In vitro, one mutant has higher integration and DNA binding activities, and another has a higher disintegration activity compared to the wild-type Cas1. Lastly, we showed that their specificity for selecting a protospacer adjacent motif is decreased. The PeDPaT technology may be used for many robust screens requiring efficient and effortless DNA transduction.Natural prokaryotic defense via the CRISPR–Cas system requires spacer integration into the CRISPR array in a process called adaptation. To search for adaptation proteins with enhanced capabilities, we established a robust perpetual DNA packaging and transfer (PeDPaT) system that uses a strain of T7 phage to package plasmids and transfer them without killing the host, and then uses a different strain of T7 phage to repeat the cycle. We used PeDPaT to identify better adaptation proteins—Cas1 and Cas2—by enriching mutants that provide higher adaptation efficiency. We identified two mutant Cas1 proteins that show up to 10-fold enhanced adaptation   Components of clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) have been identified as central to prokaryotic immune systems . VariousEscherichia coli type I-E CRISPR–Cas system, two proteins, Cas1 and Cas2, are both necessary and sufficient for acquiring new spacers , adaptation is not an efficient reaction, even when the adaptation proteins are artificially overexpressed. In such assays, only up to 20% of bacteria show spacer integration under optimal conditions after 16 h of growth  medium  and agar were from Acumedia. 2xYT medium contained 1.6% (w/v) bacto-tryptone (Acumedia), 1% (w/v) bacto-yeast extract (Acumedia) and 0.5% (w/v) NaCl (Acumedia) in distilled water. Antibiotics, lysozyme, Plasmids were constructed using standard molecular biology techniques. DNA segments were amplified by polymerase chain reaction (PCR). Standard DNA digestions and ligations were carried out according to the manufacturer’s instructions. The primers and DNA templates used for plasmid construction are listed in trxA::kan encoding T7 RNA polymerase (T7-RNAP) under an l-arabinose-induced promoter (IYB6138) was constructed as follows: E. coli IYB510 (trxA::kan from the Keio collection (BW25113Δi IYB510  encodingllection . The kanllection  resultintrxA-FLP instead of gene 1 (encoding T7-RNAP), was constructed by homologous recombination as previously described  harboring pIY168 (Cas1–2) and a mutator plasmid MP-QUR  l-arabinose, and aerated at 37°C for 16 h. The culture was then diluted 1:10 in LB and grown at 37°C for 1 h to reach the logarithmic growth phase.d MP-QUR  was diluPackaging Cas1–2 plasmids 1: A 1 μl aliquot of T7 IYPh38  at a multiplicity of infection (MOI) of ∼1 was then added to 0.5 ml of the culture from the previous step to pack the Cas1–2 plasmids. The infected culture was aerated for up to 2 h at 37°C until lysis occurred.Transduction and adaptation 1: The lysate containing the packaged Cas1–2 mutant library was diluted 1:10, and 0.3 ml was added to 0.3 ml exponentially growing E. coli BW25113  harboring pIY164 (spacer insertion into the array of these plasmids confers chloramphenicol resistance). The transduced culture was aerated for 1 h at 37°C to reach the logarithmic growth phase; 5 ml LB supplemented with 50 μg/ml streptomycin, 50 μg/ml kanamycin and 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was then added, and the culture was aerated at 32°C for 16 h to allow CRISPR adaptation.Selection and enrichment 1: A 0.1 ml sample of the overnight culture from the previous step was spread onto LB plates supplemented with 50 μg/ml kanamycin and 34 μg/ml chloramphenicol to select for colonies that underwent spacer acquisition in the pIY164 plasmid.Packaging of Cas1–2 plasmids 2: LB (3 ml) was spread on the plate to collect the resistant colonies. The collected colonies were diluted to OD600 ∼ 0.1 in 0.5 ml LB supplemented with 50 μg/ml kanamycin and were aerated at 37°C for 1 h to reach the logarithmic growth phase; 1 μl of wild-type T7 phage  at an MOI of ∼1 was then added to 0.5 ml of the culture from the previous step to pack the Cas1–2 plasmids. The infected culture was aerated for up to 2 h at 37°C until lysis occurred.Transduction and adaptation 2: The lysate containing the packaged Cas1–2 mutant library was diluted 1:10, and 0.3 ml was added to 0.3 ml of exponentially growing E. coli IYB6138  harboring pIY164. The transduced culture was aerated for 1 h at 37°C to reach the logarithmic growth phase; 5 ml LB supplemented with 50 μg/ml streptomycin, 50 μg/ml kanamycin and 1 mM IPTG was then added, and the culture was aerated at 32°C for 16 h.Selection and enrichment 2: A 0.1 ml sample of the overnight culture from the previous step was spread onto LB plates supplemented with 50 μg/ml kanamycin and 34 μg/ml chloramphenicol to select for colonies that underwent adaptation in the pIY164 plasmid; 3 ml LB was spread on the plate to collect the resistant colonies.Repetition of the steps: Steps 2–7 constitute a complete enrichment cycle. Altogether, we carried out eight cycles. In the first two cycles, to increase the variability of Cas1–2, we added a mutagenesis step in which the lysate prepared in step 5 was transduced into IYB6138/MP-QUR and then we proceeded to step 2. In the remaining six cycles, we followed the above protocol from step 2 to step 7 repeatedly.E. coli BW25113/pIY164 harboring Cas1–2 in different variations—pIY68, pIY171, pIY183, pIY184 or pIY185—were diluted to OD600 ∼ 0.01 in LB supplemented with 50 μg/ml streptomycin, 50 μg/ml kanamycin and 1 mM IPTG, and were aerated at 37°C for 16 h. Cultures were then serially diluted 1:10 to 1:107, and 10 μl of each dilution was plated on LB agar plates supplemented with either 50 μg/ml kanamycin or 50 μg/ml kanamycin and 34 μg/ml chloramphenicol. The fraction of chloramphenicol-resistant colonies was calculated by dividing the number of chloramphenicol-resistant colonies (grown on plates supplemented with kanamycin and chloramphenicol) by the total number of colonies (grown on plates supplemented with kanamycin only).Overnight cultures of E. coli NEB5α harboring Cas1–2 in different variations—pIY68, pIY171, pIY183, pIY184 or pIY185—were diluted to OD600 ∼ 0.01 in LB supplemented with 50 μg/ml kanamycin and 1 mM IPTG, and were aerated at 37°C for 16 h. A 1 μl sample from each culture then served as a template for PCR amplification using IY375Fa and IY13R. Adaptation bands were digitally captured and quantified using ImageJ software.Overnight cultures of E. coli NEB5α harboring plasmids encoding the different Cas1–2 mutants were diluted to OD600 of 0.01 in LB supplemented with 50 μg/ml kanamycin and 1 mM IPTG. Two hundred microliters of each culture was transferred into a 96-well plate in triplicates. Cells were grown with continuous shaking at 205 rpm in a Tecan Infinite M Plex plate reader at 37°C and reads were acquired every 5 min for 14 h.Overnight cultures of Adaptation assay for next-generation DNA sequencing (NGS) was carried out as described previously . Briefly600 ∼ 0.5, and then induced with 0.5 mM IPTG at 18°C for 18 h. The cells were then harvested and resuspended in lysis buffer . Sonication was used to lyse the cells and supernatant was collected after centrifugation. The cleared supernatant was subjected to a 1 ml His-Trap column  and washed with 25 column volumes of wash buffer . The bound protein was then eluted with elution buffer  and dialyzed overnight at 4°C against dialysis buffer  in the presence of TEV protease to remove the His-maltose binding protein tag. The cleaved protein was again passed through a 1 ml His-Trap column, and flowthrough containing the cleaved protein was collected. The protein was concentrated using Amicon® Ultra-4 Centrifugal Filter Units (3 kDa columns) and further purified in a Superdex 200 Increase 10/300 size-exclusion chromatography column  equilibrated with protein buffer . The fractions containing the protein were pooled and concentrated using Amicon® Ultra-4 Centrifugal Filter Units (3 kDa columns), snap frozen and stored at −80°C.Plasmids pMBP-cas1, pMBP-dcas1, pMBP-cas1-S1, pMBP-cas1-S2, pMBP-cas1-S1,S2 and pETDuet-IHF α/β were separately transformed into BL21-DE3 cells and grown in 1 l of 2xYT supplemented with 50 μg/ml kanamycin at 37°C to OD600 ∼ 0.7, and then induced with 0.5 mM IPTG at 25°C for 22 h. The cas1–cas2 integration complex was then purified as described  with 30 nM of protospacer at 37°C for 10 min. Following incubation, 200 nM of the IHF and 100 nM of linear DNA substrate were also added to the reaction mixture in a reaction buffer containing 50 mM KCl, 10 mM MgCl2, 20 mM HEPES–KOH (pH 7.5) and 1 mM DTT, and incubated at 37°C