This is primarily used to protect metallic surfaces against corrosion or to create biologically active surfaces. Gemini surfactants are known for their excellent anticorrosion, antimicrobial and surface properties; (2) Methods: In this study, we present an efficient methods of preparation of encapsulated gemini surfactants in form of alginate and gelatin capsules; (3) Results: The analysis of infrared spectra and images of the scanning electron microscope confirm the effectiveness of encapsulation; (4) Conclusions: Gemini surfactants in encapsulated form are promising candidates for corrosion inhibitors and antimicrobials with the possibility of protecting the active substance against environmental factors and the possibility of controlled outflow. Surfactants are chemical compounds that upon adsorption at the air–water interface reduce the surface tension of water. Their molecules contain at least two moieties, hydrophobic and hydrophilic. The hydrophobic part is usually a straight or branched hydrocarbon or fluorocarbon chain with 8–18 carbon atoms, whereas a hydrophilic moiety is a polar or ionic group. The head group can be cationic, anionic, amphoteric or nonionic . It affeEncapsulation is a process of enclosing or entrapping a core material (active agent) inside a solid shell or within a solid or liquid matrix. The different encapsulation techniques can provide various microcapsule morphologies  68,69].,69.68,692 (acidification), redox and enzymes or mechanical impact  ether (BDMAEE) (97%), D-glucose (99.5%) and Span 80 were obtained from Sigma-Aldrich . Sulphuric acid and isopropanol (99%) were purchased from Chempur . Acetonitrile (≥99%), methanol (≥99%), calcium chloride (99%) and phosphorus pentoxide (98%) were obtained from VWR Chemicals . Barium sulfate (95%) and gelatin (95%) were purchased from WarChem . Sodium alginate from brown algae was obtained from Biomus . Sunflower oil (food grade) was bought from Bunge Polska .1-Decanol (98%) and 1-dodecanol (98%) were obtained from Merck KGaA . Chloroacetic acid (99%), bromoacetic acid (97%), 1-bromododecane (97%), Halogenoesters  were obtained from alcohols: 1–decanol or 1-dodecanol with appropriate amount of halogenoacetic acid under acidic conditions  according to of the described esterification reaction procedure .4O10 in a vacuum desiccator.In all gemini surfactants synthesis, 1 equivalent of the appropriate diamine (TMPDA for 10(E)-3-10(E) Cl and 12-3-12; BDMAEE for 10(E)-O-10(E) Cl, 12-O-12, 12(E)-O-12(E) Cl and 12(E)-O-12(E) Br) was mixed with 2 equivalents of halogenoester (10Cl for 10(E)-3-10(E) Cl and 10(E)-O-10(E) Cl; 12Cl for 12(E)-O-12(E) Cl; 12Br for 12(E)-O-12(E) Br), or bromododecane for 12-3-12 and 12-O-12. The reactions were carried out without a solvent, at room temperature, by stirring using a magnetic stirrer until the reaction mixture solidified (max 6 h). The crude products were crystallized from a mixture of acetonitrile:methanol in the volume ratio of 10:1 and dried in an incubator (60 °C) and over PThe melting point (mp) of the products was measured on Stuart SMP30  by using a one-sealed side capillary. Measurement accuracy is up to 1 °C.1H and 13C, respectively, by using the software, VNMR VERSION 2.3 REVISION A . The 13C and 1H chemical shifts were measured in CDCl3 with TMS as an internal standard.The NMR spectra for the synthesized compounds were obtained by using a Varian model VNMR-S 400 MHz , operating at 403 and 101 MHz for The FTIR spectra of all gemini surfactants were recorded with a Thermo Scientific Nicolet iS5 FT-IR spectrometer . The samples were in a solid phase and prepared as tablets with potassium bromide, whereas the FTIR analysis of alginate and gelatin capsules was performed using a JASCO FT/IR-4600 spectrometer . The samples were tested in the form of solids with a suitable powder attachment .The elemental analysis (EA) measurements were carried out on a FLASH 2000 elemental analyzer  with a thermal conductivity detector.Digital photos were taken with Levenhuk DTX 50 Digital Microscope .Scanning electron microscopy (SEM) studies were performed using a high-resolution environmental electron scanning microscope Quanta FEG 250 FEI  in low vacuum conditions at a pressure of 70 Pa and a beam accelerating voltage of 10 kV.To fabricate gel capsules of gemini surfactants, an excess amount of suitable gemini surfactant (10(E)-3-10(E) Cl, 10(E)-O-10(E) Cl, 12-3-12, 12-O-12) higher than its critical micelle concentration was added to a mixture 50 mL of sodium alginate (0.6 g) with vigorous stirring at 70 °C for 20 min until reaching homogeneity. After that, the synthesized homogeneous viscous mixture was injected into a calcium chloride solution (2% wt) to form the capsules. Subsequently, the capsules were filtered from the solution and kept in incubator (60 °C) to dry. Empty capsules without gemini surfactant were prepared in an analogous manner.To prepare more durable capsules of gemini surfactant 12-O-12, an excess amount of was added to a mixture 50 mL of sodium alginate (0.6 g) and barium sulfate (1.2 g) with vigorous stirring at 70 °C for 20 min until reaching homogeneity. After that, the homogeneous viscous mixture was injected into calcium chloride solution (2% wt) to form the capsules. Subsequently, the capsules were collected from the solution and kept in incubator (60 °C) to dry. Empty capsules without gemini surfactant were prepared in an analogous manner.2O5. Empty capsules without gemini surfactant were prepared in an analogous manner.In total, 1.5 g of α-D-glucose was added to the aqueous gelatin solution  and allowed to react for 5 min. Then, the individual gemini surfactants (about 0.1 g) were added to the aqueous solution containing gelatin and sugar. The resulting mixture was carefully added to sunflower oil  containing Span 80 (1.5 mL). The dispersion was mixed with a magnetic stirrer to obtain an emulsion. Quick cooling to 5 °C with an ice bath hardens the gelatin droplets. After 15 min, isopropanol  was added at regular intervals to dehydrate the droplets. Stirring was continued for 2 h, followed by filtration. The microspheres were washed with isopropanol to remove any adhered oil. The microspheres prepared in this way were allowed to dry in a desiccator over PEnsuring environmental safety and the user’s safety requires the use of necessary chemical compounds with the highest activity at the lowest concentrations. Moreover, their operations should be limited to a specific place and activated by a specific stimulus, such as pH, pressure, water etc. Gemini surfactants are modern compounds with a very high biological activity and high anticorrosion efficacy. Immobilization of these compounds in alginate and gelatin microcapsules allow you to introduce them to a specific carrier and obtain smart materials. In this work, we presented effective encapsulated methods of the synthesized gemini surfactants in alginate and gelatin microcapsules. The encapsulation efficiency and the capsule morphology were confirmed by physicochemical tests. The resulting microcapsules meet the smart materials criteria and are promising candidates as corrosion inhibitors and antimicrobials.


Acute liver injury (ALI) is recognized as a serious complication of sepsis in patients in intensive care units (ICUs). S100A8/A9 is known to promote inflammation and immune responses. However, the role of S100A8/A9 in the regulation of sepsis-induced ALI remains known. Our results indicated that S100A8/A9 expression was significantly upregulated in the livers of septic mice 24 h after cecal ligation and a puncture (CLP) operation. Moreover, S100A9-KO in mice markedly attenuated CLP-induced liver dysfunction and injury, promoting the AMPK/ACC/GLUT4-mediated increases in fatty acid and glucose uptake as well as the improvement in mitochondrial function and ATP production. In contrast, treatment with the AMPK inhibitor Compound C reversed the inhibitory effects of S100A9 KO on CLP-induced liver dysfunction and injury in vivo. Finally, the administration of the S100A9 inhibitor Paquinimod (Paq) to WT mice protected against CLP-induced mortality, liver injury and mitochondrial dysfunction. In summary, our findings demonstrate for the first time that S100A9 plays an important pro-inflammatory role in sepsis-mediated ALI by regulating AKT-AMPK-dependent mitochondrial energy metabolism and highlights that targeting S100A9 may be a promising new approach for the prevention and treatment of sepsis-related liver injury. Sepsis is a common complication after infection, shock and severe trauma, and it is one of the major causes of mortality in patients in intensive care units (ICUs) ,2. Upon Increasing amounts of evidence suggest that the elevation of pro-inflammatory cytokines is closely associated with sepsis-associated liver dysfunction. S100 calcium binding protein A8 (S100A8) and A9 (S100A9)  belong to the S100 family of calcium binding proteins and can form heterodimers, which function as danger-associated molecular patterns  and display some functional features of pro-inflammatory cytokines . MoreoveSince deletion of the S100A8 gene in mice is lethal, and S100A9-deficient mice exhibit a healthy phenotype but have no S100A8 protein express . MoreoveTo determine the expression of S100A8/A9 in the livers of CLP-treated mice, we first established a sepsis-associated ALI model via CLP. qPCR assays indicated that the S100A8 and S100A9 mRNA levels were significantly enhanced in the livers at 24, 48 and 72 h after CLP compared with sham group (24 h) A. ImmunoTo examine the role of S100A9 in ALI in a mouse model of sepsis, we generated S100A9-knockout (KO) mice and subjected them to CLP A. Five dNext, we investigated whether S100A9 KO affects CLP-induced hepatic inflammation and ROS production, both of these effects being associated with sepsis-induced ALI. Twenty-four hours after CLP, S100A9-KO mice showed marked decreases in CD68-positive macrophage infiltration and ROS production (DHE staining) in their livers compared with the WT mice G,H. AccoMitochondrial dysfunction frequently triggers the apoptosis of live cells . TherefoSince AMP-activated protein kinase (AMPK) is a critical regulator of glucose and fatty acid (FA) metabolism and exerts a key role in protection against sepsis-induced hepatic injury , we then+ nuclei numbers), inflammatory responses (infiltration of CD68+ macrophages), and ROS production (DHE staining) compared with WT mice after CLP  on CLP-induced acute hepatic injury in WT and S100A9-KO mice. Consistent with the results, S100A9-KO mice exhibited significantly alleviated liver dysfunction (AST and ALT levels), necrosis, apoptosis , ATP production and MMP compared with WT mice. However, this effect was greatly reduced in CC-treated S100A9-KO mice, and there was no significant difference between CC-treated WT mice and S100A9-KO mice B–D. TogeTo demonstrate the potential clinical translational significance of our findings, we systemically administered the S100A9 inhibitor Paquinimod (Paq) to WT mice at doses of 5–20 mg/kg, subjected them to CLP, and examined them 24 h later A,B. Our + hepatic nuclei, infiltration of proinflammatory cells, especially CD68-positive macrophages, and ROS production, compared with vehicle administration  on CLP-induced liver dysfunction and injury. Our results revealed that Paq (10 mg/kg) administration significantly blocked the severity of CLP-induced hepatic injury, as indicated by necrosis, the percentage of TUNELstration F–I. Consstration J,K. Therstration C,F–K. OvThere were also changes in mitochondrial respiration function in liver tissues after Paq administration. The results showed that mitochondrial energy metabolism, ATP production and MMP were markedly higher in Paq-administered mice than in vehicle mice after CLP B,D. ThenThis study reveals a previously unknown role of S100A9 in accelerating sepsis-induced hepatic dysfunction and injury. We provided compelling