end-to-end (E2E) results, optical density (OD) consistency, dose distribution measurements in presence of tissue-air and tissue-lung interfaces with film and patient specific IMRT QA response of Gafchromic™ EBT2/3 films under magnetic field are interesting areas for research -22.In this study, we investigated the clinical feasibility of Gafchromic™ EBT3 film using multichannel dosimetry with single scan method for patient specific IMRT QA under 0.35 T magnetic field.Patient characteristics and treatment planningSeventy patients treated with ViewRay MRIdian® Linac at our department between September 2018 and June 2019 were included in this study. Most common treatment locations were lung (26%), abdominal (40%) and pelvic (34%) regions. Either expirium or inspirium (17 sec or 25 sec MR scanning times) breath hold high-resolution (0.3 x 0.3 cm) MR images were acquired for lung and upper abdominal regions. Furthermore, free breathing (173 sec MR scanning time) with 0.15 x 0.3 cm high-resolution MR images were acquired for patients treated for lower abdominal and pelvic tumors.Treatment planning computerized tomography (CT) images were acquired with Siemens Force 128 slice CT  using 0.15 cm slice thickness and similar MR simulation images were acquired in order to obtain electron density maps.All step and shoot IMRT plans were optimized under magnetic field . Monte CPrescription doses and fractionation schemes of each region are given in Table Prior to patient-specific QA evaluation with EBT3, basic scanning properties such as reflective or transmission scanning method, landscape or portrait scanning direction and scanning with or without glass cover were investigated but not published.Film batch calibration2 sheet dimensions were used in order to perform 2D gamma analysis of the IMRT plans with multichannel dosimetry by single scan method [2 dimensions for every lots separately. Calibration films were exposed to increasing dose values of 0 cGy, 50 cGy, 100 cGy, 200 cGy, 400 cGy, 600 cGy, 800 cGy and 1000 cGy under calibration reference conditions of SAD 90 cm at 5 cm depth inside the solid slab phantoms with 10 cm thickness under 0.35 T magnetic field. Ion chamber dose measurements were performed with PTW 31010 Semiflex 0.125 cc before calibration of film exposure procedure at the same depth in order to obtain consistent dose calibration of films [Four lots of EBT3 films with 20.32 x 25.40 cmn method ,24. One 2 field size at gantry angle 0° irradiation set up, the calibration films were exposed at maximum field size (24.4 x 27.4 cm2) of multileaf collimator (MLC) with equally weighted two opposite fields at 0° and 180° gantry angles under 0.35 T magnetic field in order to avoid dose heterogeneity of 6MV FFF energy and create larger homogenous dose exposure throughout film pieces, as shown in Figure In contrast to 10 x 10 cmCalibration function was fitted onto the calibration dose points. It is not recommended to use polynomial fit function in the range of 0 cGy to 800 cGy, whereas reciprocal linear relation between color channel value and absorbed dose fitting function was employed in equation 1 instead of polynomial curve fitting , 23, 24. (X(D) = a+(b/(D-c)) (1)where X is red, green and blue optical density, a, b and c are curve fitting coefficients and D is the dose in Gy. In addition, transmission mode with no color correction of positive film scanning was employed for all EBT3 film scans. We always used the same film scanning orientation during scanning ,12. MoreGafchromic™ EBT3 film dose calculation and measurement with single scan method2 sheet size Gafchromic™ EBT3 film was placed between 10 cm thickness slab phantoms at a depth of 5 cm and scanned with CT. Our intent to acquire a CT scan is to asses electron density map of phantom and to obtain accurate dose calculation.A 20.32 x 25.40 cmIMRT plans were projected to the phantom with the same gantry angles and plan isocenter was placed to center of EBT3 film. The recommended dose analysis for multichannel dosimetry ranges between 20 cGy and 800 cGy for EBT3 film , 12. TheIn order to create accurate and reproducible film dosimetry for IMRT QA, single scan method with multichannel dosimetry was applied for all EBT3 film QA irradiation procedures. Firstly, film sheets were cut into three strips with the consideration of same orientation of these strips and calibration films. One strip was used for film QA, second strip was unexposed for background dose and third strip was exposed for reference dose .The exposed reference film strip was used to re-scale the calibration curve function to fit the responses of that specific scan for each film lot. This eliminates the scan-to-scan variability from all sources and enables reliable absolute dosimetry .Multichannel scanning methodThe Epson 12000 XL scanner  was used in this study to scan calibration and QA films. Epson flatbed scanners consist of xenon cold light lamp. Therefore, scanner was warmed up at standby position and four consecutive preview scans of