And then BJP-2 was identified by SEC-MALLS-RI, GC-MS, methylation and NMR for the following characteristics: molecular weight of 6.42 × 10 At present, malignant tumors are one of the most serious diseases that threaten human health and life, and are classified into liver cancer, lung cancer, breast cancer, colorectal cancer and so on, according to their sites of development –3. Of thPlant polysaccharides, a polymeric sugar polymer carbohydrate consisting of more than 10 monosaccharides extracted from plants, has various pharmacological effects such as antitumor, anti-inflammatory, antioxidant, immunomodulatory and hypoglycemic –9. AmongZiziphus jujuba Mill.) contains abundant active ingredients such as polysaccharides, flavonoids, polyphenols and saponins, and thus has versatile health and medicinal values .Jujube  was obtained by using the earlier described technique which employed hot water extraction followed by a DEAE-cellulose 52 and Sephadex G-100 column . In thisZ. jujuba cv. Huizao fruits were provided by Guorentang Food Technology Co., Ltd. . HepG2 cells were obtained from Shandong Analysis and Testing Center . High glucose Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were provided from Gibco Biotechnology Co., Ltd. . Monosaccharide standards , 3--2,5-diphenyltetrazolium bromide (MTT), trifluoroacetic acid (TFA), Mitomycin C and NaBH4 were bought from Sigma-Aldrich .Bicinchoninic Acid (BCA) protein kit and protein extraction kit were purchased from Beyotime Biotechnology Co., Ltd. . The Annexin V-Fluorescein Isothiocyanate/Propidium Iodide (FITC/PI) Apoptotic Cell Detection Kit and antibodies  were obtained from Wanlei Biotechnology Co., Ltd. . All other chemicals and reagents were of analytical reagent grade.The chemical composition of BJP-2 was determined based on the previous method , 23. To 3 as a solvent and passed through a filter (0.45 μm). A DAWN HELEOS-II laser photometer  equipped with three tandem columns  was used for the determination under the following conditions: column temperature of 45°C, flow rate of 0.4 ml/min, injection volume of 100 μl, and mobile phase A of 0.1 M NaNO3. Data were acquired and processed using ASTRA6.1 .As described by previous studies , briefly5.00 mg of BJP-2 powder was transferred to a chromatographic sample bottle and then hydrolyzed by TFA  for 2 h at 121°C, after which it was repeatedly washed three times with methanol and blow-dried with nitrogen. The residue was re-dissolved in deionized water and purified through a 0.22 μm microporous filter before determination on an HPAEC-PAD  equipped with a Dionex™ CarboPac™ PA-20 chromatography column . Conditions: flow rate, 0.5 ml/min; injection volume, 5 μl; solvent system, B: ; gradient program, 95:5 V/V at 0 min, 80:20 V/V at 30 min, 60:40 V/V at 30.1 min, 60:40 V/V at 45 min, 95:5 V/V at 45.1 min, 95:5 V/V at 60 min . The dif4  for 3 h, respectively. The mixture was terminated by acetic acid (10 μl), followed by dialysis and freeze-drying. Next, the sample was dissolved by DMSO (500 μl) and then treated by methylation with NaOH (50 μl) and methyl iodide solutions for 30 min and 1 h, respectively. The target, obtained by dichloromethane extraction followed by nitrogen flow drying, was hydrolyzed by TFA  at 121°C for 1.5 h and then treated by ammonia  and NaBD4  at room temperature for 2.5 h. After termination by acetic acid (20 μl)and blowing dry under nitrogen, the resulting sample was acetylated for 2.5 h at 100°C using acetic anhydride (250 μl) and finally extracted with dichloromethane (500 μl). A gas chromatograph-mass spectrometer  equipped with a BPX70 GC column (30 cm × 0.25 mm × 0.25 μm) was employed. GC parameters: injection volume of 1 μl, splitting ratio of 10:1, carrier gas of high-purity helium, initial temperature of 140°C held for 2.0 min, program of 3°C/min, 230°C held for 3 min. MS parameters: a mode of full scan and a mass scan range setting of 30–600 m/z.As previously reported data , the BJP1H and 13C) and 2D NMR spectra  were performed by high-resolution AVANCE III 600 NMR spectrometer . After dissolving 30 mg of BJP-2 in D2O, it was added to the NMR tube, and then the spectra were recorded at 25°C.1D NMR spectra (2. The effects of BJP-2 on the viability of HepG2 cells were assayed by MTT assay (3 cells/well) were stimulated for 48 h with different concentrations of BJP-2  after cells were cultured overnight in 96-well plates. Reaching time, the medium was removed and 20 μl MTT was added to each well, and then placed in an incubator at 37°C and 5% CO2 for 4 h. After that, the cell supernatant was aspirated and 150 μl of Formanzan was added, and the optical density (OD) of the reaction solution at 570 nm was recorded on an microplate reader . Cell viability was computed as follows:DMEM medium containing penicillin (100 U/ml) with 10% FBS, 1% penicillin and 1% streptomycin was used to incubate HepG2 cells at 37°C with 5% COTT assay . HepG2 c5, referring to Section Cell culture and viability. After centrifugation  and aspiration of the supernatant, apoptosis was examined using Annexin V-FITC/PI Apoptosis Detection Kit based on the instructions. The flow cytometer  was utilized for the following analysis.Cells were cultured in 6-well plates at a quantity of 5 × 102 and then photographed and recorded. Mitomycin C (1 mg/ml) was employed to exclude the interference caused by proliferation on migration (Cells were scratched using a sterile pipette (200 μl), and then the cell surface was washed once with serum-free medium and observed and photographed under a microscope . Replaced with serum-free medium, cells were kept in an incubator for 24 h under 37°C and 5% COigration . The cel4 cells/well and placed in the incubator at 37°C. Transwell inserts were washed twice with PBS and fixed in 4% paraformaldehyde  at room temperature for 20 min. After that, they were stained with 0.5% crystalline violet  staining solution for 5 min, rinsed with distilled water, and observed under an inverted microscope (200 ×).The Transwell chambers  were placed in 24-well plates and coated with Matrigel gel  after overnight thawing at 4°C, and then incubated for 2 h at 37°C . Cell cuWestern blot analysis based on the method of literature . Brieflyp < 0.05.Results are denoted as mean ± standard deviation (SD). The significance of differences was evaluated using the one-way analysis of variance (ANOVA) followed by Tukey's test by SPSS software at 4 Da and the polydispersity index was 3.797. The BJP-2 was principally comprised of GalA (39.78%), Ara (31.93%), Gal (16.86%) and Rha (6.43%), and contained a small amount of Xyl (1.86%), GlcA (1.28%), Glc (1.02%), Fuc (0.61%) and Man (0.23%), showing that BJP-2 was a acidic polysaccharide (Z. jujuba cv. Huizao polysaccharide (HP-2), they both consisted mainly of GalA, Ara, and Gal, but possessed different percentages, and the molecular weight of BJP-2 (64.2 kDa) was smaller than that of HP-2 (111 kDa)  . This phpA was the most dominant linkage pattern (46.11%), followed by Araf (1 → (12.94%), → 4) Galp (1 → (11.60%) and → 5) Araf (1 → (8.31%), together accounting for 78.96% of the total methylated sugars. Meanwhile, some of the discrepancies between the above results and the analysis of monosaccharide composition were explained by the fact that the dialysis step of acidic sugars when undergoing pre-treatment could have an effect on the results. However, the qualitative and quantitative analyses of the monosaccharide composition of BJP-2 were reliable, and the qualitative results of the methylation results on the type of glycosidic bonds and the mode of attachment were also correct , δC 80.88/δH 4.14 ppm (C-2), δC 76.53/δH 3.95 ppm (C-3), δC 83.82/δH 4.04 ppm (C-4) and δC 67.91/δH (3.75/4.02 ppm) (C-5), respectively. Summarizing these NMR data, it was inferred that it belongs to → 5)-α-L-Araf -α-L-GalAp-β-D-Gal -β-D-Gal-β-D-Gal-α-L-6MeGalAp. Results showed that BJP-2 inhibited the growth of HepG2 cells within a certain range, and the effect was gradually enhanced with increasing concentration. All together, three optimal concentrations of BJP-2  were selected for subsequent experiments. BJP-2 inhibited tumor cell growth in a similar trend to other polysaccharides. For example, the Polygonum multiflorum polysaccharides at 400 μg/ml inhibited the growth of HepG-2 cells by 53.35%; and the mango pomace polysaccharide was active against HepG-2 cells in a quantitatively dependent manner . No significant effect of low doses of BJP-2 (200 μg/ml) was observed, which is consistent with the results of cell viability. After stimulation of HepG2 cells with BJP-2 of 800 μg/ml, the early (Annexin V-FITC+/PI–) and late (Annexin V-FITC+/PI+) apoptosis rates were 25.05 and 25.01 %, respectively, which were significantly higher than that of the control group at 2.24 and 2.90% (Triggering tumor decline by enhancing apoptosis of cancer cells is one of the roles of antitumor drugs . Therefond 2.90% . In summp < 0.01) and 17.86 ± 2.1% (p < 0.01) due to the treatment with high doses of BJP-2 (400 and 800 μg/ml). The numbers of cell invasion were 300 ± 35 and 172 ± 14 for BJP-2 at 400 and 800 μg/ml concentrations, respectively, which was significantly lower than that of 387 ± 38 for the control group (p < 0.01) (One of the key issues in treating cancer is to prevent tumor cells from invading and metastasizing . The scr < 0.01) . It was  < 0.01) . Various < 0.01) .p < 0.01). By contrast, the expression of Bcl-2 was significantly reduced (p < 0.01). Other polysaccharides with similar mode of antitumor effects were also found, such as those derived from Grifola frondosa, Boletus edulis and Ganoderma applanatum -α-L-Araf (1 → 4)-β-D-Gal-β-D-Gal-α-L-6MeGalAp, Shandong Province Key Research and Development Program  (2021TZXD011); Dezhou Health Food Industry Innovation and Entrepreneurship Community, and Shandong Land and Rural Revitalization Group Co., Ltd., Project Key Technology Integration and Industrialization Demonstration of the Whole Industry Chain for Efficient Green Production and High Value Utilization of Jujube.