evidence that S100A8 and S100A9 expression was significantly elevated in the livers of CLP-operated mice. The knockout of S100A9 markedly ameliorated CLP-induced liver dysfunction, hepatocyte apoptosis, inflammation, and oxidative stress partially by blocking AKT and activating AMPK-dependent mitochondrial metabolism. Furthermore, we demonstrated that administration of the S100A9 inhibitor Paq to mice also prevented sepsis-induced liver dysfunction upon CLP operation. Therefore, this study shows that S100A9 is a critical contributor to septic liver dysfunction and highlights S100A9 as a potential therapeutic target for treating sepsis and liver dysfunction.The S100A8 and S100A9 genes are located on chromosome 1q21, and the deletion or mutation of these chromosomes is associated with the occurrence of cancer. The human chromosomes S100A8 and S100A9 are composed of 93 and 113 amino acids with molecular masses of 10.8 and 13.2 kDa, respectively . S100A8/The liver is the key organ that has an important role in the regulation of immune response and host defense. It consists of multiple cell types, including hepatocytes (HCs), sinusoidal endothelial cells (SECs), and Kupffer cells (KCs), which are involved in the processes of endotoxin and bacteria scavenging, detoxification, metabolism, immune functions, and substance synthesis . HepaticSepsis-induced ALI is associated with significant alterations of mitochondrial function and energy metabolism. AMPK is a heterotrimer that consists of a catalytic α subunit and two regulatory β and γ subunits, which are allosterically activated by low ATP levels and high AMP levels ,28. ThisAccumulating data reveal that sepsis-induced liver dysfunction is related to poor clinical outcomes. However, no good therapeutic options are currently available to attenuate the most severe complications ,2. EarlyIn conclusion, this study reveals the key role of S100A9 in promoting sepsis-induced hepatic dysfunction and injury, partially by activating AKT and inhibiting AMPK-mediated mitochondrial energy metabolism. Our data also highlight that S100A8/A9 may be a new therapeutic target for sepsis-induced liver injury. Future studies will have to specifically examine the role of the myeloid-specific deletion of S100A8/A9, particularly S100A8 in the pathogenesis of sepsis-induced hepatic dysfunction, define the precise mechanism by which S100A8/A9 inhibits the activation of AMPK signaling in the liver, and determine whether targeting S100A9 presents a new opportunity for the treatment of sepsis and multiple organ failure. Further study is required to investigate how S100A9 is upregulated in CLP-treated livers.WT C57BL/6J mice were purchased from The Jackson Laboratory . Global S100A9 knockout (KO) mice (KOCMP-20202-S100A9-B6N-VA) were generated and fully backcrossed onto the C57BL/6 background more than 10 generations  as described previously . S100A9-n = 200) and S100A9-KO (n = 90) mice  were adaptively fed for 7 days, and a polymicrobial sepsis model was established by cecal ligation and puncture (CLP) as described [Male WT  and aspartate aminotransferase  were analyzed as indices of liver function; serum and tissue levels of triglyceride  and free fatty acids  were measured to evaluate lipolysis. All colorimetric assay kits were obtained from Nanjing jiancheng , and the experiments were performed based on the manufacturer’s protocols.Hepatic specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Liver sections (4-µm thick) were cut serially and stained with an H&E staining kit based on the instructions. Immunohistochemistry was performed on the sections with anti-S100A9  or anti-CD68  antibodies. Immunofluorescent staining was conducted with anti-S100A9  and anti-F4/80  antibodies to assess the localization of S100A9 in macrophages. The histological score of H&E tissue staining was determined based on four grades according to a previous study .To evaluate liver cell apoptosis, tissue cryosections at 6 µm thickness were stained by using a TUNEL Apoptosis Detection kit  based on previously described protocols . The rat® 3 system using primers specific for interleukin (IL)-1β, IL-6, TNF-α, NOX1, and NOX2. GAPDH was used as the internal control. Finally, the relative mRNA expression levels of genes were statistically analyzed with 2−ΔΔCt. All the primers for the target genes were synthesized by Sangon Biotech Co., Ltd.  and prepared to use the following primers: S100A8 (5′-GGA GTT CCT TGC GAT GGT GA-3′ and 5′-GGC CAG AAG CTC TGC TAC TC-3′), S100A9 (5′-ATA CTC TAG GAA GGA AGG ACA CC-3′ and 5′-TCC ATG ATG TCA TTT ATG AGG GC-3′), IL-1β (5′-CTT CCC CAG GGC ATG TTA AG-3′ and 5′-ACC CTG AGC GAC CTG TCT TG-3′), IL-6 (5′-GCT ACC AAA CTG GAT ATA ATC AGG A-3′ and 5′-CCA GGT AGC TAT GGT ACT CCA GAA-3′), TNF-α (5′-ATG GCC TCC CTC TCA TCA GT-3′ and 5′-CTT GGT GGT TTG CTA CGA CG-3′), NOX1 (5′-CCC ATC CAG TCT CCA AAC ATG AC-3′ and 5′-ACC AAA GCT ACA GTG GCA ATC AC-3′), NOX2 (5′-GGG AAC TGG GCT GTG AAT GA-3′ and 5′-CAA TTG TGT GGA TGG CGG TG-3′), GAPDH (5′-CAT CAC TGC CAC CCA GAA GAC TG-3′ and 5′-ATG CCA GTG AGC TTC CCG TTC AG-3′).The liver tissues were ground in a tissue-crushing apparatus . Total RNA from livers was purified using a TRlZOL reagent kit , and total 1 μg RNA was-reverse transcribed to cDNA with a PrimeScript RT reagent kit . Then, qPCR was performed on a QuantStudioThe liver tissues were removed and fully lysed using a RIPA lysis buffer. The homogenates were centrifuged at 13,300 rpm at 4 °C for 15 min. The protein concentrations in the supernatant were quantified by the BCA method . A total of 50 μg of protein per sample was resolved by 7.5–15% SDS–PAGE and transferred onto a PVDF membrane. Next, the membranes were blocked in 5% skim milk for 1 h at room temperature and incubated with the appropriate primary antibodies at 4 °C overnight, and then incubated with the secondary antibodies at room temperature for 1 h. Finally, the membranes were developed on a FluorChem M System . The intensities of protein bands were quantified with NIH Image J version 1.48, and the relative levels of the corresponding proteins were calculated in each group. The primary antibody against S100A9  was from Abcam . Antibodies against S100A8 , p-AMPK , AMPK , p-AKT , AKT , ACC  and β-actin  were from Cell Signaling Technology . Antibodies against GLUT4 , Bax  and Bcl-2  were obtained from Proteintech Group .2·6H2O, 110 mM D-sucrose, 60 mM lactobionic acid (LBA), 20 mM taurine, 10 mM KH2PO4, and 1 g/L BSA (fatty acid free). All data were recorded with DatLab acquisition software 5.2 .Mitochondrial respiratory function was detected in liver tissues with an Oroboros Oxygraph-2k  in a thermostat-controlled chamber as described . Briefly3 pieces, and were fixed in 2.5% glutaraldehyde for 2 h at 4 °C. The pieces were then post-fixed in 1% osmium tetroxide in phosphate buffer (PB) for no more than 2 h, and washed 3 times with PB. After washing, the samples were dehydrated, immersed and finally embedded overnight at room temperature. The tissues pieces were cut with an ultramicrotome. The sections (50–70 nm) were stained with uranyl acetate and lead citrate, and were then photographed with an HT7700 transmission electron microscope  at 100 kV. The total number of mitochondria and the number of abnormal or impaired mitochondria were analyzed in 5–6 cells in 3 sections from each sample with the NIH Image J software version 1.48.Liver tissues were cut into 1 mmt test was used to analyze the significance of the differences between 2 groups, and if not, the Mann–Whitney test was used. One-way ANOVA was used to analyze differences among 3 or more groups. However, when the results were not normal or equal variances were not assumed (Welch test), Kruskal–Wallis H was used. The Kaplan–Meier method and log-rank tests were conducted to analyze the survival rates of the mice after the sham or CLP operation. p < 0.05 was considered significant.All the results are presented as the mean ± standard error of the mean (SEM) and were statistically analyzed using SPSS version 25.0 . A normality test Shapiro–Wilk was used to test if the results were normally distributed. If the results followed a normal distribution, Student’s


Anaerobic ammonium-oxidizing (anammox) bacteria provide a shortcut in the nitrogen removal pathway by directly transforming ammonium and nitrite to nitrogen gas (N2). Due to its energy efficiency, the anammox-driven treatment has been applied worldwide for the removal of inorganic nitrogen from ammonium-rich wastewater. Although direct evidence of the metabolic production of N2O by anammox bacteria is lacking, the microorganisms coexisting in anammox-driven WWTPs could produce a considerable amount of N2O and hence affect the sustainability of wastewater treatment. Thus, N2O emission is still one of the downsides of anammox-driven wastewater treatment, and efforts are required to understand the mechanisms of N2O emission from anammox-driven WWTPs using different nitrogen removal strategies and develop effective mitigation strategies. Here, three main N2O production processes, namely, hydroxylamine oxidation, nitrifier denitrification, and heterotrophic denitrification, and the unique N2O consumption process termed nosZ-dominated N2O degradation, occurring in anammox-driven wastewater treatment systems, are summarized and discussed. The key factors influencing N2O emission and mitigation strategies are discussed in detail, and areas in which further research is urgently required are identified.Wastewater treatment plants (WWTPs) are important contributors to global greenhouse gas (GHG) emissions, partly due to their huge emission of nitrous oxide (N Most AOicate NO . Previoulack NOR ,31,32. Hnditions . This nofication . Putativfication ,35. Howeof NH2OH . Consideof NH2OH  and the of NH2OH , it was nditions ,31. Althnditions . Therefo process ,24,38.2O in wastewater by denitrifiers under anaerobic conditions, which begins by reducing NO3− to NO2− by nitrate reductases [2 is then reduced to NO through either haem-containing  or copper-containing  nitrite reductases, which is further reduced to N2O through NOR [2O is an intermediate product during denitrification, and part of N2O can escape from the cell before the final reduction to N2, resulting in N2O emission [2O reduction to N2 is the main sink of this powerful GHG, which is catalyzed by the enzyme nitrous oxide reductase (NOS) [3− all the way to N2 are the exception and that many denitrifiers, called incomplete denitrifiers, lack NOR or NOS and directly use NO or N2O as the end product [Heterotrophic denitrification is one of the main nitrogen removal pathways based on the reduction of NO to Nductases . The proough NOR . N2O is emission . Microbise (NOS) . It is b product .2− reduction catalyzed by NirS and NirK, which are functionally equivalent but structurally divergent [nirK- and nirS-type denitrifiers [nirS and nirK communities was observed in saline and non-saline environments, with nirS communities dominating in marine environments [nosZ gene has a higher frequency of co-occurrence with nirS than with nirK, and nirS usually co-occur with nor [nirS-type denitrifiers are more likely to be capable of complete denitrification and usually contribute less to N2O emission than nirK-type denitrifiers [nir/nor/nos gene occurrence [2O production potential in wastewater treatment systems and illustrate the importance of the microbial community structure for biotic N2O emission.In microbial processes, NO is generated via NOivergent . The genivergent ,42. Chantrifiers ,44. A cl2O-reducing microorganisms can reduce N2O to N2; therefore, their abundance and activity can strongly affect the net N2O emission from WWTPs. N2O degradation is catalyzed by members of either NosZ clade I or NosZ clade II. They can be distinguished by the signal peptide motif of twin-arginine translocation (Tat) or secretory (Sec) proteins, which govern the secretion pathway