whole active scanning area were performed in order to trim light lumen homogeneity before acquiring EBT3 film scans. Lateral nonuniformity effect of flatbed scanners are well known for long time , 24. PerIt is well known that curling of films during scanning negatively affects 2D absolute dose analysis . Films cGamma analysis of IMRT QA with multichannel dosimetryFilmQA Pro 2016  with RGB multichannel dosimetry was used for each lot of EBT3 film calibration curve and patient IMRT QA. We have evaluated, calculated and measured absolute dose maps by using gamma analysis method with dose difference % (DD) and distance to agreement (DTA) criteria -25.RGB channel images were employed to establish dose, uniformity and consistency maps from 48-bit scanned films , 24. ThiEBT3 dose map and coronal dose plan were matched according to laser markers on the EBT3 film. The matching was achieved with translational and rotational fusion.Local Gamma analysis evaluation was performed for RGB channels particularly for both 2% DD/2 mm DTA and 3% DD/3 mm DTA criteria with 0 cGy threshold. The region of interest of gamma analysis comparison was evaluated by adding extra 3 cm for x and y directions to irradiated area outline of exposed film. More than 95% of passing rate was accepted as a successful QA for local gamma comparison between TPS dose calculations and film dosimetry measurements. Passing rates between 90% and 95% were defined as acceptable. Less than 90% of passing rate was accepted as failure of QA.Gamma analysisFigure The Whisker box and scatter plots of gamma evaluation results of 70 cases for both 2% / 2 mm and 3% / 3 mm criteria in terms of triple channel passing rates are shown in Figure The mean gamma passing rate is found to be 97.3% ± 2.26%, 96.0% ± 3.27% and 96.2% ± 3.14% for RGB channels, respectively in terms of 2% / 2 mm. The median passing rate is found to be 98.2%, 97.1% and 96.95% for RGB channels, respectively, in terms of 2% / 2 mm (Table The 91% of the QA analysis was found to be more than 95% passing rate with success. The 99% of QA analysis was >90% for 2% / 2 mm. The minimum gamma passing rate is found to be 87.3%, 83.2% and 84.7% for RGB channels, respectively.The mean gamma passing rate is 99.7% ± 0.41%, 99.6% ± 0.59% and 99.5% ± 0.67% for RGB channels, respectively in terms of 3% / 3 mm. The median passing rate is 99.9%, 99.8% and 99.8% for RGB channels, respectively in terms of 3% / 3 mm. The 100% of QA analysis was more than 95% passing rate for 3% / 3 mm. The minimum gamma passing rate is 97.5%, 96.9% and 96.5% for RGB channels, respectively.MRIGRT is a new robust treatment method. QA is an integral part of safe implementation of IMRT treatments under MRI guidance , 7. In tThere are couple of studies in the literature on patient specific QA under 0.35 T magnetic field. Firstly, Li et al. reported their QA findings in 34 patients. They used EDR2 film dosimetry under 0.35 T in 30 patients . They reSecondly, Ellefson et al. reported their average gamma passing rates in 19 patients as 96.9% ± 6.8% and 84.0% ± 17.5% for 3% / 3 mm and 2% / 2 mm, respectively using ArcCHECK-MR™ , 27. HowThe EBT3 film dosimetry has several advantages such as being water equivalent, easy to use, no need for correction factors, structural flexibility, isolated for water and moisture penetration, no dependency with irradiation angle and high resolution of detection with 28 active layer thickness. However, film dosimetry has some disadvantages such as inconsistency at radiochromic film characteristics, saturation time, lot batch dependency, light sensitivity, landscape and portrait scan dependency, peripheral scanner devices limitations such as xenon lamp lifetime, scanning region calibration, lateral effect and uncertainties of film dosimetry , 28-30. Reyhan et al. reported dose perturbations using EBT2 Gafchromic™ films under 1.5 T magnetic field . Their mBoth Reyhan et al. and Reynoso et al. had investigated film characteristics of EBT2 with the presence of 1.5 T or 0.35 T magnetic field prior to EBT2 was superseded by EBT3 from manufacturer. The possible reason for their findings can be explained with asymmetric clear polyester coated layer design of EBT2. On the contrary, EBT3 active layer is coated with symmetric 125 matte polyesters , 12. TheDelfs et al. used EBT3 films for measurement of response of 6 MV photons under magnetic fields in the literature . They foAs a result, the findings of our study support the findings of the authors who studied EBT3 film characteristics under 0.35 T magnetic field and also our results show that signal scan method with multichannel analysis achieved reliable results under low tesla magnetic field -22.Gafchromic™ EBT3 film dosimetry is consistent QA tool for gamma analysis evaluation of the treatment plans even with 2% DD / 2 mm DTA under 0.35 T magnetic field presence. Our results support that IMRT QA with multichannel dosimetry is a suitable tool for implementation of safe MR-guided treatments using ViewRay MRIdian® Linac.