for 30 min. The reaction was stopped by adding an equal volume of loading dye containing 95% (v/v) formamide, 20 mM EDTA and 0.025% bromophenol blue, and boiled at 95°C for 10 min. The samples were then loaded onto a 10% polyacrylamide gel containing 7.5 M urea. Electrophoresis was carried out at 175 V for 1 h in 0.5× TBE buffer. Cy5.5 fluorescence was detected using a Sapphire biomolecular imager (Azure Biosystems).The Cy5.5-labeled 33-bp double-stranded protospacer DNA was prepared by annealing the following two strands: strand 1, CY5.5-GAAGAAGGAGATATACCATGGGGAGCGGTACAA; strand 2, CCGCTCCCCATGGTATATCTCCTTCTTCTGAGCTTGA. The DNA substrate containing the CRISPR array of Binding of the Cas1–Cas2 complex with protospacers to a plasmid encoding a CRISPR leader–repeat was studied using electrophoretic mobility shift assay (EMSA). In this assay, 400 nM of pre-annealed Cy5.5-labeled protospacers  were first incubated with 40 nM of Cas1–Cas2 complex for 10 min at 22°C in reaction buffer containing 20 mM HEPES–NaOH (pH 7.5), 50 mM KCl, 1 mM DTT, 5 mM EDTA, 0.15% (v/v) Tween 20 and 100 g/ml bovine serum albumin. Following incubation, 7.5 nM of leader–repeat-containing plasmid (pIYIT5) was added and incubated at 22°C for an additional hour. The samples were then mixed with bromophenol blue-containing loading dye and separated in a 0.6% agarose gel in 1× TAE buffer. The gel was scanned using a Sapphire biomolecular imager (Azure Biosystems) to detect the Cy5.5 fluorescence. The agarose gel was also stained with EtBr to visualize the separated plasmid DNA bands.Y-DNA was prepared by annealing four single-stranded DNAs in a stepwise manner: 95°C for 5 min, 80°C for 10 min, 70°C for 10 min, 65°C for 20 min, 60°C for 10 min, 50°C for 20 min and then slowly cooling the reaction mixture at a rate of 1°C/min to room temperature. The following strands were used for Y-DNA preparation:Strand 1: GGCCCCAGTGCTGCAATGGAStrand 2: GTGAGCGTGGGTCTCCATTGCAGCACTGGGGCC-GCCCAATTTACTACTCGTTCTGGTGTTTCTCGTACCGCGAGACCCACGCT CACStrand 3: Cy5.5Strand 4: TCGCGGTACGAGAAACACCAGAACGAGTAGTAAATTGGGCWT, Cas1-S1, Cas1-S2 and Cas1-S1,S2) incubated with 40 nM Y-DNA in a reaction buffer containing 50 mM KCl, 10 mM MnCl2, 20 mM HEPES–KOH (pH 7.5) and 1 mM DTT. Reaction mixtures were incubated for 1 h at 37°C. Equal volumes of formamide loading dye  were mixed with the reaction mixtures and loaded onto 10% polyacrylamide gels containing 7.5 M urea. Electrophoresis was carried out at 175 V for 1 h in 0.5× TBE buffer. Cy5.5 fluorescence was detected using a Sapphire biomolecular imager (Azure Biosystems).The disintegration reaction was carried out in 10 μl reaction mixture containing 210 nM purified Cas1 proteins (Cas1To identify Cas1 and Cas2 (hereafter Cas1–2) proteins with enhanced adaptation activity, we devised a system, which we term perpetual DNA packaging and transduction (PeDPaT), that results in the enrichment of adaptation proteins displaying enhanced activity. We used bacteria harboring plasmids that contain a CRISPR array and confer antibiotic resistance upon spacer acquisition . The platrxA), and another pair that lacked a different gene required for phage propagation (T7-RNAP). Each phage could propagate and package plasmids on the other bacteria. We alternated the bacteria so that packaging (which requires phage propagation) was done on the permissive strain while transduction was done on the restrictive strain . The plasmids were then transduced into an E. coli strain lacking T7-RNAP (preventing lysis). As indicated above, the transduced strain encoded a CRISPR array, providing chloramphenicol resistance only upon spacer acquisition. Thus, bacteria with active Cas1–2 that acquired spacers were selected, and those with enhanced Cas1–2 adaptation were enriched. It is important to note that only the plasmids encoding Cas1–2 carry a T7 packaging signal, and thus only they are enriched in this process. In the second transduction step, T7 phages encoding T7-RNAP were used to package the enriched Cas1–2 plasmids . These phage particles were then used to transduce the Cas1–2 plasmids into E. coli lacking trxA (preventing lysis). Here, the transduced strain also contained a CRISPR array that provided resistance upon adaptation. Thus, in this cycle, Cas1–2 proteins with enhanced adaptation efficiency were also enriched. Passaging the plasmids between the cycles in bacteria with a plasmid that induces mutagenesis  or without chloramphenicol (cm−). The percentage of bacteria acquiring resistance (on cm+ plates) out of all colonies (on cm− plates) was then calculated. We observed an over 4-fold increase in resistant colonies when Cas1-S1 was introduced, compared to the wild-type Cas1. Cas1-S2 showed an over 7-fold increase, and Cas1-S1,S2 showed a ∼10-fold increase in the percentage of resistant colonies , showed almost no resistant colonies due to lack of adaptation. We also examined the effect of substituting several residues at position 76 and determined that the V76L substitution enhances the activity more than all other tested substitutions , one had the Cas1-V76L mutation (hereafter Cas1-S2) and one had both mutations . To validate that the identified mutations enhance adaptation, we introduced them into naïve plasmids and tested their adaptation efficiency using two different assays. The first determined the resistant colonies arising with each Cas1 mutant due to adaptation, compared to the wild-type Cas1. The newly constructed plasmids, encoding the Cas1 variants and Cas2, were transformed into s Figure . As expeitutions .E. coli bacteria encoding a CRISPR array in their genome. Following overnight Cas1–2 expression, a sample of the culture served as a template for PCR of the CRISPR array. The PCR shows a larger band corresponding to the band that acquired spacers, as well as the original non-adapted band. The ratio of the intensities of these bands is proportional to the number of colonies that underwent adaptation compared to those that did not. In this assay, Cas1-S1 and Cas1-S2 showed a significant increase of over 2- and 3-fold in adaptation compared to the wild type  required for efficient and precise integration of spacers into the first position in a linear CRISPR array , a positive residue often appears in the homologous position  might be gleaned from the structure of the holo-complex of Cas1–Cas2 with a spacer and integration-site DNA (pdb 5WFE) . We findposition , furtherThe site of the substitution V76L lies at the interface between the two Cas1 domains—alpha and beta Figure . The arcin vivo of both proteins stems from two distinct mechanisms—enhanced DNA binding for Cas1-S1 and enhanced disintegration activity for Cas1-S2. The enhanced DNA binding activity allows Cas1-S1 to both retrieve more spacers and integrate them more efficiently into the CRISPR array. The increased disintegration activity of Cas1-S2 allows it to retrieve spacers more efficiently than the wild type, and thus have enhanced overall integration activity.The biochemical assays Figure  showed eWhile the specific activity of the adaptation proteins significantly increased, the specificity of PAM selection significantly decreased. The decrease in specificity suggests that a relatively low level of Cas1–2 activity is essential for maintaining specificity. In many systems, higher activity comes at the expense of higher fidelity, and vice versa. For example, a recent report showed remarkably specific cleavage activity of the Cas9 protein, accompanied by extremely low activity . This phE. coli , there is perfect homology of all residues to K-12 Cas1, except at position 76, where the valine is replaced by isoleucine, a residue that enhances the activity. This finding further suggests that position 76 plays a major role in determining adaptation activity.The driving hypothesis of this study was that some mutations in the adaptation proteins may downregulate their activity due to their possible hazardous nature. Although we cannot decisively conclude that the identified residues indeed regulate Cas1 activity, we carried out an experiment showing that several residues at position 76 enhance its activity over that of the wild type . Intereshttps://ecocyc.org/gene?orgid=ECOLI&id=G7425-MONOMER#tab=FTRS; https://www.uniprot.org/uniprotkb?query=cas1). The identified proteins may be used for applications requiring robust adaptation, such as a molecular machinery for recording biological events, or even for storing digital images and movies in DNA (To the best of our knowledge, this is the first example of a mutation enhancing Cas1 activity in any bacterial species (s in DNA ,38–42. Wgkad510_Supplemental_FileClick here for additional data file.