for N2O translocation across the cell membrane [nosZ II non-denitrifiers [nosZ II non-denitrifiers are regarded as N2O reducers, as they lack other denitrifying enzymes that specifically consume N2O [nosZ II-type non-denitrifiers could help N2O reduction in wastewater treatment systems [nosZ II non-denitrifiers in anammox-driven wastewater treatment systems associated with N2O emission mitigation deserved more attention in future studies.Nmembrane ,46. Cladtrifiers ,47. The sume N2O ,44. Henc systems . TherefonosZ gene diversity using DNA-based PCR approaches only focused on Clade I nosZ, while the abundance and diversity of Clade II nosZ are underestimated [nosZ genes in this clade [nosZ community has yet to be thoroughly investigated, and characterizing its contributions to N2O consumption will significantly enhance our understanding of N2O emission in wastewater treatment.It is noteworthy that most studies attempting to characterize stimated ,46. The is clade . The Cla2O emission rate  varies substantially among different anammox-driven reactors  [2O emission rate is a major obstacle to the sustainable application of anammox systems for wastewater treatment. Factors such as DO, NH4+, and NO2− concentrations, chemical oxygen demand (COD), and the presence of floc could significantly influence N2O emission by impacting the microbial communities and their activity in anammox-driven nitrogen removal systems.The Nreactors . The N2Oen load) ,51. The 2O emission through heterotrophic denitrification and nitrifier denitrification in PNA systems [2− rather than NO3− and indirectly yielding N2O through hydroxylamine oxidation [2− competition with NOB [2O emission.DO is a crucial operation parameter in anammox-processing systems. Maintaining a relatively low oxygen supply is suggested for PNA reactors to achieve partial nitrification by limiting oxygen availability to AOB . As most systems ,25,26. Axidation , but alswith NOB . Balanci4+ and NO2− could significantly affect the level of N2O emission during wastewater treatment [4+ can indirectly affect N2O emission through hydroxylamine oxidation or directly promote NO2− production through nitrification [4+ influx promotes NH2OH production and results in NH2OH accumulation, and part of NH2OH could leak out of the cell and enhance N2O emission during nitrification [2− is known to increase N2O emission through three main N2O production processes during wastewater treatment, i.e., hydroxylamine oxidation, nitrifier denitrification, and heterotrophic denitrification [2− not only offers a reactant for hybrid N2O formation from NH2OH via hydroxylamine oxidation but also delays the overall NH2OH abiotic decay, further stimulating the conversion of NH2OH to N2O [2− could increase N2O emission by inhibiting the N2O consumption activities of nosZ-containing denitrifiers [4+ and NO2− in anammox-driven nitrogen removal systems should be cautiously controlled to mitigate N2O emission.The concentrations of NHreatment . NH4+ cafication . A high fication . NO2− isfication . The preH to N2O . Furthertrifiers . Therefo2O mitigation has been reported in different reactors [2O emission (COD/N = 1) [2O consumption by easing the carbon limitation of N2O reduction to N2, which is the last step of denitrification [The positive effect of organic carbon on Nreactors ,59, withD/N = 1)  and imprD/N = 1) . The prefication . The enhfication ,63, cont2O emission is enhanced by NO2− accumulation from partial nitrification under low organics availability conditions [nosZ-containing and other denitrifiers could be stimulated by low influent organics under high NO2− conditions, such that N2O reduction by nosZ-containing denitrifiers could be inhibited [2O emission by affecting the number of flocs and filamentary structures around the anammox granules [2O emissions due to DO fluctuation [It is noteworthy that Nnditions ,65. Elecnhibited ,67,68,69nhibited ,70, likenhibited ,70,71,72granules ,73. The ctuation .2O emission from a high-rate anammox granular sludge reactor [2O emission in anammox granule systems [nirS was shown to much greater than that of nor in both granules and flocs, which resulted in transient NO accumulation in the anammox reactor [2O  [2− in anammox-processing systems [In the nitrification/denitrification activated sludge system, it was reported that large flocs (>200 μm), in which heterotrophic denitrification that led to the generation of N<100 μm) . Denitri<100 μm) ,82. Howe systems , so deni2O emission in anammox-driven WWTPs can be reduced by (i) lowering DO concentrations (controlling the nitrification process), (ii) adopting intermittent aeration (motivating N2O degradation), (iii) reducing NO2− concentration (controlling the nitrification and denitrification processes), and (ⅳ) increasing the C/N ratio (controlling the heterotrophic denitrification process). Additionally, regulating the microbial community composition, such as eliminating N2O producers and increasing N2O consumers, can be a potential N2O emission mitigation strategy.Based on previous analyses, N2O emission in anammox-processing systems. This strategy has been implemented in a full-scale conventional nitrification/denitrification WWTP, resulting in a 35% reduction of N2O production via the hydroxylamine oxidation pathway [2O emission by allowing heterotrophic denitrifiers to consume N2O and/or N2O precursors  during anaerobic periods, and hence is the most widely adopted approach. It was also suggested that NO2− can be maintained at relatively low levels using a recycling pump to avert N2O accumulation [2− concentration could stimulate N2O emission from nitrifier denitrification and heterotrophic denitrification processes and likely inhibit N2O reduction carried out by nosZ-containing denitrifiers [2− concentration on N2O emission during wastewater treatment could be mitigated by the addition of organic carbon, reducing NO2− influence and maintaining a neutral pH [As shown in  pathway . Insteadmulation , especiamulation ,65. It wtrifiers ,55. The utral pH .2O emission potency [2− by partly oxidizing NH4+, are essential for nitrogen removal in anammox-processing systems. However, aerobic ammonia oxidation is usually accompanied by N2O production via hydroxylamine oxidation and nitrifier denitrification [2O emissions in anammox-driven systems [2O emission potential under anoxic conditions due to the lack of NO reduction enzymes [The microbial community structure of activated sludge in WWTPs determines the nitrogen removal ability and the N potency . Ammoniafication . AOB are systems ,14,54, b enzymes . Comammo enzymes ,84,85, n2O emission, the regular elimination of flocs from anammox granule systems is an effective way to mitigate N2O emission [2O emission under constant DO conditions [2O. Therefore, a lower airflow rate is required during floc removal to maintain constant DO levels and control N2O emission from hydroxylamine oxidation.Considering that nitrifier-enriched flocs are a significant source of Nemission . It was nditions . It shounditions ,76. Alth2O emission from WWTPs. The abundance of nir genes can exceed that of nosZ by up to an order of magnitude in various environments [nirS, nirK, and nor with nosZ are expected to have a significant influence on the genetic N2O emission potential from wastewater treatment systems. Additionally, selectively inoculating and increasing N2O-consuming nosZ II non-denitrifiers in anammox-driven WWTPs is a promising N2O mitigation option [Incomplete denitrification is also a significant source of Nronments . Thus, bn option ,47,86.2O emission by effectively consuming the accumulated NO in activated sludge or granules [2O emission rates compared to flocs [2O emission control during wastewater treatment [2O emission from nitrogen removal.Besides, anammox bacteria can reduce Ngranules ,79,80. Agranules  so that reatment ,84, whic2O emission prediction models are a useful tool for evaluating the proposed N2O mitigation strategies and their effects on nutrient removal performance in full-scale WWTPs. The models typically use elements including microbial N2O generation and reduction pathways, as well as influence factors to simulate the real N2O emission and appraise mitigation strategies , and microbial communities can affect N2O emission.Biologically toxic N2O mitigation strategies for WWTPs include DO control, aeration control, NO2− limitation, C/N ratio control, and flocs removal regulation. Nonetheless, other potential strategies deserve further investigations, These include (i) increasing the biomass and activity of anammox bacteria, which are net NO consumers; (ii) the inoculation of N2O-reducing organisms, such as nosZ II non-denitrifiers with high N2O-affinity; (iii) establishing a symbiotic association of low-N2O-yield comammox and anammox.Common N2O production and consumption mechanisms by the major microorganisms present in WWTPs. Therefore, more omics studies are needed to extend our understanding of the metabolic mechanisms of N2O emission in anammox-driven WWTPs, which will help us find out and formulate effective N2O emission mitigation strategies.The feasibility and efficiency of the proposed mitigation strategies need to be verified and optimized by prediction models, such as mathematical models and deep learning models, in practical application. The development of high-throughput sequencing techniques and data analysis methods can elucidate the structure of the microbial community in WWTPs at high-resolution and low cost and can potentially uncover in great detail N


A serious global public health emergency emerged late November 2019 in Wuhan City, China, by a new highly pathogenic virus, SARS-CoV-2. The virus evolution spread has been tracked by three developing databases: GISAID, Nextstrain and PANGO to understand its circulating variants. In this study, 110 diagnosed positive COVID-19 patient’s samples, were collected from Kasr Al-Aini Hospital and the Children Cancer Hospital Egypt 57357 between May 2020 and January 2021, with clinical severity ranging from mild to severe. The viral genomes were sequenced by next generation sequencing, and phylogenetic analysis was performed to understand viral transmission dynamics. According to Nextstrain clades, most of our sequenced samples belonged to clades 20A and 20D, which in addition to clade 20B were present from the beginning of sample collection in May 2020. Clades 19A and 19B, on the other hand, appeared in the mid and late 2020 respectively, followed by the disappearance of clade 20B at the end of 2020. We identified a relatively high prevalence of the D614G spike protein variant and novel patterns of mutations associated together and with different clades. We also identified four mutations, spike H49Y, ORF3a H78Y, ORF8 E64stop and nucleocapsid E378V, associated with higher disease severity. Altogether, our study contributes genetic, phylogenetic, and clinical correlation data about the spread of the SARS-CoV-2 pandemic in Egypt. Coronaviruses (CoVs) are divided into four subgroups, two of which circulate in mammals; alphacoronavirus and betacoronavirus5. Over the past 20 years more than seven coronaviruses have crossed the species barrier causing respiratory illnesses with varying severity in humans. The pandemic of COVID-19 poses a lethal challenge due to its aggressiveness, high rate of infectivity, severity of symptoms, poor prognosis, and lack of resources. COVID-19 affects different parts of the body causing a variety of symptoms such as viral pneumonia through infecting the lower respiratory systems, and/or diarrhea and vomiting via the gastrointestinal tract, with varying severity6.The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in Wuhan China in December 2019, and since then it has spread to 228 countries and territories constituting a global pandemic causing more than 6 million deaths worldwide7. The SARS-CoV-2 genome comprises 10 functional ORFs; ORF1ab which