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Postsynaptic scaffolding proteins function as central organization hubs, ensuring the synaptic localization of neurotransmitter receptors, trans-synaptic adhesion proteins, and signaling molecules. Gephyrin is the major postsynaptic scaffolding protein at glycinergic and a subset of GABAergic inhibitory synapses. In contrast to cells outside the CNS, where one gephyrin isoform is predominantly expressed, neurons express different splice variants. In this study, we characterized the expression and scaffolding of neuronal gephyrin isoforms differing in the inclusion of the C4 cassettes located in the central C-domain. In hippocampal and cortical neuronal populations, gephyrin P1, lacking additional cassettes, is the most abundantly expressed isoform. In addition, alternative splicing generated isoforms carrying predominantly C4a, and minor amounts of C4c or C4d cassettes. We detected no striking difference in C4 isoform expression between different neuron types and a single neuron can likely express all C4 isoforms. To avoid the cytosolic aggregates that are commonly observed upon exogenous gephyrin expression, we used adeno-associated virus (AAV)-mediated expression to analyze the scaffolding behavior of individual C4 isoforms in murine dissociated hippocampal glutamatergic neurons. While all isoforms showed similar clustering at GABAergic synapses, a thorough quantitative analysis revealed localization differences for the C4c isoform . Specifically, synaptic C4c isoform clusters showed a more distal dendritic localization and reduced occurrence at P1-predominating synapses. Additionally, inhibitory currents displayed faster decay kinetics in the presence of gephyrin C4c compared with P1. Therefore, inhibitory synapse heterogeneity may be influenced, at least in part, by mechanisms relating to C4 cassette splicing. A neuron receives and integrates thousands of different synaptic inputs. At synapses of the same type, i.e., using the same neurotransmitter, subtle differences in their molecular composition could account for functional changes. Diverse synapse populations could contain different isoforms of the same protein, which are themselves generated by alternative splicing. In this work, we focused on the neuronal isoforms of gephyrin, which organizes structures at most inhibitory synapses. While overall similar properties of all isoforms were observed, an in-depth automated analysis revealed that the localization of clusters from one of the isoforms was different. Finally, this indicates that the alternative splicing of gephyrin could be one of the factors influencing the organization of postsynaptic proteins at diverse synapse populations.The postsynaptic side of neuronal synapses contains scaffolding proteins that keep neurotransmitter receptors, trans-synaptic adhesion molecules and additional functional elements with precise stoichiometries in place . GephyriA receptors (GABAARs) via protein-protein interactions and posttranslational modifications  was originally described to consist of 29 exons  in glutamatergic [Ca2+/calmodulin-dependent protein kinase II (CamKII) and sodium channel epithelial 1 subunit α (Scnn1)] and GABAergic  neurons, isolated from the cortex (SPLICECODE database). Circle and range represent mean and SD. Download The inclusion of the G2 cassette results in a dominant-negative effect on GlyR clustering and prevents the enzyme’s ability to synthesize the molybdenum cofactor . The C3 Here, we analyzed the neuronal expression and clustering of Gphn isoforms that differ in the inclusion of one of the C4 cassettes. We hypothesized that C4 isoforms could form structurally different scaffolds, which could in turn affect receptor clustering and/or synapse localization. Our results showed a relatively high abundance of C4a-containing transcripts in all major hippocampal and cortical neuron populations, while C4c and C4d were only detectable in low amounts. Interestingly, synaptic clustering in cultured neurons was similar for all C4 isoforms, suggesting that Gphn’s basic scaffolding properties remained unaltered following C4 splicing. However, an in-depth automated analysis of GABAergic synapses revealed that the addition of the C4c splice cassette resulted in more distally localized clusters. Additionally, when differentially labeled isoforms were co-expressed, we detected the largest effect between Gphn P1 and C4c variants, pointing to a unique role for the C4c-containing isoform. Functional experiments revealed that inhibitory currents displayed faster decay kinetics in the presence of gephyrin C4c compared with P1.http://n2t.net/addgene:68064; RRID:Addgene_68064) and pAAV-hSyn-mScarlet  by PCR subcloning using BamHI and EcoRI. Next, pAAV-hSyn-moxBFP and pAAV-EF1a-mCherry-IRES-Flpo  were used to generate pAAV-hSyn-moxBFP-IRES-Flpo using NEBuilder HiFi DNA Assembly (NEB). Finally, pAAV-CaMKIIa-EGFP  and pAAV-hSyn-moxBFP-IRES-Flpo were used to generate pAAV-CaMKIIa-moxBFP-IRES-Flpo using NEBuilder HiFi DNA Assembly. From this template, the control pAAV-CaMKIIa-moxBFP was generated by deleting IRES-Flpo through site-directed mutagenesis.Initially, pmEGFP-C2 was generated from pEGFP-C2 (Clonetech) by introducing the point mutation L221K through site-direted mutagenesis. Plasmids pAAV-hSyn-moxBFP and pAAV-hSyn-mEGFP were generated from moxBFP  and pAAV2/1  were a gift from James M. Wilson.The plasmids pAdDeltaF6 .in vitro (DIV) with 400 ng plasmid DNA using Lipofectamine 2000 according to the manufacturer’s instructions. After 9 DIV, cultures were infected with a total of 2 × 108 viral genome copies (GC). Recombinant AAV2/1 particles (1:1 Mix for co-infection experiments) were diluted in neurobasal medium supplemented with B-27, N-2, and L-glutamine (Thermo Fisher Scientific) before they were added to the cultures. To adjust the osmolality of the culture medium for electrophysiology experiments, 30 mm NaCl was added on DIV11.Dissociated primary hippocampal cultures were prepared from C57BL/6NRj embryos (E17.5) of either sex. After dissociation, 65,000 cells were seeded on Poly-L-lysine coated 13-mm cover slips (#1) or 75,000 cells per well of a Poly-L-lysine coated 24-well plate. Cultures were grown in neurobasal medium supplemented with B-27, N-2, and L-glutamine (Thermo Fisher Scientific). Cultures were transfected after 8 d 2O. In 10 μl, 400 ng RNA, 10 μm oligo (dT) primer, and 1 μm dNTPs were incubated at 65°C for 5 min and then chilled on ice. The RNA-mix was added to a 10 μl solution of RT buffer (Invitrogen), 25 mm MgCl2, 50 mm DTT and 1 μl SuperScript III RT . This mix was subjected to the following temperature cycle: 25°C 10 min, 50°C 60 min, 70°C 15 min, and subsequently chilled on ice. After incubation with 1 μl RNase A at 37°C for 20 min, the samples were stored at −20°C. For quantitative PCR (qPCR), 2 μl cDNA was added per well of a 384-well plate (Bio-Rad). A master mix, containing 2.5 μl ORA  mix and 0.5 μl of a 10 μm mixture of forward and reverse primer, was prepared and added to each sample. The plate was sealed with Microseal Adhesive Sealer (Bio-Rad) and spun down for 20 s at 300 × g. The plate was placed in a CFRX384 Real-Time System (Bio-Rad) and the qPCR was performed according to the ORA HighQ qPCR manual. Acquired cycle threshold (Ct) values were exported and normalized to the housekeeping control (RPL37a and Actinβ). ΔCt = Ct (gene of interest) – Ct (housekeeping gene). The fold change was calculated by dividing the 2ΔCt by 2ΔCt of the control condition.Cultures in 24-well plates were washed with PBS, harvested with a cell scraper, and resuspended in TRI reagent (Sigma). RNA was separated from other cellular components according to the manufacturer’s protocol and resuspended in 15 μl dHRecombinant AAV2/1 particles were prepared in HEK293T cells (DSMZ no. ACC 635) according to the detailed protocol by . Viral p4Cl in PBS at room temperature (RT). All subsequent incubation and wash steps (PBS) were performed at RT. After blocking/permeabilization for 1 h with 10% goat serum, 1% BSA, 0.2% Triton X-100 in PBS, the following primary antibodies (Synaptic Systems) were used: anti-vesicular GABA transporter (vGAT)  for inhibitory presynaptic terminals; anti-GABAAR γ2 , anti-GABAAR α1 , and anti-GABAAR α2  for postsynaptic GABAARs. The following secondary antibodies were used: goat anti-rabbit AlexaFluor 488 , goat anti-rabbit AlexaFluor 647 , and goat anti-guinea pig AlexaFluor 647 secondary antibodies . Coverslips were mounted with Mowiol/Dabco.Cells were fixed with 4% formaldehyde in PBS for 15 min and quenched with NHImage stacks  were acquired on a Leica TCS SP8 LIGHTNING upright confocal microscope with an HC PL APO CS2 63×/1.30 glycerol objective, equipped with hybrid detectors (Leica HyD) and the following diode lasers: 405, 488, 552, and 638 nm. LIGHTNING adaptive deconvolution using “Mowiol” setting were applied. This form of adaptive image reconstruction is capable of theoretical resolutions down to 120 nm  and 200 nm .https://imagej.net/software/fiji/) and the code is freely available online at https://github.com/FilLieb/quantitative_synapse_analysis. Maximum Z projections were used for image segmentation. Rolling ball background subtraction, median filtering, and auto thresholding methods were used: Default (for vGAT and GABAARɣ2 masks), Otsu , and mean (for neuron mask). Gephyrin cluster masks were generated by thresholding with local maxima identification using the ImageJ/FIJI find maxima and segmented particles function  using a water-immersion objective . The microscope was equipped with fluorescence and infrared differential interference contrast optics . Red flu−1 with a carbonated (95% O2 and 5% CO2) extracellular saline containing (in mm): 125 NaCl, 21 NaHCO3, 2.5 KCl, 2 MgCl2, 1.2 NaH2PO4, 10 HEPES, 5 glucose, adjusted to pH 7.2 with NaOH. For the voltage clamp recordings, glutamatergic input was blocked with 5 × 10−5m DL-2-amino-5-phosphonopentanoic acid  and 10−5m 6-cyano-7-nitroquinoxaline-2,3-dione . Na+ mediated action potentials were blocked by 10−6m tetrodotoxin . Current-clamp recordings were performed with a pipette solution containing (in mm): 140 K-gluconate, 10 KCl, 2 MgCl2, 10 HEPES, 0.1 EGTA, adjusted to pH 7.2 with KOH. For voltage clamp recordings of miniature IPSCs (mIPSCs), the patch pipette solution contained (in mm): 133 CsCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA, adjusted with CsOH to pH 7.2. The liquid junction potential between intracellular and extracellular solution for current-clamp (14.6 mV) and voltage-clamp (6.3 mV) recordings were calculated using the LJPcalc software  and compensated accordingly.During the recording, neurons were continuously superfused at a flow rate of ∼2.5 ml minA receptor-mediated currents were detected as inward currents. Data were analyzed at 50 s intervals after the recording stabilized (∼10 min after obtaining whole-cell configuration). Data analysis was performed with Spike 2 , Igor Pro 6 , and GraphPad Prism . mIPSC frequency, amplitude, and decay were determined offline. Events in voltage clamp traces were automatically detected by the Minhee Analysis program . All other data were analyzed with the indicated parametric tests. For imaging experiments, N = 15 cells per condition from three independent cultures were analyzed. For electrophysiology experiments, N = 12 for P1 and N = 13 for C4c cells from five independent cultures were analyzed.Individual data