Breast cancer (BC) has posed a great threat to world health as the leading cause of cancer death among women. Previous evidence demonstrated that germ cell-specific gene 2 (GSG2) was involved in the regulation of multiple cancers. Thus, the clinical value, biological function and underlying mechanism of GSG2 in BC were investigated in this study.The expression of GSG2 in BC was revealed by immunohistochemistry (IHC), qPCR and western blotting. Secondly, the biological function of GSG2 in BC was evaluated by MTT assay, flow cytometry, Transwell assay and wound healing assay. Furthermore, the potential molecular mechanism of GSG2 regulating the progression of BC by co-immunoprecipitation (Co-IP) and protein stability detection.Our data indicated that GSG2 was frequently overexpressed in BC. Moreover, there was a significant correlation between the GSG2 expression and the poor prognosis of BC patients. Functionally, GSG2 knockdown inhibited the malignant progression of BC characterized by reduced proliferation, enhanced apoptosis and attenuated tumor growth. Migration inhibition of GSG2 knockdown BC cells via epithelial-mesenchymal transition (EMT), such as downregulation of Vimentin and Snail. In addition, E2F transcription factor 1 (E2F1) was regarded as a target protein of GSG2. Downregulation of E2F1 attenuated the promoting role of GSG2 on BC cells. Mechanistically, knockdown of GSG2 accelerated the ubiquitination of E2F1 protein, which was mediated by E3 ubiquitin ligase MDM2.GSG2 facilitated the development and progression of BC through MDM2-mediated ubiquitination of E2F1, which may be a promising candidate target with potential therapeutic value.The online version contains supplementary material available at 10.1186/s12967-023-04358-2. Breast cancer (BC) is often referred to as the “pink killer”, and its incidence ranks first among female malignant tumors . In addiProtein kinases exhibit roles in many cellular processes such as gene expression, membrane transport, metabolism, proliferation, apoptosis, movement . Germ ceFurthermore, GSG2 inhibition delays cell cycle progression through interphase in cancer cells, which has a great potential as an anticancer therapeutic target , 17. MorIn the present study, the expression level of GSG2 in BC and its prognostic correlation were revealed. Secondly, the biological function of GSG2 in BC has been explored separately in vitro and in vivo. More importantly, the potential molecular mechanism of GSG2 regulating the progression of BC had been initially explored. Altogether, the effects of GSG2 in the development and progression of BC were investigated.The tumor tissues (n = 128) and paired normal tissues (n = 12) of human survival BC patients were purchased from Xi’an Alina Biotechnology Co., Ltd , including pathological details, such as age, gender, post-operative tumor grade and stage. None of the BC patients received radiotherapy or chemotherapy prior to biopsy sampling. The experimental procedures were approved by the Ethics Committee of Qilu Hospital of Shandong University and informed consent of the BC patients.Formalin-immobilized BC and paired normal tissues were dewaxed in xylene, rehydrated in ethanol solution, incubated with 3% hydrogen peroxide to block endogenous peroxidase and non-specific binding sites. Next, the tissues were incubated with the primary antibody ,  at 4 °C overnight and the secondary antibody HRP goat anti-rabbit IgG  at room temperature for 1 h. Afterwards, tissues were stained with 3,3-diaminobenzidine solution for 3 min and the nuclei were counterstained with hematoxylin. The staining intensity was scored according to the criteria described in the literature . The res2 at 37 °C.Human mammary epithelial cell HBL-100, BC cell lines BT549, MCF-7 and MDA-MB-231 were cultured in Dulbecco’s modified eagle medium (DMEM)  supplemented with 20% fetal bovine serum (FBS)  and maintained 5% CO6 MCF-7 and MDA-MB-231 cells were cultured for 24 h and transfected with recombinant BRV-112 vector containing shGSG2, GSG2 + NC-shE2F1, shE2F1 + NC-GSG2, GSG2 + shE2F1 and NC(OE + KD)  (1 × 108 TU/ml) at a MOI (multiplicity of infection) of 10, respectively using Lipofectamine® 3000 (Invitrogen) at 37℃. After cultured for 72 h, the expression of GFP was observed and evaluated under a fluorescence microscope . The sequences as follows: shGSG2-1, 5′-CCACAGGACAATGCTGAACTT-3′, shGSG2-2, 5′-AAGGAAACTGGTGGTGGGAAA-3′, shGSG2-3, 5′-AGGGATTGACTTAGAGCAAAT-3′; shE2F1-1, 5′- GGGCATCCAGCTCATTGCCAA-3′, shE2F1-2, 5′- CAGCTGGACCACCTGATGAAT-3′, shE2F1-3, 5′- GACCTCTTCGACTGTGACTTT-3′; shCtrl: 5′-TTCTCCGAACGTGTCACGT-3′. BRV-112, 5′-CCGGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTG-3′.Short hairpin RNAs (shRNA) target human GSG2 (shGSG2), E2F1 (shE2F1) and scrambled sequences (shCtrl) were digested using AGE I  and EcoR I  and inserted into the lentiviral vector BRV-112 that labeled with green fluorescent protein (GFP) . Meanwhile, the amplified sequence of GSG2 (GSG2) was synthesized and inserted into BRV-112 for overexpression of GSG2. Subsequently, 5 × 10RNA was extracted from MCF-7 and MDA-MB-231 cells using Trizol [MCF-7 and MDA-MB-231 cells protein was purified with RIPA (Beyotime) and the concentration was determined by bicinchoninic acid (BCA) Protein Assay Kit (Beyotime). The 20 µg/well total protein was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), transferred to polyvinylidene difluoride (PVDF) membrane (Millipore), hybridized with corresponding primary antibody  were digested by trypsin, resuspended into cell suspension, and cultured at a density of 2000 cells/well for 24 h, 48 h, 72 h, 96 and 120 h. The OD 490 nm value was detected under Microplate Reader after adding 20 µl 5 mg/ml MTT and 100 µl DMSO in turn. Finally, growth curve was drawing and analyzed by T-test method.MCF-7 and MDA-MB-231 cells were inoculated into 6-well plates (2 ml/well) for 5 days. The cells were digested by trypsin, resuspended into cell suspension, fixed with ethanol, stained with propidium iodide (PI) . Afterwards, the cells were successively washed and precipitated by PBS and 1 × binding buffer, stained with 5 µl annexin V-APC in the dark for 15 min and then stained with 5 µl PI again. The apoptosis rate was measured by flow cytometry and the results were analyzed by T-test.α–tubulin levels.In this experiment, the apoptosis antibody array kit  was used to detect the expression of proteins related to human apoptosis signaling pathway. MCF-7 cells protein was diluted with the array diluent buffer kit to 0.5 mg/ml. The protein was transferred to antibody membrane, blocked with blocking buffer for 30 min at room temperature and incubated with HRP linked streptavidin overnight at 4 °C. Finally, protein signal was observed with ChemiDoc XRS chemiluminescence and imaging system. The gray value was quantitated using Quantity One software and normalized to the MCF-7 and MDA-MB-231 cells were digested by trypsin, resuspended into cell suspension  and placed into Transwell inner chambers  (Corning) 100 µl. 500 µl DMEM medium containing 30% FBS was added in the outer chambers. After incubation 24 h at 37 °C, the migratory cells on the lower surface of Polycarbonate membrane were fixed with 4% precooled paraformaldehyde for 30 min and stained with 0.1% of crystal violet for 20 min at room temperature. Following washing with PBS, the cells in 5 fields were randomly selected under a fluorescence microscope , and the migration rate was calculated according to the number of migratory cells.MCF-7 and MDA-MB-231 were seeded into 96-well plats. After cell growing for 24 h, the complete medium was changed into medium with lower serum concentration. Then, we scratched a wound across the cell layer using a 96-wounding replicator . Cell debris were slightly rinsed with serum-free medium for 2–3 times. Images were captured at 0 and 24 h under a fluorescence microscope . Cell migration rate of each group were calculated based on the migration distance.6 cells/mouse) and divided into shCtrl (n = 10) and shGSG2 (n = 10) groups. Twelve days after cell injection, the tumor size was monitored once or twice a week and tumor volume was calculated as: π/6×L×W×W (L represented long diameter and W represented short diameter). Mice were anesthetized with 0.7% sodium pentobarbital (10 µl/g) and the tumor load was assessed by fluorescence imaging under the IVIS spectroscopic imaging system (emission wavelength of 510 nm). 