encodes the viral replication and transcription complex (RTC), 4 structural proteins—spike (S) protein, nucleocapsid (N) protein, envelope (E) protein and membrane (M) protein, and 5 accessory proteins—ORF3a, ORF6, ORF7a, ORF7b and ORF8. ORF1ab comprises two-thirds of the viral genome and encodes two polyproteins (PP1ab and PP1a). PP1ab and PP1a are then cleaved into 16 non-structural proteins nsp1–16; nsp1 (leader protein), nsp2–11 which provide supporting functions for the RTC, RNA-dependent RNA polymerase (nsp12), helicase (nsp13), 3′- to 5′ endonuclease (nsp14), endoRNase (nsp15) and 2′-O-ribose methyltransferase (nsp16). Viral entry into infected cells depends on interaction between the surface spike S protein with angiotensin-converting enzyme 2 (ACE2) host cellular receptor, followed by cleavage of S protein between the S1 and S2 domains, and subsequent internalization of the virus inside the cell. The functions of the S1 and S2 domain is to mediate the binding and downstream membrane fusion, respectively8. A subdomain of S1 folds independently and acts as a receptor binding domain (RBD)9, binding with high affinity to ACE2. The immune-dominance of the RBD makes it the primary target of natural and vaccine-elicited immunity7.Coronaviruses are single-stranded, positive-sense RNA (+ ssRNA) contained in an envelope. The coronavirus genome is approximately 26–32 kb which is currently identified as the largest RNA virus genome size10.Viral genome sequencing provides a powerful approach to monitor introductions into a country and anticipate spread and evolution of the virus. Certain variants of concern have been defined by the world health organization (WHO) as variants causing increased transmissibility, virulence, or reduced vaccine effectiveness. These variants have notable mutations affecting the spike protein, in addition to mutations in other protein coding sequences. Genetic variations in viral genome can result in a better or worse prognosis, thus studying these mutations is critical to enhance patients’ outcome11. Three other mutations accompanied the D614G substitution: a synonymous/silent C to T mutation at position 3037 in ORF1ab, a C to T mutation at position 241 in the 5` untranslated region and a nonsynonymous C to T mutation at position 14,408 in ORF1ab resulting in mutation P3715L (or P314L) in the hydrophobic cleft near the active site of the RNA-dependent RNA polymerase gene (RdRp/nsp12)12.Three software and databases were developed for the real-time tracking of the SARS-CoV-2 evolution through analysis of genomic sequences and the assignment of phylogenetic clades and lineages; Nextstrain, Global Initiative on Sharing Avian Influenza Data (GISAID) and Phylogenetic Assignment of Named Global Outbreak Lineages (PANGO). Analyses done for the S-protein on more than 28,000 spike gene sequences revealed the emergence of a non-synonymous mutation at position 614 (D614G) that was rare before March 2020 and then became more common as the pandemic spread by June 2020In this study, 110 SARS-CoV-2 viral isolates from Kasr Al-Aini Hospital and the Children’s Cancer Hospital Egypt (CCHE 57357) were sequenced using short read sequencing of total RNA content. The genomic variation and phylogenetic diversity of SARS-CoV-2 were investigated, as well as mutation patterns and correlation with clinical severity. Finally, co-occuring mutations were investigated and several groups of co-occurring mutations were identified.All procedures performed in the study involving human participants were in accordance with the ethical standards of the institutional research committee of CCHE 57357 and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. A signed informed consent was obtained from the patient (or patient’s guardian for pediatric patients) as an assent to participate in the current study, all of which were approved by the Institutional Review Board at CCHE 57357.Naso-pharyngeal swabs were collected in viral transport media and RNA was extracted using QIAamp® Viral RNA Mini kit (Qiagen). Confirmatory qualitative commercial RT-PCR kits were used for diagnosis and screening .Library preparation was performed using the TruSeq stranded total RNA . Samples were then normalized, pooled and subjected to 150-base paired-end reads sequencing using Illumina NextSeq system with a minimum of 2.4 Gb sequencing depth per sample.13 followed by host reads removal through mapping trimmed reads against Homo sapiens genome reference (GRCh38) using Burrows-Wheeler Alignment tool (BWA) mem14 then unmapped reads were extracted using SAMtools15. Unmapped reads were mapped later using BWA mem to Wuhan-Hu-1 (MN908947.3) reference genome. Both mapping runs were aligned using paired-end mode and default parameters. Mapped reads were sorted and indexed using SAMtools.Reads were processed in which low reads quality were filtered out using Trimmomatic16 starting with realign of aligned reads and indel quality assignment using Viterbi and indelqual commands from LoFreq package. Finally, LoFreq was used to call low-frequency variants.Variant calling was performed using Lofreq V2.1.217 with default parameters. Filtered variant were annotated with SnpEff18 and extracted using SnpSift19. Spearman correlation coefficient analysis between selected mutations using cor function in R 4.1.220 and corrplot R package21.Generated variants were filtered using LoFreq filter and BCFtools22 using the following parameters (--6merpair --maxambiguous 0.05 --addfragments) followed by phylogenetic tree calculation using IQ-TREE23 which was visualized by iTol24.Consensus sequence for each sample was computed using mpileup and consensus tools from Bcftools. MAFFT was used to perform multiple sequence alignment25 and Nextclade26. Downstream analysis were performed in R20 in which heatmap computed using ComplexHeatmap package27, phylogenetic tree visualized using ggtree28 and Nextclade online tool (https://clades.nextstrain.org/)26.Finally, clade assignment were performed using Ultrafast Sample placement on Existing tRee (UShER)A total of 110 diagnosed positive COVID-19 patients were included in the study; 13 from Kasr Al-Aini Hospital and 97 from the outpatient COVID clinic at the CCHE 57357. Samples were collected in the period between May 2020 and January 2021, and included 60 males (54.5%), and 50 females (45.5%). 88 patients were adults (average age 39.6), and 22 were pediatric patients (average age 10.2). Clinical severity ranged from mild  to moderate  and severe , resulting in hospitalization in 36 patients (32.7%) and a survival rate of 97.3%. Clinical data is summarized in Table https://clades.nextstrain.org/)  define clade 19B, and C3037T, C14408T (ORF1ab P4715L) and A23403G (S D614G) define clade 20A. Clades 20B and 20D are derived from clade 20A and have further clade defining mutations; GG28881-28882AA (N R203K) and G28883C (G204R) for clade 20B and C4002T and G10097A (ORF1ab T1246I and G3278S) for clade 20D. Most of our samples belong to Nextstrain clades 20A and 20D, 34.5% and 40% respectively  Fig. . NextstrFor mutation analysis, samples having mutations with coverage of less than 10X were excluded, this resulted in 71 samples. The SARS-CoV-2 sequences in our study were diverse, and included several mutations. We found 549 mutations in our strains, compared to Wuhan-Hu-1 reference (NC_045512.2) strain, including 294 non-synonymous (amino-acid changing), 224 silent mutations (non-amino acid changing), 24 mutations in intergenic regions, 4 indels and 3 stop codon gained. For further analysis, we excluded mutations that were only reported in one sample. This resulted in 9 mutations in intergenic regions, 82 non-synonymous mutations in coding regions, 66 synonymous (non-amino acid changing mutations), 1 stop codon gained, and 2 indels ; C21707T, C25624T, G28083T and A29406T which result in amino acid changes in spike H49Y, ORF3a H78Y, ORF8 E64stop and nucleocapsid E378V respectively.To analyze correlations between mutations and clinical symptoms, we excluded pediatric patients (14 samples with prefix EGY_CCHE57357_P_) as cancer co-morbidity clearly affects clinical presentation . Of the 57 adult patients included in the analysis, 42 showed mild clinical symptoms, 10 showed moderate clinical symptoms and 5 showed severe clinical symptoms. Four mutations were correlated with moderate or severe clinical symptoms , 19B (A.1-A.6), 20A (B.1), 20B and 20D (B.1.1). PANGO lineages B.1.170, B.1.466, C.17 and C.36 were mainly found in Egypt.In this study, samples collected in Dec 2020 belonged to clade 19B from patients suffering mild symptoms with some reported neutropenia. Samples belonging to clade 20A were collected from May 2020 to January 2021 with all levels of clinical severity ranges from mild to severe symptoms and reports of hypoxemia, neutropenia, and lymphopenia in some patients. Clade 20B circulated from May to August 2020 with patients showing mild to moderate clinical severity and reports of neutropenia and lymphopenia in some. The last clade 20D circulated from May 2020 to January 2021 with patients showing all levels of clinical severity from mild to severe and reports of neutropenia, lymphopenia, and hypoxemia in some patients.12 and was speculated to reduce the efficacy of the vaccines. Our results show an inverse correlation between the occurrence of D614G which occurs in most of the strains, belonging to clades 20A, 20B and 20D and other mutations in the spike N501T, H655Y and IHV68I, which appear in strains belonging to clade 19B.Clade 19B appears late in our samples and harbors D614 in the spike protein. This is in contrast to studies showing the D614 as the original ancestor, and G614 appearing late in the pandemic. D614G occurs at the B-cell epitope, and was reported to enhance the viral replication in human lung epithelial cells and primary human airway tissues thus increasing the infectivity and stability of virions31. IHV68I (or del 69–70) was also reported in the Alpha variant (B.1.1.7) and interferes with viral PCR test accuracy32. Other notable spike mutations were reported in our samples. S477N, which lies in the RBD domain, was only found in a few samples in our study and was reported to slightly increase the ACE2 binding. P681H mutation, which is near the furin cleavage site, was found in eight samples in our study belonging to Nextstrain clade 20D and PANGO lineage C.36, and is considered one of the clade defining mutations for the highly contagious delta variant—Nextstrain clade 20I. S477N and P681H were found to confer resistance to antibody therapy33, whereas contradictory reports were reported for IHV68I35. A study on 176 viral genome sequences from Egypt showed mutation patterns similar to those found in our data36.PANGO lineage A.28 was mostly reported in France, and harbored many key mutations in the spike protein; N501T, H655Y and IHV68I. The N501T spike mutation was predicted to increase the ACE2 binding37, and similar to our data E64stop mutation was associated with severity of clinical symptoms38.In our study, we identified four mutations with association to moderate and severe clinical severity, spike H49Y, ORF3a H78Y, ORF8 E64stop and nucleocapsid E378V. ORF8 gene was reported to be involved in the innate immunity evasionIn conclusion, through SARS-CoV-2 viral sequencing, our study identified several lineages circulating in Egypt between May 2020 and January 2021. We identified a large range of mutations throughout the SARS-CoV-2 genome, including four mutations, spike H49Y, ORF3a H78Y, ORF8 E64stop and nucleocapsid E378V, that were associated with higher disease severity. Additionally, we identified several mutation groups that were associated together and in specific clades. These results could provide a starting point for in vitro and in vivo analysis for the functions of these mutations, and are vital for virus tracking and the development of novel vaccines.Supplementary Information.