points, mean, confidence intervals (CIs), and standard deviation are displayed in the figures. Estimation statistics and plots were generated with the R package DABESTR . Data wehttps://www.rstudio.com/), Platform: x86 64-w64-mingw32/x64 (64-bit). Figures were prepared with OMERO (https://www.openmicroscopy.org/) and Inkscape (https://inkscape.org/).Statistical analyses and data visualization were performed using R version 4.1.0 (2021–05-18), lsmeans v. 2.30–0, emmeans v. 1.7.2, MASS v. 7.3–54, rstatix v. 0.7.0, ggpubr v. 0.4.0, svglite v2.0.0, RcolorBrewer v. 1.1–2, ggrepel v. 0.9.1, forcats v. 0.5.1, stringr v. 1.4.0, dplyr v. 1.0.7, purrr v. 0.3.4, readr v. 2.0.0, tidyr v. 1.1.3, tibble v. 3.1.3, ggplot2v. 3.3.5, tidyverse v. 1.3.1, rio v. 0.5.27, pacman v. 0.5.1, dabestr v.0.3.0 in Rstudio environment .https://www.addgene.org/): pAAV-hSyn-fDIO-mScarlet-Gphn_P1 (Addgene plasmid # 194972), pAAV-hSyn-fDIO-mScarlet-Gphn_C4a (Addgene plasmid # 194973), pAAV-hSyn-fDIO-mScarlet-Gphn_C4c (Addgene plasmid # 194974), pAAV-hSyn-fDIO-mScarlet-Gphn_C4d (Addgene plasmid # 194975), pAAV-hSyn-fDIO-mEGFP-Gphn_P1 (Addgene plasmid # 194978), pAAV-CamKIIa-moxBFP-WPRE (Addgene plasmid # 194976), pAAV-CamKIIa-moxBFP-IRES-Flpo (Addgene plasmid # 194977).Plasmids will be made available via Addgene  in neurons, we retrieved their normalized expression (reads per kilobase million) from the SPLICECODE database  and glutamate receptor kainate type subunit 4 (Grik4)] and GABAergic [somatostatin (SST)] neurons, transcript expression of 27 exons was detected . Consequently, the remainder of transcripts consisted of 10% G2 (9% in cortex), 1% C3 (1% in cortex), 19% C4a (21% in cortex), 2% C4c (4% in cortex), and 4% C4d (6% in cortex). Overall, these data suggest that in the major neuron types in the murine hippocampus and cortex in vivo , most Gpn levels .in vitro (DIV), those containing C4a, C4c, or C4d increase after 7 DIV. After 9 and 11 DIV, all transcript levels plateaued  to fill the entire cell. The expression of this construct was restricted to glutamatergic cells through the use of the CamKIIα promoter . Next, FARɣ2 clusters of ∼90% and ∼80% with vGAT clusters. No differences between the isoforms were observed (ARɣ2-positive) clusters were different among the C4 isoforms  from 60 cells (15 per condition from three independent cultures) were detected and analyzed for various parameters. All C4 isoforms showed an average colocalization with GABAobserved a,b. Furtisoforms a. Additiisoforms b.10.1523/ENEURO.0102-22.2022.f3-1Extended Data Figure 3-1a, Cumulative frequency of size for all detected synaptic (vGAT-positive and GABAARɣ2-positive) Gphn clusters. b, Number (#) of synaptic (vGAT-positive and GABAARɣ2-positive) Gphn clusters per cell are similar between isoforms; Kruskal–Wallis test, H(3) = 7.17, p = 0.067. Each data point represents the mean for an individual neuron. The filled curves indicate the resampled Δ distribution (5.000 bootstrap samples) derived from the observed data. The Δ is indicated by the black circle. The 95% confidence interval of the mean difference is illustrated by the black vertical line. Download Figure 3-1, EPS file.Quantitative analysis of synaptic mScarlet-Gphn C4 isoform clusters. AR clustering. When we selected synaptic GABAARɣ2 clusters (vGAT-positive), larger clusters were observed at Gphn C4a compared with Gphn C4d, while all other comparisons did not reveal differences  GABAARɣ2 clusters at mScarlet-Gphn C4 isoform clusters. b, Cumulative frequency of average fluorescence intensity for all detected synaptic (vGAT-positive) GABAARɣ2 clusters at mScarlet-Gphn C4 isoform clusters. c, Cumulative frequency of size for all detected synaptic (vGAT-positive) GABAARɣ2 clusters at mScarlet-Gphn positive or negative clusters. d, Cumulative frequency of average fluorescence intensity for all detected synaptic (vGAT-positive) GABAARɣ2 clusters at mScarlet-Gphn positive or negative clusters. Download Figure 4-1, EPS file.Quantitative analysis of GABAARɣ2 clusters at Gphn C4a were larger compared with clusters at C4d.Altogether, the data show that the basic scaffolding properties and association with GABAergic synapses of C4 isoforms exhibited only minor changes by C4 cassette insertion in cultured hippocampal glutamatergic neurons. The size of GABAARɣ2 clusters localized at isoform C4c compared with P1 and C4a, while the difference between C4c and C4d was not statistically significant (Extended Data To investigate the cellular distribution of synaptic Gphn clusters (vGAT-positive), we measured their distances to the neuron’s center. We observed significantly larger distances for isoform C4c compared with P1 and C4a, while the difference between C4c and C4d was not significant a. In agr10.1523/ENEURO.0102-22.2022.f5-1Extended Data Figure 5-1a, Distances of GABAARɣ2 (Gphn-positive) clusters from the center of the neuron’s soma are increased for isoform C4c compared with P1 and C4a F = 4.54, p = 0.006, Tukey’s post hoc test; *p < 0.05. b, The fraction of synaptic (vGAT-positive and GABAARɣ2-positive) Gphn clusters are changed for C4a, C4c, and C4d. Data were analyzed with a two-way mixed ANOVA. There was a statistically significant two-way interaction between Gphn isoform and distance on fraction of synaptic clusters, F = 4.683, p = 0.002. Considering the Bonferroni adjusted p-values, the simple main effect of distance was significant at C4a (p < 0.0001), C4c (p < 0.001), and C4d (p = 0.0014) but not at P1 (p = 0.352). Pairwise comparisons show that the mean fraction of synaptic clusters was significantly different in <20 versus 20–40 μm (p < 0.001) and 20–40 versus >40 μm (p = 0.0014) comparison at C4a; in <20 versus >40 μm (p < 0.001) and in 20–40 versus >40 μm (p < 0.001) at C4c; in <20 versus >40 μm (p = 0.0010) and in 20–40 versus >40 μm (p = 0.0017) at C4d. Each data point represents the mean for an individual neuron. The filled curves indicate the resampled Δ distribution (5.000 bootstrap samples) derived from the observed data. The Δ is indicated by the black circle. The 95% confidence interval of the mean difference is illustrated by the black vertical line. Download Figure 5-1, EPS file.Quantitative analysis of cluster distances. p < 0.01). Specifically, the fraction of synaptic clusters was reduced for C4c versus C4a for the somatic and perisomatic clusters (r < 20 μm) and increased for C4c versus P1 and C4a for distally located clusters  and distally located clusters (r > 40 μm) compared with the intermediate location (20–40 μm). Both synaptic C4c and C4d clusters showed a higher fraction of distally located clusters (r > 40 μm) compared with the other locations , 20 to 40 μm radius and >40 μm radius . The analysis revealed that the fraction of synaptic clusters at these distances differed for the Gphn isoforms  compared with all other conditions  synaptic (vGAT-positive) mEGFP-tagged Gphn P1 clusters from 60 cells (15 per condition from three independent cultures) were inspected. First, we studied whether P1 clustering was affected by additional C4 isoform expression. Neither cluster size nor intensity of mEGFP-Gphn P1 was altered in the presence of mScarlet-Gphn P1, C4a, C4c, or C4d a,b. Nextnditions c. This rnditions d and is nditions c,d, show10.1523/ENEURO.0102-22.2022.f7-1Extended Data Figure 7-1a, Cluster sizes (average per cell) of synaptic (vGAT-positive) C4 isoform clusters remain unchanged upon P1 co-expression. F = 1.75, p = 0.167. Each data point represents the mean for an individual neuron. The filled curves indicate the resampled Δ distribution (5.000 bootstrap samples) derived from the observed data. The Δ is indicated by the black circle, which is horizontally aligned with the mean of the test group. The 95% confidence interval of the mean difference is illustrated by the black vertical line. b, Individual fluorescence intensities at synaptic (vGAT-positive) Gphn P1 clusters. Data were analyzed with Spearman correlation. P1 ρ = 0.92 (95% CI [0.91–0.93]), p < 0.001; C4a ρ = 0.92 (95% CI [0.91–0.93]), p < 0.001; C4c ρ = 0.78 (95% CI [0.75–0.80]), p < 0.001; C4d ρ = 0.86 (95% CI [0.84–0.87]), p < 0.001. Download Figure 7-1, EPS file.Quantitative analysis of simultaneously expressed mEGFP-tagged Gphn P1 and mScarlet-tagged Gphn C4 isoforms. Together, our results imply that the subcellular distribution of Gphn C4c shows differences from the other C4 isoforms. On average, fewer Gphn C4c molecules get recruited to P1 predominating clusters. The fluorescence intensities of the corresponding isoforms at each individual synaptic cluster demonstrate this relationship in detail. Spearman correlation analysis revealed monotonic relationships between mEGFP and mScarlet fluorescence intensities. Highest correlation coefficients were obtained for P1 ρ = 0.92 (95% CI [0.91–0.93]) and C4a ρ = 0.92 (95% CI [0.91–0.93]), while the coefficient for C4d was smaller ρ = 0.86 (95% CI [0.84–0.87]) and the smallest for C4c ρ = 0.78  2/1 carrying Flp dependent mScarlet-tagged Gphn P1 or C4c variants as transgene. Cells were immunostained for GABAARα1 and GABAARα2 after 15 DIV. Representative confocal images (with adaptive image reconstruction) of neurons expressing moxBFP-IRES-Flpo and mScarlet-tagged Gphn. Scale bars: 25 μm and 2.5 μm in insets. b–e, Estimation plots of GABAARα1 and GABAARα2 cluster sizes and fluorescence intensities at P1 and C4c clusters. b, GABAARα1 cluster sizes (average per cell) are not different at P1 and C4c clusters; unpaired two-samples Wilcoxon test, W = 128, p = 0.539. c, GABAARα1 cluster intensities (average per cell) are not different at P1 and C4c clusters; unpaired two-samples Wilcoxon test, W = 105, p = 0.775. d, GABAARα2 cluster sizes (average per cell) are not different at P1 and C4c clusters; unpaired two-sample t test t(28) = 1.296, p = 0.206. e, GABAARα2 cluster intensities (average per cell) are not different at P1 and C4c clusters; unpaired two-samples Wilcoxon test, W = 102, p = 0.683. Each data point represents the mean for an individual neuron. The filled curves indicate the resampled Δ distribution (5.000 bootstrap samples) derived from the observed data. The Δ is indicated by the black circle, which is horizontally aligned with the mean of the test group. The 95% confidence interval of the mean difference is illustrated by the black vertical line. Download Figure 8-1, TIF file.Expression and analysis of mScarlet-tagged Gphn P1 and C4c isoforms in CamKIIα-expressing murine hippocampal neurons stained for GABAARɣ2 clusters were increased at exogenous Gphn clusters  contains the cassette G2 (SPLICECODE database), which alters the protein’s trimerization behavior and molybdenum cofactor biosynthesis . This caThe presence of ∼1% C3-containing transcripts (SPLICECODE database) could be