49 days after the subcutaneous injection, the mice were sacrificed , the tumor was removed and weighed. Finally, IHC staining was used to detect the expression of KI67  in tumor tissues of each group.The experimental procedures performed on the mice were approved by the Ethics Committee of Qilu Hospital of Shandong University and accordance with Guide for Care and Use of Laboratory animals . The total of 20 BALB/c nude SPF mice   were kept in a sterile environment and fed normally. Lentivirus-transfected MDA-MB-231 cells were digested with trypsin and injected into the right forearm of mice with 500 µl (8 × 10RNA was purified from MDA-MB-231 cells and sequenced using Affymetrix human Gene Microarray Prime View (Affymetrix Scanner 3000 scan). The differentially expressed genes (DEGs) was recognized with criterion of |Fold Change| ≥ 1.8 and false discovery rate (FDR) ≤ 0.05 and presented as hierarchical clustering. Significant enrichment of DEGs in disease and function was investigated based on Ingenuity Pathway Analysis (IPA).http://ubibrowser.ncpsb.org). In order to reveal the impact of GSG2 knockdown on downstream genes and signaling pathways, we used the database for Annotation Visualization and Integrated Discovery  to analyze the Kyoto Encyclopedia of Genes and Genomes (KEGG).The human E3 ubiquitin ligase-substrate interaction network was explored on UbiBrowser  and P value < 0.05 as statistically significant. The significance difference between two groups were determined using the two-tailed Student’s T test or One-way ANOVA analysis.According to the analysis of BC and normal tissue samples in the TCGA database, the expression level of GSG2 in BC was significantly higher than that in normal tissue P < 0.001; Fig. ; Fig. 1AIn order to determine the clinical significance of GSG2 in BC, we analyzed the GSG2 levels in 128 BC patients. Representative images of IHC staining for BC patients’ tissues were shown in Fig. Next, we further revealed the difference in the expression of GSG2 in BC cells and normal mammary epithelial cells at the cellular level. As illustrated in Fig. Based to the results of MTT assay, we found that MCF-7 and MDA-MB-231 cells in shGSG2-1 and shGSG2-1 groups exhibited slower proliferation rate than that in shCtrl group (P < 0.001)   for 0–8 h and the protein stability of E2F1 was tested, indicating that GSG2 knockdown accelerated the degradation of E2F1 protein Fig. D. NotablConsidering that ubiquitination is a multi-step reaction process involving the participation of multiple ubiquitin ligases . AccordiAdditionally, the high expression of E2F1 in BC tissues and cell lines suggested that E2F1 may play a key role Fig. A and B. In addition, the results of KEGG indicated that GSG2 knockdown resulted in significant enrichment of p53 signaling pathway  in BC. Moreover, GSG2 knockdown distinctly promoted E2F1 ubiquitination. A large number of studies have revealed and demonstrated that targeted ubiquitination has great therapeutic potential in a variety of cancers –39. As wFurthermore, the results of KEGG indicated that GSG2 knockdown resulted in significant enrichment of p53 signaling pathway. We found that p53 may be involved in the process of GSG2 promoting BC cell progression. Previous study reported that p53 suppressed E2F1 expression upon DNA damage by forming p53-E2F1-DNA complex . On the In summary, GSG2 facilitated the development and progression of BC through MDM2-mediated ubiquitination of E2F1, which may be a promising candidate target with potential therapeutic value.Additional file 1: Fig. S1. Effective sequence screening targeting GSG2 and the effect of GSG2 knockdown on apoptosis-related protein expression. Fig. S2. Functional enrichment analysis and differential gene expression detection after GSG2 knockdown. Fig. S3. The knockdown of E2F1 and the overexpression and pathway enrichment of GSG2 in tumor cells were determined. Table S1. Primer sequence for PCR. Table S2. Antibodiesused in western blotting and Co-IP. Table S3. Cox multivariate analysis of GSG2expression in BC and other clinical features.Additional file 2. Summary of differential expression gene after GSG2 knockdown.


Ploidy changes were examined by morphological, cytological, macroscopic, and microscopic methods. Leaf thickness, chlorophyll contents, stomatal sizes, and chloroplast numbers of polyploid seedlings stimulated with mutagens increased but their stomatal densities decreased. Flow cytometry (FC) analyses were performed on 50 samples selected by morphological and microscopic preliminary determinations. In FC analyses, 1 tetraploid seedling and 4 mixoploid seedlings from Fercal offspring and 1 mixoploid seedling from 41 B offspring were verified. The nuclear DNA content of tetraploid and mixoploid seedlings were increased by 2.00 and 1.34-fold, respectively, when compared to their diploid parents. Chromosome counts in root tip samples propagated in vitro from the tetraploid Fercal offspring confirmed a 2-fold increase compared to the diploid parent. In polyploidy induction studies, it was deemed appropriate to use FC analysis and chromosome count together to confirm the ploidy levels of mutants. Oryzalin and N2O applications at different doses and exposure times were found to be effective for inducing polyploidy in 41 B and Fercal grapevine rootstocks.In this study, the mutagenic effects of different doses and exposure times of oryzalin and Nitrogen Protoxide (N Due to its socio-economic value, viticulture is carried out in various regions and climates all over the world, and therefore, diversity is needed in both grape and rootstock varieties. Over 25000 grape varieties and 1435 grape rootstocks have been registered in the world . HoweverFollowing the destructive effect of phylloxera and the necessity to use rootstocks, rootstock breeding was started in France and 862 vine rootstocks were identified until today, and 171 vine rootstocks were identified in Italy, which started later. Grapevine rootstock breeding is also studied in countries such as Germany, Hungary, and Czech Republic. Although the vineyards are mostly established on their own roots in China, which has the largest vineyard area today, rootstock breeding has intensified in the last 10 years . Also, rDue to the heterozygous nature of the grapevine, more than 274 billion different possible combinations of genotypes are formed when hybridized. Therefore, the prediction of hybridization results is difficult, and it is extremely low to reach defined chromosome combinations by crossing .Polyploidy is the presence of more than two genomes per somatic cell in the organism. Polyploid organisms, it may occur spontaneous with chromosome copies of somatic cells (mitotic ploidy) or in meiosis due to the failure to separate homologous chromosomes that cause diploid gametes . PolyploWith the increased interest in the tetraploid grape varieties, polyploidy studies intensively are conducted in China , Brazil 2O can easily penetrate the plant and promote polyploidy due to its small molecule structure. In addition, N2O is not as harmful to nature as colchicine  × (Vitis berlandieri Planch.)] and Fercal [(Vitis vinifera L. × Vitis berlandieri) × 333 EM (Cabernet-Sauvignon × Vitis berlandieri)] grapevine rootstocks in a moist perlite at +4°C for 90 days were used as a plant material. With the onset of the first germination, N2O applied seeds at various doses  and exposure times (48-h and 96-h) were planted in germination pans. In addition, seeds that were not treated with N2O were sown in plastic viol and 25 and 100 μM oryzalin were applied with a Pasteur pipette for 2 days (48 h) and 4 days (96 h) twice a day (08:30 and 18:00) during the formation of cotyledon leaves. Control plants were not mutagenized during the seed germination period in which N2O was applied and the cotyledon leaves developed when oryzalin was applied. Only pure water was dripped onto the growth zones with a Pasteur pipette for 4 