Our previous big data analyses reported a strong association between CHI3L1 expression and lung tumor development. In this present study, we investigated whether a CHI3L1-inhibiting natural compound, ebractenoid F, inhibits lung cancer cell growth and migration and induces apoptosis. Ebractenoid F concentration-dependently  and significantly inhibited the proliferation and migration of A549 and H460 lung cancer cells and induced apoptosis. In the mechanism study, we found that ebractenoid F bound to CHI3L1 and suppressed CHI3L1-associated AKT signaling. Combined treatment with an AKT inhibitor, LY294002, and ebractenoid F synergistically decreased the expression of CHI3L1. Moreover, the combination treatment further inhibited the growth and migration of lung cancer cells and further induced apoptosis, as well as the expression levels of apoptosis-related proteins. Thus, our data demonstrate that ebractenoid F may serve as a potential anti-lung cancer compound targeting CHI3L1-associated AKT signaling. Chitinase-3 like 1 (CHI3L1), of the glycoside hydrolase 18 (GH18) family of chitinases, is secreted by macrophages, neutrophils, synoviocytes, chondrocytes, epithelial cells, and smooth muscle cells ,3. Many CHI3L1 has been reported to activate its downstream signaling pathways, such as protein kinase B, also known as AKT, and Wnt/β-catenin, as well as MMP signaling pathways, which have been reported to be significantly associated with lung cancer cell growth . FurtherDespite extensive evidence regarding the role of CHI3L1 and its downstream signals in lung tumor growth, knowledge regarding compounds disrupting CHI3L1-specific downstream signaling, and consequently, preventing lung cancer growth, is limited. Herein, we screened several natural compounds using a luciferase activity assay, and selected ebractenoid F as a candidate compound, because it showed strong inhibitory activity against CHI3L1 and has notable cytotoxic effects against lung cancer cells. Ebractenoid F is one of the active ingredients of Euphorbia ebracteolata. The roots of E. ebracteolata are well known as traditional drugs for treating chronic inflammation, including bronchitis, tuberculosis, and psoriasis . A studyWe screened 58 natural compounds, of which we selected ebractenoid F, using the cell viability assay and CHI3L1 luciferase assay. Ebractenoid F (#.31) effectively inhibited A549 lung cancer cell growth . The tra50 value of 60 µM in A549 cells and 54 µM in H460 cells for 24 h, respectively. At 72 h of treatment with ebractenoid F, the IC50 value was approximately 38 µM in A549 cells and H460 cells  on lung cancer cell proliferation, which was analyzed using the MTT assay. We also found that ebractenoid F inhibited lung cancer cell growth in a concentration-dependent manner, with an IC60 cells A,C. Lung60 cells B,D. We a60 cells . These dTo evaluate the inhibitory effect of ebractenoid F on the migration of lung cancer cells, we conducted a cell migration assay. We evaluated the migrating ability of A549 and H460 cells treated with various concentrations of ebractenoid F using the wound-healing assay. We found that ebractenoid F suppressed the migration of A549 and H460 cells in a concentration-dependent manner A,B. SimiTo determine the cell cycle stage affected by ebractenoid F, A549 and H460 lung cancer cells were treated with different concentrations of ebractenoid F for 24 h; then, the proportion of cells in each phase of the cell cycle was measured using flow cytometry system. When treated with 70 µM of ebractenoid F for 24 h, 10-fold and 20-fold increases in the proportion of A549 and H460 cells, respectively, in the sub-G1 ratio were observed, compared with cells in the respective control groups A,B. The We performed a TUNEL staining assay to determine whether the inhibition of cell growth by ebractenoid F was due to the inhibition of apoptosis, and we used DAPI to stain the nucleus and then analyzed labeled cells with fluorescence microscopy. Consistent with cell growth inhibitory effects, apoptotic cell death was significantly increased in ebractenoid F-treated A549 and H460 cells. The number of apoptotic (DAPI-positive TUNEL-stained cells) A549 and H460 lung cancer cells increased to approximately 23% and 40%, respectively, at an ebractenoid F concentration of 70 μM A,B. The Previous big data analysis studies have shown that CHI3L1 is a target protein close to several cancer outbreaks. In addition, CHI3L1 is associated with cell growth, invasion, and metastasis. To determine whether ebractenoid F binds to CHI3L1 and inhibits lung cancer cell growth by blocking the CHI3L1 signal, we first investigated the expression of CHI3L1 on ebractenoid F treatment. CHI3L1 expression was decreased by ebractenoid F in a concentration-dependent manner A,B. To dCombination therapy is a promising strategy that shows the synergistic effects of two or more drugs used to treat lung cancer simultaneously. These approaches potentially provide therapeutic anticancer effects, such as reduction in tumor growth and metastatic potential, the suppression of mitotically active cells, and the induction of apoptosis ,16. To fAKT signal is associated with CHI3L1. CHI3L1 regulates apoptosis, pyroptosis, inflammasome activation, oxidative injury, antimicrobial responses, melanoma metastasis, and TGF-1 development through activation of the MAPK, AKT, and Wnt/-catenin signaling pathways . Thus, ATo further demonstrate the involvement of CHI3L1 and the AKT signaling pathways in ebractenoid F-induced lung cancer cell growth inhibition and apoptosis, we compared the effects of combination treatment with ebractenoid F and the AKT inhibitor LY294002 and treatment with ebractenoid F or the AKT inhibitor alone. Lung cancer cells were pretreated with the AKT inhibitor 30 min before their treatment with ebractenoid F; then, the expression levels of proteins related to cell growth and apoptosis and CHI3L1 were analyzed A,B. The Our previous GWAS/OMIM/DEG analysis results demonstrated a substantial correlation between CHI3L1 and lung cancer development . Using aCHI3L1 is overexpressed in lung cancer and tumor-associated macrophages, and its high expression is detected in the serum of metastatic lung cancer patients ,20,21. TEbractenoid F has an anti-lung cancer effect, although the exact mechanisms by which it affects CHI3L1 and CHI3L1-associated signaling are yet to be elucidated. CHI3L1 downstream signaling pathways can be induced by CHI3L1 expression. CHI3L1 activates the AKT signaling pathway, thereby regulating cancer cell apoptosis and melanoma metastasis . NotablyEbractenoid F was isolated from the dried roots of Euphorbia fischeriana, as described previously . The str2 humidified environment. According to the manufacturer’s instructions, the A549 and H460 cells were transiently transfected with pcDNA 3.1-CHI3L1 plasmid or CHI3L1 siRNA using Lipofectamine 3000 (for plasmid DNA) and RNAiMAX (for siRNA) reagent in Opti-MEM. We bought CHI3L1 siRNA from OriGene .A549 and H460 cells were obtained from the American Type Culture Collection . The cells were cultured in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 g/mL penicillin, and 100 g/mL streptomycin. The cell cultures were maintained in a 37 °C incubator with a 5% COIC50 value was calculated using the MTT assay data.The A549 and H460 lung cancer cells were plated in 96-well plates and then treated with ebractenoid F at 0, 17, 35, and 70 μM for 24 h. According to the manufacturer’s instructions, the MTT  assay  was used to determine the cells’ viability after treatment. Briefly, MTT (5 mg/mL) was added, and the plates were incubated at 37 °C for 4 h before 150 μL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the absorbance of each well was read at a wavelength of 570 nm using a microplate reader. Experiments for each cell line were performed in triple experiments. The CHI3L1 promoter dual-reporter (HPRM 12518-LvPG04) and negative control (NEG-LvPG04) lentiviral plasmid vectors were purchased from GeneCopoeia . pcDNA3.1+6Myc-tagged CHI3L1 was cloned (Bionics). According to the manufacturer’s instructions, the A549 cells were plated in 12-well plates and transfected with a mixture of plasmid and Lipofectamine 3000 in Opti-MEM medium for 18 h to evaluate the transcriptional luciferase activity . After an appropriate time had passed since the transfection, the medium was harvested to measure the luciferase activity for the analysis of dual-luciferase activity. Luciferase activity was measured by using the Secrete-Pair™ Dual Luminescence Assay kit (GeneCopoeia). The data were read using a luminometer .For the wound-healing assay, the A549 and H460 cells were seeded in an SPLScar Block cell culture dish . This was composed of a 500 um thick wall to artificially generate a cell-free gap. The cells were incubated for 24 h after the culture medium was immediately replaced with RPMI containing ebractenoid F. Using an Olympus light microscope, migrated cell pictures were observed and analyzed using ImageJ software (NIH).4 cells per well and incubated at 37 °C, 5% CO2, in a humidified incubator. The cells were then fixed with 4% formaldehyde for 5 min and permeated with 100% methanol for 15 min before being stained with 0.1% crystal violet for 20 min. In the upper chamber, non-migrated cells were removed with a cotton swab. Using an Olympus light microscope, migrated cell pictures were observed and analyzed using ImageJ software (NIH).For the trans-well assay, the A549 and H460 cells were seeded on upper chamber inserts . The ebractenoid F-treated lung cancer cells were plated at 2.0 × 10To examine the cell cycle arrest of lung cancer cells, ebractenoid F-treated A549 and H460 cells were seeded on a 12-well plate, harvested by trypsin, pelleted, re-suspended in PBS, and fixed in 70% ethanol. At least 1–2 h before the flow cytometric analysis, the cells were washed in PBS and stained with 500 μL of PI/RNase (Propidium Iodide) solution. The flow cytometric analysis was performed with the flow cytometry system .TM Fluorometric TUNEL System  was used for the TUNEL assay to detect apoptotic cells. Lung cancer cells (2 × 104 cells/well) were