because of imperfect NOVA splicing . In addiThe remaining 28% of transcripts differ in their C4 cassettes, with the majority of transcripts containing C4a (20%) and minor amounts having C4c (3%) or C4d (5%) incorporated (SPLICECODE database). This implies that the majority of neuronal alternatively spliced gephyrin transcripts are generated through differential incorporation of C4 cassettes. These insertions might provide additional protein-protein binding sites, change binding affinities, or alter the structure, thereby modifying the function of the protein isoforms.The C4a cassette translates into 19 residues . In a reAR function is differently affected by C4c versus P1 scaffolding.Three different residues within the 14 amino acid long C4c cassette can be phosphorylated; in a bioinformatic prediction, multiple kinases such as PKA, PKC, CamKII, GSK3, RSK, and cdc2 have been identified as potential candidates . The intSimilarities between the 21 residue C4d sequence and the α-helical dimerization domain 1B of the human keratin 8 were proposed as an additional binding site for protein-protein interactions . In all Limitations of our study are the exogenous Gphn expression and the use of dissociated cultured hippocampal neurons. While artificial Gphn aggregation, which is well-known in the field, was reduced with our AAV expression system, we still introduced additional transcript copies. These could promote the association of exogenously expressed Gphn molecules to any preexisting synaptic cluster rather than to a specifically-targeted synapse, simply because of increased protein levels. While our P1-targeting approach underline this view, the results demonstrate that C4c-containing variants were less abundant at P1 clusters, which is in line with the preferred distal dendritic localization of this variant.in vivo.Regarding a second limitation, dissociated neuron cultures may not retain their full set of naturally occurring synaptic partners. For instance, different types of interneurons can preferentially form synapses in specific regions of the postsynaptic neuron; SST-expressing neurons predominantly from synapses on dendrites, while parvalbumin-expressing neurons form synapses in the perisomatic region . While sImportantly, in our experimental paradigm, exogenous Gphn expression started roughly after DIV11, which is later than the onset of synaptogenesis . TherefoOverall, cassette exon splicing of the C4 cluster occurred in GABAergic and glutamatergic neurons of the hippocampus and cortex (SPLICECODE dataset) and other studies have suggested that this is predominantly a neuron-specific process . Exogeno


Several recent studies have found that Osteocalcin (OCN), a multifunctional protein secreted exclusively by osteoblasts, is beneficial to glucose metabolism and type 2 diabetes mellitus (T2DM). However, the effects of OCN on islets function especially islet ɑ cells function in patients with type 2 diabetes mellitus characterized by a bi-hormonal disease are still unclear. The purpose of this cross-sectional study was to investigate the relationship between serum OCN and the secretion of islet β cells and ɑ cells in Chinese patients with type 2 diabetes mellitus.204 patients with T2DM were enrolled. Blood glucose , insulin , C-peptide , and glucagon  levels were measured on 0 h, 0.5 h, 1 h, 2 h, and 3 h after a 100 g standard bread meal load. Early postprandial secretion function of islet β cells was calculated as Δcp0.5h = CP0.5-FCP. The patients were divided into low, medium and high groups  according to tertiles of OCN. Comparison of parameters among three groups was studied. Correlation analysis confirmed the relationship between OCN and pancreatic secretion. Multiple regression analysis showed independent contributors to pancreatic secretion.ins3h) in T3 Group were significantly lower than T1 Group . GLA1h in T3 group was lower than T1 group (p < 0.05), and GLA0.5 h in T3 group was lower than T2 and T1 groups (p < 0.05). Correlation analysis showed OCN was inversely correlated with Homeostatic model of insulin resistance (HOMA-IR), INS3h, AUCins3h (p < 0.05), and was still inversely correlated with FCP, GLA0.5 h, GLA1h, area under the curve of glucagon (AUCgla3h)  after adjustment for body mass index (BMI) and alanine aminotransferase (ALT). The multiple regression analysis showed that OCN was independent contributor to Δcp0.5h, GLA0.5h and GLA1h .FBG, and PBG2h were the lowest while Δcp0.5h was the highest in the highest tertile group . INS3h, area under the curve of insulin (AUCHigher serum OCN level is closely related to better blood glucose control, higher insulin sensitivity, increased early-phase insulin secretion of islet β cells and appropriate inhibition of postprandial glucagon secretion of islet ɑ cells in adult patients with type 2 diabetes mellitus. Type 2 diabetes mellitus (T2DM) is a multifaceted disease and the regulation of glucose metabolism relies on the interplay of multiple hormones that act in many target organs. In 1975 Unger et al. .Osteocalcin (OCN) is a multifunctional protein secreted exclusively by osteoblasts . There a−/− mice which is a gain-of-function model of osteocalcin activity, display increased insulin secretion, and reduced fat mass /22.5. Homeostasis model of β cell function (HOMA-β) = 20 × fasting insulin (mIU/L)/(fasting plasma glucose(mmol/L)-3.5); insulin and C peptide at all time points, AUC for insulin and C-peptide in 3 h  years. The BMI of all patients was 25.28 .p > 0.05). Grouped by BMI, serum OCN in BMI < 24 kg/m2 Group was significant higher than 24 kg/m2 ≤ BMI < 28 kg/m2 group and BMI ≥ 28 kg/m2 group  , age (youth group (18–44 years old), middle-aged group (45–59 years old), and elderly group (≥ 60 years old)) and diabetes duration (diabetes duration ≤ 5 years and diabetes duration > 5 years), there was no difference in osteocalcin levels between the groups . Serum uric acid (SUA) is lower in T2 group (14.02 ≤ OCN < 19.78 ng/mL) than group T1 (299.08 ± 76.35 vs 340.91 ± 104.92 p < 0.05). There were no significant differences in gender composition, age, diabetes duration, systolic blood pressure (SBP), diastolic blood pressure (DBP), alanine aminotransferase (ALT), aspartate aminotransferase AST, urea nitrogen (UN), creatinine (Cr), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), HbA1c among three groups  (Table As Table p < 0.05). PBG2h and AUCglu3h was lower in T3 group than group T2 and group T1  . Make pairwise comparison of those indexes and discover that the INS3h, FCP, AUCins3h in T3 Group were significantly lower than T1 Group  vs. 27.0 ; 1.26  vs. 1.85 ; 53.45  vs. 78.48 , p < 0.05), while Δcp0.5h in T3 Group were significantly higher than T1 Group and T2 Group  vs. 0.36  vs. 0.24 , p < 0.05). There were no significant differences in HOMA-β, INS0.5h, INS1h, INS2h, CP0.5h, CP1h, CP2h, CP3h, AUCcp3h among three groups. . Compared with the T1 group, GLA1h in the T3 group was lower . GLA0.5h in T3 group was lower than T2 and T1 groups . There were no significant differences in GLA0, GLA2h, GLA3h, AUCgla3h among three groups. . Serum OCN was also inversely correlated with FINS, FCP, AUCins3h . Meanwhile, Serum OCN was inversely correlated with alpha cell secretory function indexes such as GLA0.5h, GLA1h, AUCgla3h . The test also explored that serum OCN was inversely correlated with HOMA-IR   remained significant after adjustment for age, sex, the disease durations, BMI and serum UN, serum Cr, SUA, ALT, AST by partial correlation analysis  OCN and UN were independent contributors to Δcp0.5h , and the other variables such as sex, age, BMI, duration of diabetes, SBP, DBP, ALT, AST, Cr, SUA, TG, TC, HDL, LDL, HbA1c, FBG and PBG0.5h failed to enter the final model; (2) OCN, age and ALT were independent contributors to GLA0.5h ,and the other variables such as sex, BMI, duration of diabetes, SBP, DBP, AST, UN, Cr, SUA, TG, TC, HDL, LDL, HbA1c, FBG and PBG0.5h failed to enter the final model; (3) OCN was independent contributor to GLA1h , and the other variables such as sex, age, BMI, duration of diabetes, SBP, DBP, ALT, AST, UN, Cr, SUA, TG, TC, HDL, LDL, HbA1c, FBG,PBG0.5h and PBG1h failed to enter the final model (Table A growing number of clinical studies have observed that osteocalcin is associated with blood glucose control and metabolic syndrome , 7. Our In the present cross-sectional study most of the patients were overweight or obese type 2 diabetes, and insulin resistance is significant in this population. There were no significant differences in serum osteocalcin levels among groups by sex, age and duration of diabetes. This was different from previous studies that serum osteocalcin may be affected by gender and age , 36, andSerum osteocalcin in normal weight patients with type 2 diabetes was significantly higher than in overweight or obese patients with type 2 diabetes. The serum osteocalcin level was significantly negatively correlated with BMI and HOMA-IR. The relationship between BMI and OCN has been confirmed by multiple studies in different populations. A meta-analysis of 28 cross-sectional studies found an overall significant inverse association between serum osteocalcin and BMI in healthy adults, especially in metabolic syndrome patients . MoreoveSimilar to previous studies , 38, 39,In the highest serum OCN levels group, fasting insulin, postprandial plasma insulin at the third hour and area under 3-h insulin curve of 100 g standard bread experiment were significantly reduced, as well as FCP. Therefore, this reduced fasting, postprandial and total insulin secretion, alleviated hyperinsulinemia in overweight or obese type 2 diabetes. Conversely, early pancreatic beta cell secretion, Δcp0.5h, was significantly increased in the highest OCN group, and multiple regression analysis showed that serum OCN was independent contributors to Δcp0.5h. This is consistent with the results of previous studies , 40, 41.Our study still has certain limitations. This is only a cross-sectional observational study. The number of cases included is not enough, and all participants are inpatients, so there may be deviations in the study results. Prospective studies can be carried out in the later recruiting a wider population to further clarify the effects of serum osteocalcin on islet α cells secretion function and treatment of diabetes, further more cytological experiments will be done to explore the specific action mechanism of osteocalcin on islet α cells.In summary, this research has showed higher serum OCN level is closely related to better blood glucose control, higher insulin sensitivity, increased early-phase insulin secretion of islet β cells and appropriate inhibition of postprandial glucagon secretion of islet ɑ cells in adult patients with type 2 diabetes mellitus.