days (96 h). Mutagen-treated plant materials were planted in peat:perlite (3:1) medium in the greenhouse. N2O and oryzalin mutagen agents were applied to a total of 1200 plants in the same amount for each of 41 B and Fercal rootstocks. The effects of mutagens were investigated by the survival rates of plants, stomatal density (number mm–2), stoma size (μm), chloroplast numbers in stoma guard cells (number stoma–1), leaf thickness and chlorophyll amount  in developing plants. In addition, FC analysis, nuclear DNA contents and chromosome counting were determined within the scope of cytological examinations.The seeds of 41 B  was added and left in an environment with light isolation for 5 minutes. Subsequently, samples were analyzed in FC  Duncan multiple comparison test, dose and duration applications JMP 7 statistical programs with Student’s t test at p < 0.05 significance level.The data obtained because of oryzalin and N2O treated plants were lower than the control. The lowest viability rate in oryzalin applications was determined at 100 μM- 96-h application to 41 B grapevine rootstock , Fercal (2n), 41 B (2n + 4n), Fercal (2n + 4n), Fercal (4n) and Kyoho (4n)], whose ploidy level was detected by FC analysis with our mutagen stimulation, were significant. Leaf thicknesses of selected seedlings and control plants were measured as 190.05 ± 4.78 μm, 190.44 ± 4.54 μm, 214.68 ± 4.76 μm, 217.68 ± 7.27 μm, 226.38 ± 4.65 μm and 229.50 ± 8.08 for 41 B (2n), Fercal (2n), 41 B (2n + 4n), Fercal (2n + 4n), Fercal (4n) and Kyoho (4n), respectively . The lea–1 for 41 B (2n), Fercal (2n), 41 B (2n + 4n), Fercal (2n + 4n), Fercal (4n) and Kyoho (4n), respectively  were determined as 194.58 ± 5.92, 194.94 ± 5.40, 153.27 ± 6.13, 150.94 ± 3.24, 146.66 ± 4.68 and 140.24 ± 4.99 for 41 B (2n), Fercal (2n), 41 B (2n + 4n), Fercal (2n + 4n), Fercal (4n) and Kyoho (4n), respectively  and 1 tetraploid (10 bar-96-h) seedling were detected. Four of the mixoploid plants were Fercal and one was the 41 B offspring. Only one Fercal offspring was confirmed to be tetraploid . In FC aThe nuclear DNA content of rootstock offspring’s, which was confirmed by FC analysis, was significantly different. Nuclear DNA contents of 41 B (2n), Fercal (2n), 41 B (2n + 4n), Fercal (2n + 4n), Fercal (4n) and Kyoho (4n) plants were at the levels of 1.034, 1.115, 1.477 (0.99 + 1.97), 1.510 (1.02 + 2.00), 2.110 and 2.144 pg, respectively . While t2O application) offspring, whose increase in ploidy level was confirmed by FC analysis, were counted in preparations prepared by root tip crushing technique. The Fercal offspring, which was determined to be polyploid (tetraploid) by FC analysis, was propagated by in vitro nodal culture, and chromosomes were counted in fresh root tip samples. In addition, diploid Fercal grapevine rootstock was also propagated same method, and chromosomes were counted for control purposes. The chromosome number of diploid Fercal was 2n = 38  has often been used as a critical parameter for chemical mutagenic agents  Lindl was significantly less than the original diploids.In addition, in previous polyploid studies, it was reported that the chlorophyll content of tetraploid plants increased compared to diploids  plants. Significant increases were determined in leaf thickness stomatal sizes, chloroplast numbers, and chlorophyll content but decrease stomatal densities of polyploid individuals, which induced by oryzalin and NZK and OD planned to research. OD carried out seed germination and mutagen applications. ZK and OD analyzed morphological features. OD performed FC analysis and chromosome count. ZK and OD did statistical analysis and wrote the article. ZK supervised OD’s PhD study.


TGF-β upregulates the expression of the enzymes of the de novo serine/glycine synthesis pathway in lung fibroblasts through mTORC1 and ATF4-dependent transcriptional programs. SHMT2, the final enzyme of the de novo serine/glycine synthesis pathway, transfers a one-carbon unit from serine to tetrahydrofolate (THF), producing glycine and 5,10-methylene-THF (meTHF). meTHF is converted back to THF in the mitochondrial one-carbon (1C) pathway through the sequential actions of MTHFD2 (which converts meTHF to 10-formyl-THF), and either MTHFD1L, which produces formate, or ALDH1L2, which produces CO2. It is unknown how the mitochondrial 1C pathway contributes to glycine biosynthesis or collagen protein production in fibroblasts, or fibrosis in vivo. Here, we demonstrate that TGF-β induces the expression of MTHFD2, MTHFD1L, and ALDH1L2 in human lung fibroblasts. MTHFD2 expression was required for TGF-β-induced cellular glycine accumulation and collagen protein production. Combined knockdown of both MTHFD1L and ALDH1L2 also inhibited glycine accumulation and collagen protein production downstream of TGF-β; however knockdown of either protein alone had no inhibitory effect, suggesting that lung fibroblasts can utilize either enzyme to regenerate THF. Pharmacologic inhibition of MTHFD2 recapitulated the effects of MTHFD2 knockdown in lung fibroblasts and ameliorated fibrotic responses after intratracheal bleomycin instillation in vivo. Our results provide insight into the metabolic requirements of lung fibroblasts and provide support for continued development of MTHFD2 inhibitors for the treatment of IPF and other fibrotic diseases.A hallmark of Idiopathic Pulmonary Fibrosis is the TGF-β-dependent activation of lung fibroblasts, leading to excessive deposition of collagen proteins and progressive scarring. We have previously shown that synthesis of collagen by lung fibroblasts requires  Idiopathic Pulmonary Fibrosis (IPF) is a progressive, fatal disease, which has a median survival of 3.5 years and affects approximately 150,000 people in the United States . IPF is de novo synthesis of glycine, the most abundant amino acid in collagen protein is required for production of collagen downstream of TGF-β , is an entry point for carbon into the mitochondrial one-carbon (1C) pathway . To. Tode noof TGF-β . TGF-β sof TGF-β , S1A–C.MTHFD1 transcript levels were increased by TGF-β, its protein expression was reduced compared with untreated cells . In. In22). ed cells , 1C, S1DMTHFD2 and ALDH1L2 was inhibited by ATF4 knockdown, while MTHFD1L and MTHFD1 were unaffected . Cells were cultured in media containing 13C2-glycine (glycine labeled with 13C on both carbon atoms) to allow us to determine the relative contribution of extracellular (M+2) glycine as well as glycine synthesized de novo from either extracellular glucose or serine (M+0) to total cellular glycine pools (de novo-synthesized (M+0) glycine in NHLFs , which has been used to demonstrate the contribution of 1C metabolism to cellular NADPH homeostasis , suggesting an involvement of MTHFD2 in disease progression , we measured lung aeration at day 7 and day 14 after bleomycin instillation. All mice had similar levels of lung aeration prior to and at day 7 after bleomycin , S10G. Mde novo serine and glycine synthesis  has recently been shown be required for proline synthesis  supplemented with 0.1% bovine serum albumin and 2mM glutamine for 24 hours prior to treatment with 1ng/mL TGF-β . DS18561882  was added at the time of TGF-β treatment. For serine and glycine labeling experiments, MEM  was supplemented with MEM vitamin solution . For glycine labeling, 400μM 13C2 glycine  and 400μM unlabeled L-Serine  were added. For serine labeling, 400μM 2,3,3-D3-L-Serine  and 400μM unlabeled .Normal human lung fibroblasts (NHLFs) ) were maintained in Fibroblast Growth Medium 2 . Cells were plated at 1×106 NHLFs were transfected with 250 pmol ON-TARGETplus siRNA (Dharmacon) using an Amaxa Nucleofector 2b set to program A-024. Cells were plated on 10cm dishes for 24 hours and then replated for experiments as above. Dharmacon product numbers: ATF4 , MTHFD2 , MTHFD1L , ALDH1L2 , MTHFD1 , SHMT2 .For siRNA knockdowns, 1×10Cells were lysed, and electrophoresis was performed as we previously described . Wells wRNA as isolated using GenElute Mammalian Total RNA Miniprep Kit (Sigma RTN350) and reverse transcribed using iScript Reverse Transcription Supermix . Quantitative mRNA expression was determined by real-time RT-PCR using iTaq Universal SYBR Green Supermix . Primers used were: MTHFD2 , ALDH1L2 , MTHFD1L , MTHFD1 , RPL13 .2O)). The solution was vortexed and centrifuged at 21,000 × g for 20 minutes. 