cultured on 8-chamber slides after being were treated with ebractenoid F. The cells were washed with PBS and fixed by 4% paraformaldehyde in PBS for 20 min at room temperature. They were then permeabilized by 0.1% Triton X-100 in PBS for 5 min at room temperature. The slides were incubated with mounting media for fluorescence-containing DAPI  for 15 min, at room temperature and in the dark. After this, the cells were visualized using the ZEISS Axio Observer fluorescence microscope system . Digital images were analyzed using ZEN 2.1 software (Carl Zeiss).According to the manufacturer’s instructions, the DeadEndhttp://www.swissdock.ch, 4 June 2021) web service based on EADock DSS was used to predict the interaction of CHI3L1 with ebractenoid F. For molecular docking studies, the crystal structure of the CHI3L1 available at the PDB (Protein Data Bank) database was retrieved and prepared. The PDB file format of the CHI3L1 structure (PDB ID 1NWR) was downloaded from the PDB database . Before the docking study, the water molecules were deleted, and polar hydrogens were added to the structures. The Discovery studio  was used to prepare the removal of water molecules and addition of hydrogen molecules, and for building the structure of ebractenoid F. The detailed experimental method was carried out according to the following references [The SwissDock  was used to dissolve 1 mg of ebractenoid F. The CNBr-activated Sepharose 4B and epoxy-activated Sepharose 6B were swelled and washed in 1 mM of HCl through a sintered glass filter, then washed with the coupling buffer. Beads were incubated in the coupling buffer containing ebractenoid F overnight at 4 °C. Three cycles of alternating pH wash buffers  were used to wash the ebractenoid F-conjugated Sepharose 4B and 6B. Ebractenoid F-conjugated beads were then equilibrated with a binding buffer . The control unconjugated CNBr-activated Sepharose 4B and epoxy-activated Sepharose 6B beads were made as previously described. Ebractenoid F-conjugated Sepharose 4B and 6B were mixed with the lysate of myc-tagged human CHI3L1 transfected cells and incubated at 4 °C for 24 h. The beads were then washed with TBST three times. SDS loading buffer was used to elute the bound proteins. The proteins were then resolved by SDS-PAGE, followed by immunoblotting with antibodies against anti-myc .Ebractenoid F was conjugated with cyanogen bromide-activated Sepharose 4B and epoxy-activated Sepharose 6B . In brief, coupling buffer  and visualized by using a FUSION Solo S chemiluminescence detection system .Western blotting was performed as described previously . Lung cat-test. Multiple comparisons were performed using a one-way analysis of variance, followed by Tukey’s tests. Differences between groups were considered significant at p-values of < 0.05.The statistical analysis was performed as described previously . Statist


A high dose of regular vitamin D protected non-obese diabetic (NOD) mice against type 1 diabetes (T1D), when initiated at birth and given lifelong. However, considerable controversy exists on the level of circulating vitamin D D3) needed to modulate the immune system in autoimmune-prone subjects and protect against T1D onset. Here, we evaluated the impact of two doses of dietary vitamin D supplementation (400 and 800 IU/day), given to female NOD mice from 3 until 25 weeks of age, on disease development, peripheral and gut immune system, gut epithelial barrier function, and gut bacterial taxonomy. Whereas serum 25(OH)D3 concentrations were 2.6- (400 IU/day) and 3.9-fold (800 IU/day) higher with dietary vitamin D supplementation compared to normal chow (NC), only the 800 IU/day vitamin D-supplemented diet delayed and reduced T1D incidence compared to NC. Flow cytometry analyses revealed an increased frequency of FoxP3+ Treg cells in the spleen of mice receiving the 800 IU/day vitamin D-supplemented diet. This vitamin D-induced increase in FoxP3+ Treg cells, also expressing the ecto-5’-nucleotidase CD73, only persisted in the spleen of mice at 25 weeks of age. At this time point, the frequency of IL-10-secreting CD4+ T cells was also increased in all studied immune organs. High-dose vitamin D supplementation was unable to correct gut leakiness nor did it significantly modify the increased gut microbial diversity and richness over time observed in NOD mice receiving NC. Intriguingly, the rise in alpha-diversity during maturation occurred especially in mice not progressing to hyperglycaemia. Principal coordinates analysis identified that both diet and disease status significantly influenced the inter-individual microbiota variation at the genus level. The abundance of the genera Ruminoclostridium_9 and Marvinbryantia gradually increased or decreased, respectively in faecal samples of mice on the 800 IU/day vitamin D-supplemented diet compared to mice on the 400 IU/day vitamin D-supplemented diet or NC, irrespective of disease outcome. In summary, dietary vitamin D reduced T1D incidence in female NOD mice at a dose of 800, but not of 400, IU/day, and was accompanied by an expansion of Treg cells in various lymphoid organs and an altered intestinal microbiota signature.The hormonally-active form of vitamin D, 1,25-dihydroxyvitamin D The increase in the incidence of type 1 diabetes (T1D), the most common chronic immune-mediated disease in young children and adolescents, over the last 40 years cannot be explained by genetic drift but is probably related to one or more environmental exposures. The ‘hygiene hypothesis’ proposes that fewer early childhood infections , 3. Inte3 2D3), has powerful anti-inflammatory and immunomodulatory properties and functions by binding to the nuclear vitamin D receptor (VDR), present in most immune cell types [reviewed in (+ T (Treg) cells, and stimulate production of antimicrobial peptides [reviewed in D3) concentrations showed a strong correlation with the relative abundance of Lachnospiraceae genera D3 were associated with peripheral and gut immunity  cells), intestinal barrier function, and gut microbiota composition in diabetes-prone NOD mice. We found that only the 800 IU/day vitamin D-supplemented diet, which increased 25(OH)D3 serum concentration up to a mean value of 193.9 nmol/L, delayed disease onset, significantly reduced T1D incidence, and was accompanied by an increased frequency of Treg cells in the studied immune organs, and an altered intestinal microbiota composition favouring Ruminiclostridium_9 and diminishing Marvinbryantia at the genus level.The complex interplay between vitamin D, the immune system, and the gut microbiota is clearly understudied and not much is known about how vitamin D supplementation affects the intestinal characteristics in autoimmune-prone subjects. In the current study, we evaluated the impact of two different doses of vitamin D supplementation (400 and 800 IU/day), administered lifelong ® R/M-H maintenance diet containing 1% calcium, 0.7% phosphorus, and 1,000 IU vitamin D/kg diet , meeting the recommended concentrations for rodents according to the criteria described in the ‘Nutrient Requirements of Laboratory Animals’  and venous blood . Mice were diagnosed as diabetic  when having positive glycosuria and two consecutive blood glucose measurements above 200 mg/dL. The remaining mice that maintained normal blood glucose concentrations were labelled as non-progressor (NP) mice (The NOD Leuven strain is bred in specific pathogen free (SPF) environment and maintained under semi-barrier conditions in the animal facility of the KU Leuven since 1989. For this study, 93 female NOD mice were randomly assigned at the time of weaning to a grid cage and group housed (5/cage) per dietary condition to either a vitamin D-sufficient diet  or two different doses (400 or 800 IU/day) of a vitamin D-supplemented diet until 25 weeks of age. In NC the natural-ingredient was ssniffAnimals’ . Based o3, 1,25(OH)2D3 and both serum and plasma 24,25(OH)2D3 levels were analysed using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method  Dham, MA) . Serum cSingle-cell suspensions of spleen, pancreatic lymph nodes (PLN), and mesenteric lymph nodes (MLN) were prepared by mechanical disruption from mice at 8 and 25 weeks of age. The following antibodies were used: CD4, CD25, CD39, CD49b, CD73, lymphocyte-activation gene 3 (LAG3), and IL-10 . Intracellular staining was performed with FoxP3/Transcription Factor Staining Buffer Set (eBioscience). Cells were acquired on a Canto II AIG flow cytometer  and analysed with FlowJo software . All analyses were performed on fixable viability dye negative singlet population as outlined in the gating strategy  were food and water-starved overnight and kept in a cage without bedding to limit the coprophagic behaviour. FITC-dextran  dissolved in PBS (100 mg/mL) was administered to each mouse (44 mg/100 g body weight) by oral gavage. After 4 hours, mice were sacrificed, serum was collected and FITC-dextran measurements were performed in duplicate by fluorimeter . Serial dilutions of FITC-dextran in PBS were used to calculate a standard curve.Dextran sulfate sodium  was added at 5% (w/v) in drinking water Faecal pellets were collected longitudinally from mice at both 3 and 8 weeks of age and were stored within 2 hours after sampling at −80°C until processing. The data set comprised 38-paired samples, 18 originated from P mice and 20 from NP mice. Nineteen samples (9 P mice and 10 NP mice) belonged to mice receiving NC, while 10 (5 P mice and 5 NP mice) and 9 (4 P and 5 NP) samples belonged to mice receiving the 400 and 800 IU/day vitamin D-supplemented diet, respectively.DESeq2 package . Samples for paired-end Illumina MiSeq  were constructed using a two-step PCR amplicon approach targeting the V4 region of the 16S rRNA gene as described . The 16S package  in R.Data were plotted as mean ± SEM and statistics calculated with GraphPad Prism 8 software . Diabetes incidence was evaluated by Kaplan–Meier survival analysis with Mantel-Cox log-rank test. For all other data derived from mice receiving NC or vitamin D-supplemented diets throughout life, differences were estimated by a Student’s two–tailed t test or Mann–Whitney U test if