In this study, the effects of perfusion culture strategies when using different media on the process stability, product titer, and product quality were investigated in 3-L bioreactor. The results indicated that continuous perfusion could maintain higher levels of cell density, product titer, and quality in comparison with those of the intermittent perfusion culture. Next, the perfusion culture conditions with different perfusion rates and temperature reduction methods were further optimized. When combining the high perfusion rates and delayed reduction of culture temperature at day 6, the product titer reached a higher level of 16.19 g/L with the monomer relative abundant of 97.6%. In this case, the main peak of the product reached 56.3% and the total N-glycans ratio was 95.2%. To verify the effectiveness of the optimized perfusion culture in a larger scale, a 200-L bioreactor was used to perform and the final product titer reached the highest level of 16.79 g/L at day 16. Meanwhile, the product quality  could also be well maintained. This study provided some guidance for the high-efficient production of monoclonal antibody by CHO cells  Recently, monoclonal antibodies (mAbs) and recombinant biopharmaceutical proteins (rBPs) have revolutionized the pharmaceutical industry . Since 22 partial pressure (pCO2), and perfusion rate could affect the performance of recombinant protein production in CHO cells , and 1.00 g/kg poloxamer 188 (HyCloneTM). The feed medium I (FM-I) consists of 79.60 g/kg CD efficient feed C AGT nutrient supplement , 15.00 g/kg sheff-CHO plus PF ACF , and 13.30 g/kg 10 N NaOH solution. The feed medium II (FM-II) consists of 4.75 g/kg L-tyrosine, and 7.95 g/kg L-cystine. The chemical composition of basic medium II (BM-II) was 24.30 g/kg CD CHO AGT , 0.58 g/kg glutamine, 15.81 mg/kg hypoxanthine monosodium, and 3.88 mg/kg thymidine. The basic medium III (BM-III) consists of 22.23 g/kg Eden B600S, 0.58 g/kg glutamine, 15.49 mg/kg hypoxanthine monosodium, 3.80 mg/kg thymidine, and 2.13 g/kg NaHCO3. The feed medium III (FM-III) consists of 177.05 g/kg Eden F600aS. The feed medium IV (FM-IV) consists of 71.84 g/kg Eden F600bS and 25.70 g/kg NaOH. The basic medium IV (BM-IV) consists of 22.31 g/kg Eden B501S, 0.58 g/kg glutamine, 15.49 mg/kg hypoxanthine monosodium, 3.80 mg/kg thymidine, and 2.18 g/kg NaHCO3. The feed medium V (FM-V) consists of 179.69 g/kg Eden F500aS. The feed medium VI (FM-VI) consists of 77.47 g/kg Eden F200bS and 29.05 g/kg NaOH. The feed medium VII (FM-VII) consists of 20.01 g/kg Eden B600S, 0.52 g/kg glutamine, 13.49 mg/kg hypoxanthine monosodium, 3.42 mg/kg thymidine, 1.92 g/kg NaHCO3, and 17.71 g/kg Eden F600aS. The commercial media including Eden B600S, Eden F600aS, F600bS, Eden B501S, Eden F500aS, Eden F200bS, and Eden B501S were purchased from Bio-Engine Co., Ltd., Shanghai, China.The CHO cell line, which could produce anti-PD-1 mAb, was used in this work. Here, several media were chosen for CHO cells culture. The chemical composition of basic medium I (BM-I) was 24.30 g/kg Dynamics serum-free medium , 15.49 g/kg hypoxanthine monosodium, 3.80 mg/kg thymidine, 0.57 g/kg glutamine, 0.70 g/kg cell boost 5  concentration was maintained at 40 ± 15% of air saturation. The initial culture temperature was controlled at 36.5°C during the growth stage and then decreased to 31.0°C at day 5  or day 6 (run #6 and runs #8-#9). The cultures were ended at day 14 or day 16. The perfusion rates  and culture conditions of runs #1-#8 were set as shown in In this study, eight runs (#1-#8) were performed by using 3-L bioreactor  for the optimization of perfusion culture strategy to produce anti-PD-1 mAb. Furthermore, run #9 was performed by using a 200-L bioreactor  for verifying the effectiveness of perfusion culture for mAb production with a larger scale. The alternating tangential flow filtration (ATF) technology was used to develop a concentration culture by using the hollow fiber membranes (50 kDa). The schematic diagram of the CHO perfusion culture for the production of anti-PD-1 mAb was shown in CHO cells were sampled from bioreactor every day, and the cell density and viability were then measured with an automatic cell counter . The mAb titer was measured by a POROS™ A20 column  with an HPLC system (Agilent 1260 Infinity II). Antibody purity and aggregation analysis were performed by the analytical size-exclusive chromatography  with the column of TSKgel G3000SWXL  . The chaThe oligosaccharide profiles were ascertained for the quantitative assessment of the mAb products by an HPLC-based method . For simp < 0.05.In this study, to ensure the data accuracy and reproducibility, all analytical methods used were validated prior to the measurements. The data are represented as mean ± SD of three biological independent experiments. Significant differences between perfusion cultures were confirmed by a two-tailed Student’s t-test using Microsoft Excel 2016. The statistical significance was considered as 2 during perfusion cultures by using different media in 3-L bioreactor were shown in 6–17×106 cells/mL although using different media for perfusion culture  generally occupies high ratio over the total production cost. Therefore, several cultures using relative low-price medium to replace the high-price and commercial media  were performed in a 3-L bioreactor to improve the overall economics. In this study, some culture media purchased from Bio-Engine Co., Ltd.  with low-prices were selected as the perfusion culture media for evaluation of the production performance of anti-PD-1 mAb. The perfusion culture conditions for runs #1-#4 were summarized in  culture . When uscells/mL . In addicells/mL . The rescells/mL . As show2 levels were shown in Besides CHO cells growth, the key metabolites including glucose and lactate in culture broth were also analyzed. The glucose concentration during the period of day 6–14 in run #1 ranged 3.73–4.53 g/L, which was lower than that of runs #2-#4 . As showp < 0.05, The profiles of mAb titer, monomer and aggregate relative abundance, variant contents, and percentages of N-glycans under perfusion cultures of run #1-#4 were displayed in In order to improve the overall performance of mAb production, several cultures of runs #5-#8 were further performed, and the culture conditions/perfusion rates were summarized in 6 cells/mL at day 7 and final VCD was 45.6×106 cells/mL at day 16. In contrast, when reducing the culture temperature to 31.0°C from day 5 to day 6 (run #6), the VCD of CHO cells was higher than that of run #5 during the period of day 8–16. It was found that adjustment of the perfusion rates in day 2–6 (run #7) could not increase the VCD of CHO cells compared with runs #5-#6 . The results revealed that the selected perfusion media  could not affect the aggregation relative abundance significantly. The contents of main peak, along with the acidic and basic variants produced from the four perfusion runs were shown in p > 0.05). When increasing the perfusion rate in run #8, the acidic variant also maintained 23.8% with p > 0.05, while the basic variant was reduced to a lower level of 19.9% (p < 0.05). Compared with the main peak in run #5 (53.8%), the contents of main peak was 54.9% in run #6 (p > 0.05), 55.1% in run #7 (p > 0.05), and 56.3% in run #8 (p < 0.05), respectively. Furthermore, the biological and physiological properties of proteins could be affected by N-glycans. As shown in The mAb production obtained from runs #5-#8 was shown in Generally, it is considered that large-scale bioproduction is the key to a successful biopharmaceutical manufacturing. However, the mAb production performance under large-scale bioreactor was not desirable due to the lower cell viability and productivity . Here, t6 cells/mL at day 5 rapidly and then the cells growth rate was reduced gradually. The highest VCD reached 73.0×106 cells/mL at day 7, which was lower than that of run #8 . It was mainly attributed to the higher lactate accumulation . Furthermore, the overall production performances of different cultures including run #1, #2, #8, and #9 were summarized in The mAb production profiles were displayed in 6 cells/mL could be achieved with perfusion cultivations and the recombinant human Erythropoietin (rhEPO) was stabilized expressed with a volumetric productivity of 0.6 g/L/day (Perfusion culture processes have been widely used for enhancing the production performance by CHO or human cell lines. A CHO cell line expressing a humanized anti-CD52 mAb was cultivated in perfusion mode, which results in a higher productivity and stabilized product quality. In the perfusion process, the maximal volumetric productivity reached up to 0.55 g/L/day, which was 2.2-fold higher than that of the fed-batch culture . Recentl g/L/day . Additio g/L/day . In summ g/L/day . It shouIn this study, the continuous perfusion culture was verified as an optimized method for mAb production by CHO cells with low-priced perfusion media. Then, the perfusion culture rates and culture temperature were also optimized for evaluation of mAb titer and quality in 3-L bioreactor. The results indicated that the product titer reached a higher level of 16.19 g/L when combining the high perfusion rates and delayed reduction of culture temperature at day 6. In addition, the effectiveness of the proposed perfusion culture strategy was successfully verified in a 200-L bioreactor, which produced 16.79 g/L mAb at day 16 and the product quality was also maintained. This study provides some guidance for the mAb production by CHO cells with process engineering methods.