400 μLof each extract was transferred to a new tube and dried under nitrogen. Dried metabolites were derivatized in 16μL Methoxamine reagent  for 1hr at 37°C. 20 μL of 1% N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide  for 1hr at 60°C. Derivatized samples were analyzed with an 8890 gas chromatograph with an HP-5MS column (Agilent) coupled with a 5977B Mass Selective Detector mass spectrometer (Agilent). Helium was used as the carrier gas at a flow rate of 1.2ml/min. One microliter of each sample was injected in split mode (1:8) at 280°C. After injection, the GC oven was held at 100°C for 1 min and increased to 300°C at 3.5°C/min. The oven was then ramped to 320°C at 20°C/min and held for 5 minutes. The MS system was operated under electron impact ionization at 70eV and the MS source was operated at 230°C and quadrupole at 150°C. The detector was used in scanning mode, and the scanned ion range was 100–650 m/z. Peak ion chromatograms for metabolites of interest were extracted at their specific m/z with Mass Hunter Quantitative Analysis software (Agilent Technologies). Ions used for quantification of metabolite levels were as follows: Glycine m/z 246, Serine m/z 390, Proline m/z 258. Mass isotopomer distributions were determined by integrating appropriate ion fragments for each metabolite using IsoCor  and metabolites were extracted in 600μL ice cold 80% Methanol , W6  following manufacturer’s instructions.Formate content of media was measured using a Formate Assay Kit  on 50 μL of culture media following manufacturer’s instructions. For hydroxyproline measurements, 50 mg of cryogenically pulverized lung tissue was homogenized in 500 μL ultrapure HThe protocol for the use of animals was approved by the University of Chicago Institutional Animal Care and Use Committee. Male C57Bl/6J mice  were intubated and bleomycin  was instilled intratracheally. Mice were sacrificed on the indicated days and perfused lung tissue was either snap frozen for hydroxyproline measurement or processed for histology. Mice receiving MTHFD2 inhibitor were IP injected with DS18561882  beginning on day 8 after bleomycin instillation. Drug was administered at 100mg/kg in 150μL of 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline.MicroCT was performed on an X-Cube CT imager . Animals were anesthetized via inhalation of 2% isoflurane. MicroCT scan was performed using a respiratory-gated protocol to reduce motion artifacts. A window delay of 30% and window width of 50% was used to capture the end-expiration phase of the breathing cycle. Voltage was set to 50 kVp and current at 350 mAS. For quantitative 3D assessment of lung area, HU ranges  were considered as normally-aerated lung tissue, and HU ranges  were defined as poorly-aerated. Volumetric microCT images were reconstructed in an 800 × 800 × 749 format with isotropic voxel dimensions of 50 × 50 × 50 μm3. Images were processed using VivoQuant 4 patch 1 (InviCRO).The collection and use of human lung specimens were approved by the University of Chicago Institutional Review Board. After deparaffinization and rehydration, tissue sections were treated with antigen retrieval buffer  in a steamer for 20 min. Rabbit polyclonal anti-MTHFD2 antibody  was applied on tissue sections for a 1-h incubation at room temperature in a humidity chamber. Following a TBS wash, tissue sections were incubated with biotinylated anti-rabbit IgG  for 30 min at room temperature. The antigen-antibody binding was detected by Elite kit  and DAB  system. Tissue sections were briefly immersed in hematoxylin for counterstaining and were covered with cover glasses. Whole slide images were analyzed using ImageJ.Processed gene expression datasets from GSE32537 were downloaded from Gene Expression Omnibus. Available clinical data was correlated to individual gene expression profiles using Pearson’s correlation analysis using GraphPad Prism v10.0.2.Data were analyzed in Prism 10 . All data are shown as mean ± standard error of the mean (SEM). Significance was determined by unpaired two-tailed Student’s t test (for comparisons between two samples), or by one or two-way ANOVA using Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.001.1


To circumvent these hurdles, herein, phosphorus (P) atom embedded three-dimensional marigold-shaped 1T MoS2 structures combined with the design of S vacancies (Sv) are synthesized via the oxygen-assisted solvent heat method. The oxygen-assisted method is utilized to aid the P-embedding into the MoS2 crystal, which can expand the interlayer spacing of P-MoS2 and strengthen Zn2+ intercalation/deintercalation. Meanwhile, the three-dimensional marigold-shaped structure with 1T phase retains the internal free space, can adapt to the volume change during charge and discharge, and improve the overall conductivity. Moreover, Sv is not only conducive to the formation of rich active sites to diffuse electrons and Zn2+ but also improves the storage capacity of Zn2+. The electrochemical results show that P-MoS2 can reach a high specific capacity of 249 mAh g−1 at 0.1 A g−1. The capacity remains at 102 mAh g−1 after 3260 cycles at a current of 0.5 A g−1, showing excellent electrochemical performance for Zn2+ ion storage. This research provides a more efficient method of P atom embedded MoS2-based electrodes and will heighten our comprehension of developing cathodes for the ZIBs.Structural unsteadiness and sluggish diffusion of divalent zinc cations in cathodes during cycling severely limit further applications of MoS As expected, the P-MoS2 electrode achieves a remarkably high capability of 249 mAh g−1 at 0.1 A g−1, which is five times higher than the specific capacity of pristine MoS2. In comparison to the pristine P-MoS2 counterpart with minimal capacity delivery, P-MoS2 can achieve a high reversible capacity of 105 mAh g−1 at 0.5 A g−1 with 3260 cycles and excellent capacity retention of 70 mAh g −1 at 1 A g−1. This study will provide more efficient avenues for investigating more electrode materials with poor intercalation kinetics in ZIBs.Herein, an oxygen-assisted(O) strategy is used to aid the P-embedding into the MoS2: In a representative procedure, 1 mmol (1236 mg) ammonium molybdate tetrahydrate ((NH4)6Mo7O24·4H2O, AR) and 30 mmol (2284 mg) thiourea  were added into 35 mL deionized water on the basis of a beforehand report with slight adjustment [2PO2·4H2O, AR, 400 mg) was dissolved into a mixed solution with magnetic stirring for 2 h. Then, the precursor solution was heated to 180 °C for 24 h in a 50 mL Teflon-lined stainless steel autoclave. After cooling the reaction system to air temperature, which was collected by centrifugation, cleaned multiple times with water, and then dried at 80 °C. As a contrast, pristine MoS2 was also prepared without NaH2PO2·4H2O following a similar synthetic route.Synthesis of P-MoSjustment . After b2 adsorption/desorption isotherms were measured with an automated SSA and porosity analyzer (asap2460). Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) adsorption techniques were used to determine the SSA and mesopore size distribution of each sample. All samples’ crystal phases and compositions were determined using X-ray diffraction  and X-ray photoelectron spectroscopy . Raman spectra at 532 nm were measured with a Horiba Scientific LabRAM HR Evolution Raman spectrometer. The TGA/DTA was tested by thermogravimetric analysis equipment  from room temperature to 750 °C at a heating rate of 10 °C/min. A hydrophilicity test was measured using a contact angle test device .The morphology of materials was evaluated with a scanning electron microscope  and a transmission electron microscope . An energy dispersive spectrometer  connected to the SEM was used to study the elemental composition and elemental analyses of the composites. At −196 °C, N3SO3)2 served as the electrolyte in CR2016-type coin cells used to investigate the Zn2+ storage properties of P-MoS2. To manufacture the working electrode, P-MoS2 (70 weight percent), super p carbon (20 weight percent), and polyvinylidene fluoride (10 weight percent) were thoroughly blended in N-methyl-2-pyrrolidone for 15 min. Finally, the aforementioned slurry was distributed over a clean stainless steel mesh and dried for 24 h at 60 °C. On a Neware battery tester (CT4008), galvanostatic charge/discharge measurements and galvanostatic intermittent titration technique (GITT) experiments were carried out continuously between 0.25 and 1.25 V (vs. Zn/Zn2+). An electrochemical workstation was used for the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments (CHI-660D). When the batteries were completely charged, impedance measurements were taken (zinc extraction). At room temperature, all tests were conducteddw.Zn served as the anode, glass fiber membrane served as diaphragms, and 3 M Zn and displays severe aggregation in the absence of a well-tuned morphology , all peaks of the P-MoS2 are broadened due to nanoscale effects and crystal structural defects. The weakening of the (002) diffraction peak of P-MoS2 indicates a low stacking height along this direction [2 slightly shifts to a lower angle (14.2° → 13.96°), indicating the formation of sulfur vacancies in MoS2 [2. According to Bragg’s law, the lattice spacing of the stacking layer at 9.8° is consistent with the spacing in TEM. Due to P’s larger atomic radius than S, P atoms were introduced into the MoS2 matrix, causing the lattice to expand and contributing to the widening of interlayer spacing [2. Three typical Raman scattering peaks at 280 cm−1 (E1g), 234 (J2) cm−1, and 334 cm−1 (J3) are attributed to the octahedral coordination of metal 1T-MoS2 [2 exhibits two typical characteristic peaks at 376 cm−1 and 394 cm−1, corresponding to the in-plane (E12g) and out-of-plane (A1g) modes of 2H MoS2, which are clearly distinct from pristine MoS2. The distance between the E12g and A1g peaks is 18 cm−1 for P-MoS2 and 23 cm−1 for bulk MoS2. Thus, the moving peak of A1g might represent the substantial out-of-plane vibration, indicating that the decreased van der Waals force, together with the enlarged interlayer spacing of MoS2 caused by P-embedding, is favourable for Zn2+ movement and storage [12g peak in P-MoS2 is weaker compared to other samples, thereby proving the presence of Sv in the crystal structure [The structural information of the two obtained samples was studied by X-ray diffraction (XRD) analysis a. Comparirection ,35. Mean in MoS2 ,37. In a spacing ,38,39. F 1T-MoS2 ,41,42. P storage . The weatructure .2 shown in 2 is probably caused by water insertion [2 structure, with the unsaturated S atoms resulting In the reaction 2MoS2 + 7O2 → 2MoO3 + 4SO2 [2, which is confirmed by XRD analysis. Moreover, the cumulative pore volume and BET surface area of P-MoS2 and pristine MoS2 were also measured. As shown in 2 and pristine MoS2 exhibited typical type IV isotherms with hysteresis loops, indicating mesopores between MoS2 nanosheets. Nevertheless, the hysteresis of P-MoS2 starts at a lower pressure region (P/P0 ≈ 0.5) than that of pristine MoS2 (P/P0 ≈ 1), showing their different porous nature. Furthermore, the wide hysteresis of P-MoS2 indicates its increase in porosity. According to the pore volume–pore size distribution curve, P-MoS2 has more abundant mesoporous structures, which is conducive to electron transport. The increased specific surface area of P-MoS2 is 51.572 m2 g−1, which is significantly more than pristine MoS2 (11 m2 g−1). The increased specific surface area improves the interaction between the material’s exposed active sites and the electrolyte, which favors increasing the number of active sites for Zn2+ storage. These results clearly show that the P-embedding strategy can greatly increase the surface area and pore volume of MoS2. To evaluate the hydrophilicity of P-MoS2, we performed a contact angle test. Water contact angles of P-MoS2 drop from 54.46° to 38.8°  and 162.7 eV (S 3d1/2 of S2−) attributed to 1T MoS2, while peaks at 162.0 and 163.1 eV ascribed to 2H-MoS2 [2  reveals detailed valence states and chemical contents of manufactured materials. The survey XPS spectra for P-MoS 2H-MoS2 ,48,49. T 2H-MoS2 . Further 2H-MoS2 . Meanwhi-MoS2 [2 c. It can30.06 eV d, confir lattice ,51. Furtacancies ,45.2+ storage capacity of the prepared samples as stand-alone cathodes for AZIBs, CR2016 coin cells were manufactured in the air environment . Cyclic voltammetry (CV) curves in a voltage window of 0.25 to 1.25 V of 0.1 mV s−1 are shown in 2, P-MoS2 has two redox peaks: the cathodic peak at 0.65 V linked to Zn2+ insertion (possibly overlapping or merging with the reduction in Mo6+/Mo4+) and an anodic peak at 0.98 V is associated to Zn2+ inlay removal [2 is considerably larger than that of pristine MoS2, showing that the enlarged layer spacing and Sv can significantly boost volume capacity. The P-MoS2 charge/discharge (CD) curves are comparable with the CV data , significantly better than the well-known Zn2+ intercalation host, i.e., the Chevrel Phase Mo6S8 (60 mA h g−1 at 0.06 A g−1) [2 materials is compared at 0.1 A g−1 in 2, which are 249, 158, 125, 97, and 75 mAh g−1 at 0.1, 0.2, 0.5, 1, and 2 A g−1, respectively, and 2 capacitance achieves 143 mAh g−1 (91% capacity retention) when it reaches 0.2 A g−1. These hint at the excellent rate characteristic and rapid dynamics of P-MoS2. In contrast, pristine MoS2 offers rather low capacities (0–45 mAh g−1) at a variety of current densities. P-MoS2 demonstrates a consistent capacity of 70 mAh g−1 after 200 cycles at 1 A g−1 to further illustrate its outstanding working life  ,53. Meaning life f. The st2 maintains a capacity of 102 mAh g−1 after 3260 cycles, achieving capacity retention of 70%, with the efficiency of the electrode being close to 100%. However, the cycling stability of the P-MoS2 electrode seems rather poor after 2000 cycles, which is attributed to the irreversible structural damage, volume changes, and unstable 1T phase during the continuous charging and discharging process [2 has almost zero capacity, which is related to its poor conductivity and unstable structure [P-MoS process ,55. In ctructure ,23.2 outperforms pristine MoS2 in terms of reversible capacity, cycle stability, and rate capacity. To better understand the electrochemical reaction behavior of the P-MoS2 electrode, CV curves at varied scan speeds (0.1–1.0 mV s−1) are tested  vs. log (b) graph. In particular, b = 0.5 indicates that the discharge-specific capacity belongs to the diffusion process control, and b = 1 implies the pseudocapacitance control. When b is between 0.5–1, there is both diffusion and pseudocapacitance behavior [b-values of peak 1 and peak 2, which are 0.76 and 0.70, showing that the redox reaction of the P-MoS2 electrode consists of diffusion and capacitive processes. Additionally, the capacitive contribution and diffusion contributions of P-MoS2 may be evaluated based on the equation proposed by Dunn [In between, behavior . Obvious by Dunn ,58:(2)i(v), i is the total current response, where v are diffusion-controlled and capacitance-dominated contributions. −1 to 50.1% at 1 mV s−1. The pseudocapacitance effect is slowly dominant, indicating that the surface reaction is faster than the internal diffusion reaction, which accelerates the intercalation and extraction of Zn2+ in the material. Moreover, the diffusion characteristics of P-MoS2 are more obvious, which can be attributed to the marigold-shaped structure and wide layer spacing [2 and pristine MoS2 is evaluated by EIS, as shown in 2 is measured at 42.82 Ω, which is lower than the pristine MoS2 under the same conditions. In addition, Rcts of pristine MoS2 are markedly over 180 Ω, indicating that expanded layer spacing and S vacancies improve charge-transfer dynamics. Because of the activation of the active material, the charge transfer impedance of P-MoS2 was lowered during the first ten cycles.At a certain voltage  and extensively analyzed their performance as cathode materials for ZIBs. Meanwhile, the electrochemical investigation revealed that the S vacancy and interlayer spacing generated by P-embedding are particularly advantageous to the rapid diffusion of Zn2+ in P-MoS2 and boost the Zn2+ storage capacity of the marigold-shaped nanosheets. In comparison to the pure MoS2 equivalent, P-MoS2 nanosheets have a great specific capacity and display outstanding continuous cycle capabilities in ZIBs. When the current density is 0.1 A g−1, the discharge capacity can reach 249 mAh g−1. In particular, it displayed a high specific discharge capacity of 105 mAh g−1 with a capacity retention of 70% after 3260 cycles at the current density of 0.5 A g−1 and an excellent capacity retention of 70 mAh g−1 at 1 A g−1. This work offers new ideas for designing MoS2 as cathode materials for ZIBs.In summary, marigold-shaped 1T-MoS