the data did not assume Gaussian distribution. However, when two or more groups were compared, normally distributed data sets were analysed by one–way ANOVA with Bonferroni’s multiple-comparison test, whereas the Kruskal–Wallis test with subsequent Dunn’s multiple-comparison test was used for non-normally distributed data.post-hoc Dunn’s test, to determine which diets were driving the differences in the corresponding features. The contribution of metadata variables on genus-level microbiota community variation was determined using multivariate distance-based redundancy analysis (dbRDA) using the Bray-Curtis dissimilarity index as implemented in the R package vegan. Microbiome inter-individual variation was visualized by canonical correspondence analysis (CCA) on the genus-level VS transformed abundance matrix using the ggplot2 v.3.3.5 R package  of daily lifelong dietary vitamin D supplementation on T1D development in the NOD mouse model and evaluated whether serum vitamin D concentrations correlated with disease outcome.3, the major circulating form of vitamin D, was recorded in mice receiving the 400  or 800  IU/day vitamin D-supplemented diet respectively, compared to mice receiving NC (49.5 ± 2.8 nmol/L). Serum values of 25(OH)D3 further increased 1.5-fold with the 800 IU/day vitamin D-supplemented diet compared to values obtained under the 400 IU/d vitamin D-supplemented diet. The amount of circulating 1,25(OH)2D3, the active form of vitamin D, is strictly regulated in a renal negative feedback loop and is independent of circulating 25(OH)D3. We observed that serum concentrations of 1,25(OH)2D3 increased 6.9- and 6.8-fold in mice receiving a 400  or 800  IU/day vitamin D-supplemented diet respectively, compared to mice receiving NC (237.1 ± 26.4 pmol/L). Serum 24,25(OH)2D3 concentrations serve as an indicator of vitamin D catabolic status and depend on the amount of 25(OH)D3. We found that serum 24,25(OH)2D3 concentrations increased 11.5- and 11.4-fold in mice receiving a 400  or 800  IU/day vitamin D-supplemented diet respectively, compared to mice receiving NC (45.9 ± 1.7 nmol/L).We first determined the effects of oral vitamin D supplementation on circulating vitamin D metabolites 5 weeks upon dietary intervention (at 8 weeks of age)  or 800  IU/day vitamin D-supplemented diet compared to mice receiving NC (2.53 ± 0.02 mmol/L), with a delay in disease onset of 5 weeks compared to mice receiving NC  at both 8 and 25 weeks of age using multi-colour flow cytometry.Treg cells are the central component for maintaining peripheral tolerance. Here, we studied FoxP3+ Treg cells in all studied immune organs were similar between mice receiving the 400 IU/day vitamin D-supplemented diet compared to those on NC at 8 and 25 weeks of age. However, the percentages of FoxP3+ Treg cells in mice receiving the 800 IU/day vitamin D-supplemented diet were increased in the spleen, PLN, and MLN, compared to mice receiving the 400 IU/day vitamin D-supplemented diet at 8 weeks of age  by CD4+ Treg cells that subsequently degrade the extracellular ATP pool and catalyse the formation of adenosine, which has the ability to dampen aberrant immune reactions . At the genus level, NOD mice receiving NC showed an increase in the abundance of Lachnospiraceae UCG-001 and a decrease in Helicobacter between 3 and 8 weeks of age (We first demonstrated that the composition of the gut microbiome at 3 weeks of age (weaning) in mice receiving NC was indeed significantly different from that at 8 weeks of age, a time when all mice were still normoglycaemic. We observed that the Shannon’s alpha-diversity index (genera diversity in terms of genera richness and relative abundance) and the Chao1 index (genera richness) significantly increased between 3 and 8 weeks of age in NOD mice receiving NC . At the genus level, we demonstrated that the differential abundance observed over time in Lachnospiraceae UCG-001 and Helicobacter was only significant in the NP mice receiving NC . On the other hand, we identified two bacterial genera that discriminated the 800 IU/day vitamin D group including Ruminiclostridium_9 and Marvinbryantia from both the 400 IU/day vitamin D and NC groups at 8 weeks of age  should be avoided to prevent skeletal defects (3 concentrations are needed to interfere with the initiation and progression of inflammatory and autoimmune pathologies including T1D. Our previous study indicated that serum 25(OH)D3 concentrations reaching a mean value of 290 nmol/L at the end of the study, in 35-week-old NOD mice, was linked to T1D protection. These concentrations are way beyond the 50 and 75 nmol/L that are respectively recommended by the Institute of Medicine (IOM)   and the ne (IOM)  for maxi0 nmol/L . Behind isk-free , 50, 51.3, but not 1,25(OH)2D3 and 24,25(OH)2D3, concentrations positively correlated with T1D outcome later in life. Although 1,25(OH)2D3 is the active  form of vitamin D, its circulating values are 1,000-fold less compared to 25(OH)D3 values and it has a short half-life (4-6 hours), limiting its utility as biomarker for vitamin D status. Both 25(OH)D3 and 1,25(OH)2D3 undergo further metabolism, primarily by renal 24-hydroxylase, to generate 24,25(OH)2D3  immune systems, also the effects of vitamin D on different characteristics of the gut , we hoped to acquire insights into the mechanisms by which vitamin D elicits disease protection in NOD mice. We demonstrated that only the 800, and not the 400, IU/day vitamin D-supplemented diet could significantly delay and prevent T1D development in NOD mice by 25 weeks of age. We also found that at 5 weeks on the diet (at 8 weeks of age), serum 25(OH)D5(OH)2D3 . The proentation , and its2D3 functions directly via its receptor, being present on almost all cells of the immune system 1 towards a tolerogenic response via the induction or expansion of Th2 cells and Treg cells e system . Many imeg cells , 54. Whieg cells , others eg cells . Althougnication . Active or FoxP3 , 59. In gnalling .+ Treg cells endowed with the expression of the cell surface ecto-enzymes CD39 and CD73 that subsequently regulate pericellular adenosine accumulation from extracellular nucleotide catabolism  and LAP, in addition to neuropilin-1  increased in NOD mice during aging (from 3 until 8 weeks of age), specifically in NOD mice not further progressing to overt hyperglycaemia. A greater intestinal microbiota diversity is conducive to enrichment of bacterial taxa that can produce bile acids and short chain fatty acids (SCFA) like acetate, butyrate, and propionate, which are beneficial for not only gut barrier function, and intestinal immunomodulation, but also help maintain homeostasis and health during the whole lifespan  trafficking, could trigger T1D initiation l mucosa , 65. Altl mucosa . We conflifespan –68. The elopment , while o1D onset . Still, Ruminiclostridium_9 and a lower relative abundance of the genus Marvinbryantia compared to the 400 IU/day vitamin D-supplemented and NC groups at 8 weeks of age. We also observed a trend of a higher relative abundance of Ruminiclostridium_9 in mice not further progressing to T1D. Little is known about the physiological and pathogenic roles of Ruminiclostridium_9 and Marvinbryantia in the gastrointestinal tract. A recent report found a decrease in Ruminiclostridium_9 to be associated with exacerbated insulitis in NOD mice  could delay disease onset and significantly reduce T1D incidence in NOD mice. In an attempt to characterise the mechanisms of T1D protection elicited by this dosing regimen, we identified a peripheral expansion of FoxP3https://www.ebi.ac.uk/ena/browser/view/PRJEB51877.The original contributions presented in the study are publicly available. This data can be found here: All animal experiments were approved by the Ethics Committee of the KU Leuven Animal Care and Use Committee and compiled with Belgian animal protection law under animal experiment license 114/2015.P-JM, JC-L, DC, and DE designed and performed research as well as analysed and discussed the data and wrote the manuscript. LV did experiments and analysed data. LV, AV, and JR offered resources. JR discussed the data. CM and CG conceptualized the research goals, acquired major funding, designed research, analysed and discussed the data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was mainly supported by KU Leuven (C16/18/006) and the Fonds voor Wetenschappelijk Onderzoek Vlaanderen . This publication was supported by the Open Access Publication Fund of the KU Leuven.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.


In Huang et alThe corrected Figure The corrected Figure 7 legend is given below:Figure 7 RGS16 played an oncogene role in glioma cell lines. (A) The expression of RGS16 protein in the human astrocytes (HA) cell line and glioma cell lines. (B) The real‐time quantitative PCR (qPCR) assay of RGS16 mRNA expression in LN229, U87, and U251 cell lines after infection with RGS16 siRNA or negative control. (C) The Western blot analysis of RGS16, TCF8, N‐cadherin, and β‐catenin protein expression in LN229, U87, and U251 cell lines after applying RGS16 siRNA or negative control.  Transwell assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control.  Cell scratch assay of LN229 and U87 cell lines treated with RGS16 siRNA or negative control.  Clonogenic assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control.It earlier reads as:RGS16 played an oncogene role in glioma cell lines. (A) The expression of RGS16 protein in the human astrocytes (HA) cell line and glioma cell lines. (B) The real‐time quantitative PCR (qPCR) assay of RGS16 mRNA expression in LN229, U87, and U251 cell lines after infection with RGS16 siRNA or negative control. (C) The Western blot analysis of RGS16, TCF8, N‐cadherin, and β‐catenin protein expression in LN229, U87, and U251 cell lines after applying RGS16 siRNA or negative control.  Transwell assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control. (D) Cell scratch assay of LN229 and U87 cell lines treated with RGS16 siRNA or negative control. (E) Clonogenic assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control.The authors apologize for these errors.