However, several of the non-genomic effects of CS seem to be mediated by putative non-classic membrane receptors characterized by pharmacological properties that are different from those of classic cytosolic GR. Since pre-clinical findings suggest that inhaled CS (ICS) may also regulate the bronchial contractile tone via putative CS membrane-associate receptors, the aim of this review was to systematically report and discuss the impact of CS on human airway smooth muscle (ASM) contractility and airway hyperresponsiveness (AHR). Current evidence indicates that CS have significant genomic/non-genomic beneficial effects on human ASM contractility and AHR, regardless of their anti-inflammatory effects. CS are effective in reducing either the expression, synthesis or activity of α-actin, CD38, inositol phosphate, myosin light chain kinase, and ras homolog family member A in response to several pro-contractile stimuli; overall these effects are mediated by the genomic action of CS. Moreover, CS elicited a strong bronchorelaxant effect via the rapid activation of the Gsα–cyclic-adenosine-monophosphate–protein-kinase-A pathway in hyperresponsive airways. The possibility of modulating the dose of the ICS in a triple ICS/long-acting β Generally, the effects elicited by corticosteroids (CS) are generally mediated by the binding and activation of cytosolic glucocorticoid receptors (GR) that, in turn, translocate towards the nucleus, interact with glucocorticoid response elements (GRE), and ultimately elicit genomic effects that modulate protein expression ,2. IndeeHowever, a wide range of non-genomic effects of CS seems to be mediated by putative non-classic membrane receptors characterized by pharmacological properties that are different from those of classic cytosolic GR . InteresTherefore, the aim of this review was to systematically report and discuss the impact of CS on human airway smooth muscle (ASM) contractility and airway hyperresponsiveness (AHR).The question of this systematic review was to assess the impact of CS on human ASM contractility and AHR that was not related to the anti-inflammatory effects of CS.The protocol of this synthesis of the current literature was performed in agreement with the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) , with thThe PICO  framework was applied to develop the literature search strategy and question, as previously reported . Namely,A comprehensive literature search was performed for clinical studies, ex vivo studies, and in vitro studies written in English, characterizing the impact of CS on ASM contractility and AHR. The search was performed in MEDLINE in order to provide for relevant studies available with no time limit up to 25 July 2022. The research string was as follows:  OR “adrenal cortex hormones”[All Fields] OR “corticosteroid”[All Fields] OR “corticosteroids”[All Fields] OR “corticosteroidal”[All Fields] OR “corticosteroide”[All Fields] OR “corticosteroides”[All Fields]) AND ((“airway”[All Fields] OR “airway s”[All Fields] OR “airways”[All Fields]) AND  OR “smooth muscle”[All Fields] OR (“smooth”[All Fields] AND “muscle”[All Fields]))) AND (“human s”[All Fields] OR “humans”[MeSH Terms] OR “humans”[All Fields] OR “human”[All Fields]) AND (“contractilities”[All Fields] OR “contractility”[All Fields] OR “muscle contraction”[MeSH Terms] OR (“muscle”[All Fields] AND “contraction”[All Fields]) OR “muscle contraction”[All Fields] OR “contractile”[All Fields] OR ((“airway”[All Fields] OR “airway s”[All Fields] OR “airways”[All Fields]) AND (“hyperresponsive”[All Fields] OR “hyperresponsiveness”[All Fields] OR “hyperresponsivity”[All Fields]))). Citations of previously published relevant and recently published reviews or editorials were examined to select further pertinent studies, if any . Two revData from the included studies were extracted in agreement with Data Extraction for Complex Meta-anALysis (DECiMAL) recommendations  and chec2-adrenoceptor agonists (LABA) and long-acting muscarinic antagonists (LAMA).The endpoint of this systematic review was the impact of CS on human ASM contractility and AHR that was not related to the anti-inflammatory effects of CS. The effects of CS were assessed both alone and in combination with long-acting βData from original papers were extracted and reported via qualitative synthesis. The summary of the risk of bias for each included RCT was analyzed via the Jadad score  and CochOf the 155 potentially relevant records identified in the initial search, 21 studies were deemed eligible for a qualitative analysis. Data were obtained from eight studies conducted in vitro on human ASM cells (hASMC) ,18,19,20An in vitro study on hASMC investigated whether CS transcriptionally regulated the expression of CD38, a ~45-kDa glycosylated transmembrane protein having a central role in intracellular calcium homeostasis and AHR . In hASMp < 0.05) reduced the gene overexpression of CD38 in hASMC exposed to TNF-α 10 ng/mL  ) and TNF-α 10 ng/mL (from 0.84 ± 0.06 to 0.39 ± 0.03 [RhoA to β-actin ratio]). PSL significantly (p < 0.01) reduced RhoA promoter activity elicited by IL-13 and TNF-α [Goto et al.  investignd TNF-α .p ≤ 0.01) reduced the overexpression of α-actin (from 7.12 ± 1.77 to a maximum of 3.05 ± 0.98) and the short isoform of MLCK [In primary hASMC exposed to TGFβ 1 ng/mL, co-incubation with DEX 0.1–1 μM and salmeterol (SAL) 1 nM for 6 days significantly ( of MLCK . Combini of MLCK .p ≤ 0.01) reduced the overexpression of α-actin from 7.12 ± 1.77 to 3.46 ± 1.02-fold increase, while FP 100 nM plus SAL induced a numerical decrease [p ≤ 0.01) reversed the TGFβ-induced shift in MLCK expression from the long to the short isoform. Combining FP with SAL did not result in a change in hASMC proliferation [In primary hASMC exposed to TGFβ 1 ng/mL, co-incubation with FP 10 nM and SAL 1 nM for 6 days significantly (decrease . Adding feration .p < 0.05) reduced when incubated for 16 h with FP 10 μM combined with formoterol (FOR) 1nM by ≃31.5% or with olodaterol (OLO) 1 nM by ≃36.3% [p < 0.05) reduced spontaneous contraction by 23.2% and 38.9%, respectively [In hASMC-embedded collagen gels, spontaneous contraction was significantly (y ≃36.3% . When huectively .p ≤ 0.05) inhibited the protein overexpression of α-actin from 96.7 ± 1.5% to 2.3 ± 0.6% [In human bronchial myofibroblasts stimulated with TGFβ 5 ng/mL, co-incubation with FP 1 pM and SAL 10 nM for 24 h significantly (3 ± 0.6% . Within 3 ± 0.6% .p > 0.05) modulate the FcεRI-dependent ASM contractility [One study  evaluateactility .p > 0.05) reduce the histaminergic contractile tone in non-sensitized tissues [In two ex vivo studies ,25, over tissues , in pass tissues ,25, and max) of 33.41 ± 3.47% and a potency  of 4.79 ± 0.15. BDP was significantly (p < 0.001) more effective in passively sensitized medium bronchi, with an Emax of 43.61 ± 2.06% and a pEC50 of 5.09 ± 0.23. Pre-treatment with the Gsα subunit G protein antagonist NF449 and the cyclic-adenosine-monophosphate (cAMP)-dependent protein kinase A (PKA) inhibitor KT5720 significantly (p < 0.001) suppressed the non-genomic bronchorelaxant action of BDP in passively sensitized tissues, but not in non-sensitized ones, suggesting that the CS effect was dependent from the activation of Gsα–cAMP–PKA cascade [p < 0.001) greater in passively sensitized tissues (Emax 63.89 ± 5.09% and pEC50 7.23 ± 0.27) than in non-sensitized ones (Emax 31.94 ± 3.01% and pEC50 7.27 ± 0.37) [Cazzola et al.  investigp > 0.05) modulate the ASM contractile tone. In airways challenged with CM 1:10 v/v for 60 min, BDP administered at 1 μM and 10 μM, but not at 0.1 μM, significantly (p < 0.05) lowered the ASM contractility by −52.49 ± 10.97% and −66.98 ± 7.90%, respectively [A recent ex vivo study  performeectively .p < 0.01) synergistic relaxant effect in medium bronchi pre-contracted by His, both in non-sensitized and passively sensitized tissues [max, whereas for concentrations eliciting ≥50% Emax the extent of synergism was strong [Combining cumulative concentrations of BDP with FOR at a 100:6 concentration ratio induced a significant ( tissues . In non- tissues . A syners strong .p < 0.001) synergistic bronchorelaxation. The maximal synergistic interaction was achieved by BDP/FOR 1/0.06 ng/mL in non-sensitized tissues (+20.41 ± 4.10% vs. expected additive effect) and by BDP/FOR 10/0.06 ng/mL in passively sensitized airways (+20.04 ± 2.18%). In non-sensitized PCLS, the synergistic interaction was greater at higher concentrations; the magnitude of interaction was strong at concentrations inducing 25–50% Emax, and very strong at higher concentrations. In passively sensitized PCLS, BDP/FOR produced a greater synergistic interaction when administered at lower concentrations; the extent of synergism was very strong over the range of concentrations inducing 15–25% Emax and strong for concentrations inducing 75% Emax [In non-sensitized and passively sensitized PCLS pre-contracted by His, increasing concentrations of BDP/FOR induced a significant (75% Emax .50) was 2.31 ng/mL, while in PCLS, it was 4.59 ng/mL [max of 86.90%, whereas in PCLS the bronchorelaxant effect was only partial, with an Emax of 51.83% [In medium bronchi collected from COPD donors, the concentration of BDP/FOR administered at a 100:6 concentration ratio that reduced by 50% the ASM contractility elicited by CCh (Ab EC59 ng/mL . The potf 51.83% .p < 0.05) bronchorelaxant effect and the maximal synergistic interaction was +17.61 ± 8.34% greater than the expected additive effect. The magnitude of synergistic interaction was strong to very strong [p < 0.05) bronchorelaxant effect on the histaminergic contractile tone, reaching an Emax +20.97 ± 7.47% greater than the expected additive effect, and the magnitude of synergistic interaction was very strong. When high concentrations of MF were combined with IND, the synergism significantly (p < 0.001) increased, reaching an Emax +27.36 ± 12.40% greater than the expected additive effect and the magnitude of interaction ranged from middle to very strong [In passively sensitized medium bronchi pre-contracted by His, combining high, rather than medium concentrations of MF with the LABA indacaterol (IND) at a 100:90 molar ratio induced a significant (y strong . In passy strong .max (EC30) for 30 min did not induce synergism [30, the bronchorelaxant response was 64.71 ± 1.60% in passively sensitized medium bronchi and 73.30 ± 5.39% in PCLS [In non-sensitized medium bronchi and PCLS pre-contracted by His, BDP combined with glycopyrronium (GLY) at low concentrations of each monocomponent inducing 30% of the Eynergism . In passp < 0.05) synergistic bronchorelaxant effect in passively sensitized medium bronchi and PCLS submaximally contracted by His [max. In passively sensitized PCLS, the maximal synergistic interaction was achieved with BDP/FOR/GLY 10/0.6/1.25 ng/mL (+24.95 ± 7.85% vs. expected additive effect). When administered at concentrations inducing 25–75% Emax, BDP/FOR/GLY produced a very strong synergism, while at concentrations inducing 90% Emax, synergism was strong [In an ex vivo model of bronchial asthma, combining BDP with the LABA FOR, and the LAMA GLY at a 100:6:12.5 concentration ratio produced a significant  synergistic bronchorelaxant effect. Synergism was observed with BDP/FOR administered at concentrations of 0.318–31.8 ng/mL plus GLY at concentrations of 0.0375–3.75 ng/mL. The maximal synergistic interaction was reached when BDP/FOR 1.06 ng/mL was combined with GLY 0.125 ng/mL, reaching an improved bronchorelaxation of +32.00–35.00%, compared to the expected additive effect [A more recent ex vivo study  confirmee effect .p < 0.05) synergistic bronchorelaxant response resulted when BDP/FOR administered at concentrations of 1.06–31.8 ng/mL was combined with GLY at concentrations of 0.125–3.75 ng/mL. Maximal synergism was detected for BDP/FOR 10.6 ng/mL combined with GLY 1.25 ng/mL, leading to an improved bronchorelaxant effect of +36.00–37.00%, compared to the expected additive effect [In PCLS collected from COPD donors and submaximally contracted with CCh, BDP/FOR/GLY administered at a 100:6:12.5 concentration ratio induced a supra-additive effect, as resulted from the analysis of interaction via Bliss model; according to the Loewe and HSA models, a significant (e effect .p < 0.05) synergistic bronchorelaxant effect and the Emax achieved was +22.94 ± 13.81% greater than the additive effect [p < 0.001) increased when high MF concentrations were combined with IND and GLY at a 100:37:90 molar ratio, and the Emax achieved was +28.73 ± 2.59% greater than the additive effect. The magnitude of synergistic interaction was always very strong, irrespective of MF concentration. In passively sensitized PCLS, treatment with medium concentrations of MF, IND, and GLY at a 100:37:45 molar ratio produced a significant (p < 0.001) synergistic bronchorelaxant response, reaching an Emax +45.00 ± 4.41% greater than the additive effect. The synergistic interaction was further significantly (p < 0.001) enhanced when high concentrations of MF were combined with IND and GLY at the 100:37:90 molar ratio, and the Emax was +53.72 ± 9.10% compared with the expected additive effect. The magnitude of synergistic interaction was always very strong, irrespective of MF concentration [In passively sensitized medium bronchi and PCLS pre-contracted by His, combining medium concentrations of MF with the LABA IND and the LAMA GLY at a 100:37:45 molar ratio produced a significant (e effect . Synergintration .p < 0.05) increased the MCh provocative dose causing a 20% fall in FEV1 (PD20), by producing a doubling dose improvement of 1.6 (95% CI 0.8–2.3) vs. the baseline [Currie et al.  performebaseline .p < 0.01) increased the provocative concentration causing a 20% decrease in FEV1 (PC20) to MCh vs. the baseline  in non-smoking asthmatic patients. In contrast, FP was not significantly (p > 0.05) effective on AHR in patients who smoke. The response to FP was significantly (p < 0.01) different between current smokers and non-smokers  [In another RCT , two-wee51–3.56) .p < 0.01) reduced the AHR to MBS and MCh to a similar extent, shifting the dose–response curve of each agonist to the right by 1.06-dd (0.34–1.78) and 1.17-dd (95% CI 0.34–2.00), respectively. BUD induced a further significant (p < 0.01) reduction in the AHR to AMP vs. placebo and the other challenges, shifting rightward the dose–response curve by 2.92-dd (2.12–3.72) [O’Connor et al.  investig12–3.72) .p < 0.05) increased the PC20 to MCh at day 8 of treatment pre-allergen challenge and prevented the allergen-induced AHR at day 11 of treatment.Kelly et al.  conductep < 0.05) increased the PC20 to MCh from 3.7 ± 2.7 mg/mL to >16 mg/mL vs. the baseline.According to a clinical study  performep > 0.05) increased MCh PC20 vs. the baseline and vs. the placebo. Across the 15 genes modulated by treatment with PSL, the FAM129A and SYNPO2 genes resulted to be significantly (p < 0.01) correlated with AHR  [Yick et al.  evaluatectively) .Of the six clinical studies ,31,32,33The findings resulting from this systematic review indicate that, generally, CS have significant genomic/non-genomic beneficial effects on human ASM contractility and AHR, regardless of their anti-inflammatory effects .More specifically, CS are effective in vitro in reducing either the expression, synthesis or activity of α-actin, CD38, IP, MLCK, and RhoA in response to several stimuli  that increase the contractile tone of hASMC ,36,37; o2-adrenoceptor agonist (SABA) reliever [The evidence that, when administered in combination with a LABA, CS induced significant synergistic bronchorelaxant effects in passively sensitized small and medium human bronchi fully supports the current global initiative for asthma  approach in which an ICS are recommended for Steps 1–5 . Effectireliever .1 compared to the SABA terbutaline [ICS-FOR as a MAintenance and Reliever Therapy (MART), currently recommended at Step 3–5 , resultebutaline .Triple ICS/LABA/LAMA FDC is currently considered an alternative treatment at Step 4 as well as a preferred therapy at Step 5 . As a maAnother important point arising from this systematic review is that ICS can be ineffective in preventing AHR in smoking patients with asthma, a condition related to the presence of relative steroid resistance due to the impairment of histone deacetylase 2 (HDAC2) ,46. In tThis systematic review has certainly some limitations, generally intrinsic to primary publications. First, although the large body of evidence resulting from in vitro and ex vivo research supported the acute direct effects of ICS against human ASM contractility and AHR, ad-hoc translational studies are still missing for most of the ICS. However, we can speculate that the beneficial effects of ICS on airway contractility is an effect of class not specific for each single molecule. Second, the trial by Williams et al.  was not In conclusion, while the genomic effects of CS have been well-characterized with respect to the expression, synthesis, and activity of pro-contractile factors, current evidence suggests that CS may also elicit rapid non-genomic effects on human airways via the Gsα–cAMP–PKA pathway, a cascade activated by one or more specific CS membrane-associated receptors ,53. Howe


ATG5 and ATG7) and immune-related genes  compared to mock. However, the virus cultured at 30°C showed significantly decreased expression of autophagy-related genes (ATG5 and ATG7), but not significantly different major immune-related genes  compared to mock. Importantly, expression of the transcription factor FOXO1, which controls autophagy- and immune-related gene expression, was significantly lower at 30°C. Moreover, FOXO1 inhibition in temperature-optimized MDCK cells enhanced HuNoV replication, highlighting FOXO1 inhibition as an approach for successful virus replication. In the temperature-optimized cells, various HuNoV genotypes were successfully replicated, with GI.8 showing the highest replication levels followed by GII.1, GII.3, and GII.4. Furthermore, ultrastructural analysis of the infected cells revealed functional HuNoV replication at low temperature, with increased cellular apoptosis and decreased autophagic vacuoles. In conclusion, temperature-optimized MDCK cells can be used as a convenient culture model for HuNoV replication by inhibiting FOXO1 and providing adaptability to different genotypes.Second, the expression levels of autophagy-, immune-, and apoptosis-related genes at 30°C and 37°C were compared to explore factors affecting HuNoV replication. HuNoV cultured at 37°C showed significantly increased autophagy-related genes ( Caliciviridae family, is classified into seven genogroups (GI–GVII) based on the capsid viral protein (VP)1 sequence [Norovirus, a non-enveloped single-stranded positive-sense RNA virus belonging to the sequence , 3. GI oThe study of HuNoV transmission and the development of effective antiviral interventions are mainly hindered by a lack of an appropriate in vitro culture system. Many cell lines derived from humans and animals cannot support HuNoV replication , 5; for FOXO1) also plays a key role in regulating immune response, and is further associated with the replication of different viruses, including hepatitis B, vesicular stomatitis, and Newcastle disease viruses [FOXO1 expression in HuNoV replication has not been reported yet.Temperature affects viral replication in host cells by modulating either the rate of synthesis of viral proteins and genomes or the host cell immune response -19. Temp viruses , 22. HowFOXO1 signaling.Hence, the present study was designed to investigate whether temperature optimization would facilitate HuNoV replication in MDCK cells. Our study demonstrated that MDCK cells can be used as an in vitro model to cultivate HuNoV by modulating temperature via 2. For viral infection, the cells were cultured in DMEM supplemented with 100 μg/ml streptomycin, 100 U/ml penicillin, and trypsin treated with 2 μg/ml Np-tosyl-L-phenylalanine chloromethyl ketone . Stock solutions of AS1842856  were prepared in dimethyl sulfoxide (Sigma-Aldrich) and stored at −20°C.MDCK cells were purchased from the Korean Cell Line Bank . The cells were cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin  at 37°C and 5% COFour HuNoV genotypes , isolated separately from HuNoV-positive fecal samples, were obtained from the Gwangju Institute of Health and Environment. All patient information related to the specimens was anonymized for research purposes. The experimental protocol adopted in this study was approved by the Bioethics Review Committee of Chonnam National University (IRB No. 1040198-170420-HR-038-05).4 cells/ml at 37°C for 24 h. The cells were infected with 1.91 × 102 genome copies of HuNoV GII.4 for one h, and the cells were then washed with PBS to remove unbound virus. After complete removal, the infected cells were incubated for 72 h at 27°C, 30°C, 34°C, and 37°C in an atmosphere of 5% CO2. The infected cells were scraped