GBA1 gene, which produces the glucocerebrosidase (GCase) protein. There are more than 500 mutations reported in GBA1, among which L444P (p.Leu444Pro) and F213I (p.Phe213Ile) are the most common in the Chinese population, while the function of F213I mutation remains elusive. This study aims to establish the GD mouse model of partially humanized Gba1 gene with F213I mutation. In vitro GCase activity assays showed that the product of partially humanized Gba1 gene, in which the mouse exons 5-7 were replace by the corresponding human exons, displayed similar activity with the wild-type mouse Gba1, while the F213I mutation in the humanized Gba1 led to significant decrease in enzyme activity. ES cell targeting was used to establish the mice expressing the partially humanized Gba1-F213I. Gba1F213I/+ mice did not show obviously abnormal phenotypes, but homozygous Gba1F213I/F213I mice died within 24 h after birth, whose epidermal stratum corneum were abnormal from the wild-type. The GCase activity in Gba1F213I/F213I mice greatly decreased. In conclusion, our results showed that the partially humanized GD mouse model with the F213I mutation was developed and homozygous F213I mutation is lethal for newborn mice.Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the  GBA1). Due to the deficiency of glucocerebrosidase (GCase) activity, glucosylceramide (GlcCer) accumulates in the lysosomes and is metabolized to produce glucosylsphingosine, sphingosine and then sphingosine-1-phosphate (S1P) (Gaucher disease (GD) is one of the most common lysosomal storage diseases. GD is an autosomal recessive hereditary disease caused by mutations in the gene encoding β- Glucocerebrosidase (te (S1P) . GD is cte (S1P) . Accordite (S1P) . Gaucherte (S1P) . Type 2 te (S1P) .GBA1 is located in human chromosome 1q21-22, 7.2 kb long, composed of 11 exons and 10 introns. More than 500 types of mutations linked to GD have been found in GBA1, including splice site mutation, point mutation, coding frameshift mutation, insertion or deletion mutation , F213I  and N188S   . F213I mpatients . The F21patients .Gba1 mutations in GD patients have been established, such as L444P mice, D409V and N370S mice  were generated by using the ES-cell-based gene targeting technology at Shanghai Model Organisms Center in Shanghai, China. Briefly, the ES cell targeting vector was constructed by fusions, containing 3.0 kb 5′ homologous arm, hGBA1 exon 5-7 with F213I mutation, PGK-Neo-poly A, 3.0 kb 3′ homologous arm and MC1-TK-polyA, a negative screening marker. The vector was linearized and transferred into JM8A3 ES cell by electroporation. After PCR identification, the positive ES cell clones were amplified and injected into the blastocysts of C57BL/6J mice to obtain chimeric mice. The Neo-removed Gba1+/F213I mice were obtained by mating chimeric mice with mice with Flp gene. In this way, the exon 5-7 site of the mouse Gba1 gene was replaced by the human GBA1 exon 5–7. The genotype of each mouse was determined by PCR analysis of genomic DNA prepared from tail biopsies. PCR was performed by using the forward primer mGba1-F and the reverse primer mGba1-T for wild type or the forward primer hGBA1-F and the reverse primer hGBA1-T for mutants. The 5′ homology arm and the 3′ homology arm were amplified for sequencing. Sequencing was performed by the Tsingke Biotechnology Co., Ltd. in China. Primer mGba1-5F and hGBA1-5T were designed for 5′ homology arm, while hGBA1-3F and mGba1-3T were designed for 3’ homology arm. The primers are listed in The Institutional Animal Care and Use Committee of Fudan University, China approved all protocols. Mice with partially humanized Gapdh-RF and the reverse primer mGapdh-RT for Gapdh or the forward primer mGba1-RF and the reverse primer mGba1-RT for Gba1. The primers are listed in Total RNA was extracted from tissues using TRIzol reagent (Thermo Fisher) according to the manufacturer’s protocol. RNA degradation and contamination were assessed on 1% agarose gels, and the RNA concentration was measured by using a NanoDrop 1,000 spectrophotometer (Thermo Scientific). cDNA was synthesized by using the Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR , and the integrity of the synthesized cDNA was confirmed by using glyceraldehyde 3-phosphate dehydrogenase (Gapdh) as the endogenous control. Real-time PCR was carried out using SYBR Premix Ex Taq TM II  and measured by using an ABI 7500 instrument. qRT-PCR was performed by using the forward primer mGBA1. Infected cells were pooled by using puromycin selection (1ug/ml), and after 7 days, the cells were conducted for other assays. Partially humanized cDNA was constructed by Tsingke Biotechnology Co., Ltd. Single mutagenesis was inserted by overlapping PCR. Wild type mouse cDNA was synthesized by taking mRNA from wild type mice as template. The sequences are listed in GBA1 was knocked down by CRISPR-Cas9. Cells were harvested for following GCase activity assays and western blotting forty-eight hours after the transient transformation.Crispr/Cas9 system was used to reduce the interference of the endogenous GCase activity, as we performed previously . BrieflyPerkinElmer, EnSpire). During the measurement, blank controls without GCase were set to remove background value. According to the manufacturer’s instructions, specific sample Gucosylceramidase activity = B/(30 × V × P) × D = pmol/min. mg ≡ µU/mg, where B is 4-MU amount from the standard curve (pmol), 30 is the reaction time (min), V is sample volume added into the reaction well (ml), P is initial sample concentration in mg-protein/ml (mg/ml), D is sample dilution factor. One unit of Glucosylceramidase activity is the amount of enzyme that generates 1.0 µmol of 4-Methylumbelliferone per min at pH 4.5 at 37°C. The weight of each mouse tissue has been carefully weighed, and the relative quantity of GCase per mg tissue was calculated, respectively.The GCase enzyme activity assay on the homogenate samples including sample preparation was performed according to the manufacturer’s instructions of Glucosylceramidase Activity Assay Kit . Fluorescence intensity (Ex/Em = 360/445 nm) was detected in a Multimode Plate Reader  supplied with a protease inhibitor cocktail (Roche Diagnostics). Protein samples of 20 ug each sample were separated on a 10% polyacrylamide gel and analyzed by Western blot using anti-HA  and anti-Gapdh  antibodies. Peroxidase-conjugated rabbit immunoglobulin G  was used as the secondary antibody. Western blots were developed using ImmobilonTM Western Chemiluminescent HRP Substrate (Merck Millipore), and analysis was performed with a Luminescent Image Analyzer . The results were quantified by using the ImageJ software.Tissues were immersion fixed with 4% neutral-buffered paraformaldehyde, embedded tissues in paraffin blocks and prepared 5 µm sections. Sections of the different tissues were stained with hematoxylin and eosin as reported previously . Sectiont-test, as reported in the figure legends. The results were significant at p values under 0.05. All statistical tests were performed using Prism software .Statistical significance was assessed using Student’s GBA1 gene is highly conserved in mice and humans. Both of them have 11 exons and exons matched one-to-one (had the same boundaries in both genomes), and matching encoding exons are highly similar (84% sequence identity). F213I, the second popular point mutant GBA1 allele in Chinese GD patients, lies in the exon 6 of human and mouse GBA1 genes. In order to establish the GD mouse model with partially humanized Gba1 gene carrying F213I mutation, we planned to replace mouse Gba1 exons 5-7 with human exons 5-7 carrying the F213I , partially humanized Gba1 (mhGba1) and mhGba1 with F213I (mhGba1-F213I). GCase activity assays showed that there was no significant difference in GCase activity between mGba1 and mhGba1 while the activity of mhGba1-F213I was greatly reduced , spinous (stratum spinosum), granular (stratum granulosum), and cornified (stratum corneum)—were identified in the epidermis. The Gba1F213I/F213I cornified layer appeared abnormal organization. Compared with wildtype littermate controls, the stratum corneum of newborn Gba1F213I/F213I mice was more compact between layers and more basophilic , and the results showed that the expression of Gba1 transcripts had no significant difference between Gba1F213I/F213I mice and wild type mice  coding for GCase, which lead to the accumulation of glucosylceramide in lysosomes  mice  mice . F213I aGBA1 gene is highly conserved in human and mouse. In this research, the mouse Gba1 exon 5-7 was replaced by the human counterparts to generate the human-mouse chimeric Gba1 gene (mhGba1) and in vitro GCase assays showed that the partial humanization had little effect on the activity of GCase, while the F213I mutation in mhGba1 (mhGba1-F213I) greatly reduced the activity. To generate the mhGba1-F213I mice, the mouse genomic fragment containing exons 5-7 were replaced by human corresponding region carrying F213I mutation, and the correctly spliced mature mRNA of mhGba1-F213I was generated. GCase activity assay revealed that the GCase activity decreased in both central and peripheral tissues of Gba1F213I/F213I mice. However, like the previously reported Gba1 point mutation mice, homozygous Gba1F213I/F213I mice died within 24 h after birth  can solve the problem of early postnatal death  up-regulated cellular enzyme activity of some GCase mutants in cultured GD fibroblasts, including F213I, N188S, G202R and N370S  and substrate reducing therapy (SRT) are used as clinical therapeutic methods, and AAV-mediated gene addition has been investigated by other researchers and our team . Gene edal death . It willnd N370S , so it wGBA1 mutation in homozygous or heterozygous form is associated with an approximately 20-fold increase in the risk for Parkinson disease (PD)  . F213I wase (PD) . Up to nGBA1 has a pseudogene GBAP1, acting as competing-endogenous RNA (ceRNA) to regulate GBA1 expression (GBAP1 is involved in the pathogenesis of PD and GD, and manipulation of GBAP1 may have potential therapeutic effects on the diseases. A research also designed specific easy-to-use CRISPR-Cas9 gene editing strategy to correct the common GBA1 N370S mutation and to ensure the integrity of this pseudogene (Moreover, pression . It is peudogene . Mice lain vivo.In summary, our research revealed that F213I mutation caused early postnatal lethality and this partially humanized mouse GD model has the potential for future gene repairing researches