to extract RNA for virus quantification. To observe the passage of the virus, MDCK cells were cultured in concentrations of 6.25 × 104 cells/ml in 0.75-cm2 dishes (48-well plate), 1.375 × 105 cells/ml in 21.50-cm2 dishes (60-mm culture dish) and 5.156 × 105 cells/ml in 75-cm2 dishes (75T-Flask) for three consecutive passages. The cells were infected with HuNoV by incubation for 72 h in the 1st and 2nd passages and for two weeks in the 3rd passage. For every subsequent passaging, the whole supernatant from the previous passage was used as an inoculum source. To test the adaptability of the culture system for the replication of different genotypes, the cells were infected with 2.28 × 102 virus genome copies of four different genotypes of HuNoV, including GI.8, GII.1, GII.3, and GII.4, and incubated at 30°C for 72 h.HuNoV specimens isolated from fecal samples were diluted to 10% in PBS (pH 7.4) and stored at −80°C until use. For the infection of MDCK cells, the HuNoV specimens were diluted in infection media consisting of DMEM containing 1% penicillin/streptomycin and 2 μg/ml TPCK. All experiments, excluding a test for adoptability of the culture system to cultivate different HuNoV genotypes, were performed with GII.4 genotype of HuNoV in 48-well plates. The cells were cultured in a 48-well plate at 6.25 × 10VP1 gene primer pair JJV2F (5′-CAA GAG TCA ATG TTT AGG TGG ATG AG-3′) and COG2R (5′-TCG ACG CCA TCT TCA TTC AC-3′) and probe primer RING2P (5′-FAM-TGG GAG GGC GAT CGC AAT CT-Black Hole Quencher) [2 = 0.9974 was calculated from the HuNoV standard curve. The fold change of HuNoV copy number was calculated relative to the initial inoculum of the virus. The viral genome in the initial inoculum was detected after 1 h incubation and washing with PBS.To detect HuNoV genome copies in the culture medium by quantitative RT-PCR, 300 μl of the supernatant was collected 72 h after infection. Total RNA was extracted from the supernatant and cells using 700 μl of RNAiso Plus (Takara), according to the manufacturer’s protocol. For cDNA synthesis, 10 μl of RNA and 1 μl of 6-mer random primer  were mixed and incubated at 65°C for 10 min; 4 μl of 5 × Moloney murine leukemia virus (M-MLV) reverse transcriptase reaction buffer, 2 μl of 5 × dithiothreitol, 2 μl of dNTP, 0.5 μl of RNase inhibitor , and 1 μl of M-MLV were then added, followed by incubation at 37°C for 1 h and 95°C for 5 min using a PCR Thermal Cycler Dice Gradient system. The cDNA was prepared using the HuNoV uencher) . QuantitNorovirus-infected cells were detached from the plates using a cell scraper, then collected by centrifugation, and the cell pellets were fixed in 2.5% glutaraldehyde  in 0.1 M phosphate buffer (pH 7.2) for 3 h at 4°C. The pellet was then washed in several changes of 0.1 M phosphate buffer to remove excess glutaraldehyde. Fixed samples were post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences) in 0.1 M phosphate buffer for 1–2 h in the dark at 4°C. After osmium fixation, the pellets were washed 2–3 times with 0.1 M phosphate buffer and dehydrated through a graded series of ethanol . Dehydrated samples were exposed to two changes of propylene oxide, and then immersed in 2:1 (1 h), 1:1 (overnight), and 1:2 (1 h) mixtures of propylene oxide and Epon 812 (Electron Microscopy Sciences) embedding medium, respectively. The pellets were transferred to 100% epoxy resin and incubated overnight. Infiltrated samples were embedded in fresh pure epoxy resin and polymerized for 24–48 h at 70°C. Using a Leica EM UC6 Ultramicrotome (Leica Microsystems GmbH), polymerized blocks were cut into thin slices and mounted on 200- mesh carbon-coated grids. The grids were post-stained with 2% uranyl acetate and 1% lead citrate at room temperature for 15 and 5min, respectively. Zeiss LEO912AB 120 kV TEM (Carl Zeiss) and FEI Tecnai G2 Spirit Twin 120 kV TEM (FEI Company) were used for transmission electron microscopy analysis [® Real-Time System III (TaKaRa). The primer sequences are given in Table S1.Quantitative RT-PCR was also used to measure the gene expression levels in response to HuNoV replication in the MDCK cells. RNA was extracted using 200 μl of RNAiso Plus (TaKaRa) added to the cells at the infection endpoint, according to the manufacturer's manual. For cDNA synthesis, 10 μl of RNA and 1 μl of 6-mer random primer (TaKaRa) were mixed and reacted at 65°C for 10 min. Then, the cDNA synthesis kit  was added, and left to react at 37°C for 1 h and then at 95°C for 5 min. The cDNA synthesis was performed using the PCR Thermal Cycler Dice Gradient (TaKaRa). The levels of replication-related genes in the prepared cDNA were quantified with Thermal Cycler Dicet-test. A value of p < 0.05 was considered statistically significant.All experiments were performed in triplicate. Statistical data analysis was performed using IBM SPSS v.23 software . Significant differences between groups were determined by one-way analysis of variance followed by Duncan’s post-hoc test. Comparisons between two groups were performed with Student’s 4 cells/ml and incubated in a 48-well plate at 27°C, 30°C, 34°C, and 37°C for 72 h (2 HuNoV genome copies) in a 48-well plate. The HuNoV genome copy number was increased with increasing virus concentration, except for at 5.6 HuNoV genome copies , 2.8×101 and 5.6 × 101 HuNoV genome copies, respectively  of the three consecutive passages, respectively. These results indicated that the passaging of HuNoV and scaling up of the virus production is possible in our culture system. Additionally, to identify the adaptability of the culture system, the replication of different HuNoV genotypes including GI.8, GII.1, GII.3, and GII.4 (2.28 × 102 HuNoV genome copies) were observed at 30°C for 72 h -infected MDCK cells (hereafter denoted as ‘infected cells’) were seeded at a density of 6.25 × 10for 72 h . HuNoV rfor 72 h . The hig genotypee copies . The higectively . To conffor 72 h . IncreasATG5 was decreased by 1.9-fold at 30°C; however, the level was increased by 1.6-fold at 37°C in infected cells compared to the mock (ATG7 (2.84-fold decreased at 30°C and 1.5-fold increased at 37°C) A, IFNB, IFN-stimulated gene (ISG)15, and nuclear factor-kappa B (NF-κB) was upregulated by 2.0-, 2.3-, 1.7-, and 1.6- fold, respectively, at 37°C compared to that in their respective mock (BAX), was decreased at 37°C and exceptionally increased at 30°C compared to mock. Even though other apoptosis-related genes, including cytochrome c (CYCS) and caspase (CASP-3) were significantly increased at both temperatures compared to mock, the pattern of the expression level was higher at 30°C compared to that at 37°C  HuNoV obtained from fecal sample. The expression level of autophagy-related gene the mock . A similat 37°C) . Our res reduced . In contperature . Moreoveive mock . Upregul at 37°C . Since Fed genes , 29 and  to mock .Overall, these results suggest that at 37°C, autophagy- and immune-related genes were upregulated in infected cells to promote the antiviral response to HuNoV infection; however, these genes were either inhibited or unchanged at 30°C, thereby permitting viral replication.FOXO1 inhibition on HuNoV replication was investigated by treating the infected cells with a FOXO1 inhibitor (AS1842856) [FOXO1 inhibition only induced HuNoV replication at 30°C.Next, the effect of 1842856)  at 30°C FOXO1 inhibition on the cellular morphology of infected cells, ultrastructural analysis of FOXO1 inhibitor (AS1842856)-treated HuNoV-infected and uninfected cells was performed by transmission electron microscopy. Uninfected cells exhibited well-preserved cellular morphology with evenly distributed cell constituents, such as an intact nucleus (N), Golgi apparatus (G), and mitochondria (M)  . The inf at 37°C . However at 30°C . Interesn groups . Overall at 37°C .et al. reported that HBGA expression alone is not sufficient to enable virus replication in cell culture [In this study, a temperature-optimized cell culture model was developed and used to successfully replicate HuNoV using MDCK cells. A mechanism for replication in the model was also proposed. The successful replication of HuNoV in B cells and HIE in vitro were reported previously, with the cells requiring the additional cofactor HBGA and bile salt, respectively. However, Karst  culture ; other s culture , 34. The culture , 35. Theet al. demonstrated successful replication of different genotypes in the HIE model in the presence of bile salt. However, a jejunal HIE model could only replicate GII genotypes [2 HuNoV genome copies) in inoculum increased by 12.6-fold the virus copy number in our model, indicating that the HuNoV replication also depends on viral concentration of inoculum. Moreover, modifying the cell culture conditions by lowering the temperature significantly increased HuNoV replication . However, the role of autophagy was inconsistent in viral replication; viruses can manipulate autophagy signaling in either a suppressive or supportive manner [All tested autophagy-related genes, including eostasis  to viral or cellular proteins to inhibit virus replication [ISG15 significantly increased the replication of influenza A, Sindbis, and herpes viruses [IFNs and ISGs is critical for virus replication [In the present study, upregulation of immune-related genes, including rine NoV . On the rine NoV , 49. Furlication , 51. On lication . Similarlication , 20. Inh viruses , and inhlication . Moreovelication . Consistlication .CASP3 and CYCS at 30°C [CASP3 [BAX was exceptionally induced at 30°C in the infected cells, resulting in apoptosis possibly through activation of  at 30°C , 58. SimC [CASP3 . MoreoveFOXO1 inhibition at low temperature induced HuNoV replication. Consistent with our result, FOXO1 inhibition also induced HIV and Kaposi’s sarcoma-associated herpes virus replication in cells by inducing intracellular ROS levels [FOXO1 expression was not observed previously. Notably, FOXO1 inhibition at low temperature may play a crucial role in virus replication because it modulates autophagy- and immunity-related genes and associated pathways [Finally, S levels , 60, 61.pathways .FOXO1, which facilitated HuNoV replication in the cells. Thus, temperature-optimized MDCK cells can serve as a useful model for further studies to elucidate the pathogenic mechanisms of HuNoV infection.In conclusion, this study demonstrated that HuNoV infection at 30°C can support virus replication in MDCK cells. The study revealed an unexpected link between temperature and the expression of