The critical size for the complete desolvation of [TEA(AN)]+ was 4.7 Å, and the partial desolvation size ranged from 4.7 to 4.8 Å. The critical size for the complete desolvation of [SBP(AN)]+ was 5.2 Å, and the partial desolvation size ranged from 5.2 to 5.5 Å. As the ionic radius of the quaternary ammonium cation decreased, the desolvation size showed a positive trend. A density of states (DOS) analysis of the desolvated quaternary ammonium cations embedded in the hydroxyl-flat pore structure showed that the conductivity of the hydroxyl-flat pore was enhanced after gaining electrons. The results of this paper provide some help in selecting organic electrolytes to improve the capacity and conductivity of supercapacitors.Supercapacitors have a wide range of applications in high-technology fields. The desolvation of organic electrolyte cations affects the capacity size and conductivity of supercapacitors. However, few relevant studies have been published in this field. In this experiment, the adsorption behavior of porous carbon was simulated with first-principles calculations using a graphene bilayer with a layer spacing of 4–10 Å as a hydroxyl-flat pore model. The reaction energies of quaternary ammonium cations, acetonitrile, and quaternary ammonium cationic complexes were calculated in a graphene bilayer with different interlayer spacings, and the desolvation behavior of TEA Eint2 < 0 for dAA < 5.5 Å indicates that the AN molecule can exist alone in AA-stacked graphene bilayer hydroxyl-flat pores. The energy value of Eint1 (−8.42 eV) compared to Eint3 (−8.50 eV) is similar when the AA-stacked graphene bilayer hydroxyl-flat pores size dAA = 4.7 Å, that is, 4.7 Å is the critical point for the desolvation of [TEA(AN)]+ in AA-stacked graphene bilayer hydroxyl-flat pores. When the pores are smaller than 4.7 Å and Eint1 < Eint3 < 0, then TEA+ is more stable than [TEA(AN)]+, and [TEA(AN)]+ can undergo complete desolvation. The energy value of Eint4 (−3.58 eV) is similar to that of Eint5 (−3.55 eV) when the AA-stacked graphene bilayer flat pore size dAA = 4.5 Å, that is, 4.5 Å is the critical point for the desolvation of [TEA(AN)]+ in AA-stacked graphene bilayer flat pores. When these pores are smaller than 4.5 Å and Eint4 < Eint5 < 0, TEA+ is more stable than [TEA(AN)]+, and [TEA(AN)]+ can be completely desolvated.+ in AB-stacked graphene bilayer hydroxyl-flat pore and flat pore models. Eint2 < 0 for dAB < 5.4 Å indicates that AN molecules can exist alone in the AB-stacked graphene bilayer hydroxyl-flat pores. The energy value of Eint1 (−8.97 eV) is similar to that of Eint3 (−9.05 eV) at dAB = 4.8 Å, that is, 4.8 Å is the critical point for the desolvation of [TEA(AN)]+ in the AB-stacked graphene bilayer hydroxyl-flat pores. When these pores are smaller than 4.8 Å and Eint1 < Eint3 < 0, TEA+ is more stable than [TEA(AN)]+, and [TEA(AN)]+ can be completely desolvated. The energy value of Eint4 (−4.43 eV) is similar to that of Eint5 (−4.36 eV) when the AB-stacked graphene bilayer flat pore size dAB = 4.6 Å, that is, 4.6 Å is the critical point for the desolvation of [TEA(AN)]+ in AB-stacked graphene bilayer flat pores. When these pores are smaller than 4.6 Å and Eint4 < Eint5 < 0, TEA+ is more stable than [TEA(AN)]+, and [TEA(AN)]+ can be completely desolvated. In summary, the two different stacked forms of AA and AB hydroxyl-flat pores and flat pores can exist in solution at the same time. Therefore, the pore size required for the partial desolvation of [TEA(AN)]+ is 4.7 Å and 4.7–4.8 Å in bilayer graphene hydroxyl-flat pores, and for the complete desolvation of graphene bilayer flat pores, it is 4.5 Å and 4.5–4.6 Å. TEA+ consumes less energy to enter the graphene bilayer hydroxyl-flat pores and is easier to access than the graphene bilayer flat pores. The increase in TEA+ in the graphene bilayer hydroxyl-flat pores leads to a rise in supercapacitor capacity. The hydroxyl group increases the adsorption capacity of the flat pores to TEA+ and enhances the ability of the flat pores to store TEA+.+. To obtain the critical size of the desolvation pore size of [SBP(AN)]+, the reaction energies of SBP +, AN, [SBP(AN)]+ in the hydroxyl-flat pores, and SBP+ and [SBP(AN)]+ in the flat pores were calculated and expressed as Eint1, Eint2, Eint3, Eint4, and Eint5, respectively. Eint2 < 0 when dAA < 5.7 Å, indicating that the AN molecule can exist alone in the AA-stacked graphene bilayer hydroxyl-flat pores. The energy value of Eint1 (−9.65 eV) is similar to that of Eint3 (−9.64 eV) when the AA-stacked graphene bilayer hydroxyl-flat pore size dAA = 5.5 Å, that is, 5.5 Å is the critical point for the desolvation of [SBP(AN)]+ in AA-stacked graphene bilayer hydroxyl-flat pores. At AA-stacked graphene bilayer hydroxyl-flat pores smaller than 5.5 Å, Eint1 < Eint3 < 0, meaning SBP+ is more stable than [SBP(AN)]+ in the pores, so when the pore size is smaller than 5.5 Å, [SBP(AN)]+ can be completely desolvated in AA-stacked graphene bilayer hydroxyl-flat pores. The energy value of Eint4 (−6.13 eV) compared to Eint5 (−6.08 eV) is similar when the AA-stacked graphene bilayer flat pore size dAA = 5.1 Å, that is, 5.1 Å is the critical point for the desolvation of [SBP(AN)]+ in AA-stacked graphene bilayer flat pores. At AA-stacked graphene bilayer flat pores smaller than 5.1 Å and Eint4 < Eint5 < 0, meaning SBP+ is more stable than [SBP(AN)]+ in the pores, so [SBP(AN)]+ can be completely desolvated in AA-stacked graphene bilayer flat pores when the pore size is smaller than 5.1 Å.+. Eint2 < 0 for dAB < 5.8 Å, indicating that AN molecules can exist alone in the AB-stacked graphene bilayer hydroxyl-flat pores. The energy value of Eint1 (−9.63 eV) is similar to that of Eint3 (−9.65 eV) when the AB-stacked graphene bilayer hydroxyl-flat pore size dAB = 5.2 Å, that is, 5.2 Å is the critical point for the desolvation of [SBP(AN)]+ in these pores. When these pores are smaller than 5.2 Å and Eint1 < Eint3 < 0, then SBP+ is more stable than [SBP(AN)]+, and [SBP(AN)]+ can be completely desolvated. The energy value of Eint4 (−5.68 eV) is similar to that of Eint5 (−5.71 eV) when the pore size of AB-stacked graphene bilayer flat pores dAB = 5.0 Å, that is, 5.0 Å is the critical point for the desolvation of [SBP(AN)]+ in these pores. When these pores are smaller than 5.0 Å and Eint4 < Eint5 < 0, then SBP+ is more stable than [SBP(AN)]+, which can be completely desolvated. Therefore, the pore size for the complete desolvation of [SBP(AN)]+ in graphene bilayer hydroxyl-flat pores is 5.2 Å, and the partial desolvation pore size is 5.2–5.5 Å. SBP+ consumes less energy than the bilayer and so it is easier for it to enter the hydroxyl-flat pores than the flat pores. The increase in the number of SBP+ ions allows the supercapacitor capacity to be increased. The hydroxyl group increases the adsorption capacity of the flat pores to SBP+ and enhances their capacity to store it.+ and 2.09 Å for SBP+) + is more significant than that of [TEA(AN)]+ in both FP and HFP for quaternary ammonium cations with a larger electric capacity when AN is used as the solvent system. The critical size for the desolvation of the quaternary ammonium cation depends mainly on the ionic radius, which is consistent with the findings of Janes et al. + was 4.7 Å, and the partial desolvation pore size ranged from 4.7 to 4.8 Å. The complete desolvation flat pore size of [SBP(AN)]+ was 5.0 Å, and the partial desolvation pore size ranged from 5.0 to 5.1 Å. The complete desolvation hydroxyl-flat pore size of [SBP(AN)]+ was 5.2 Å, and the partial desolvation pore size ranged from 5.2 to 5.5 Å. The adsorption of hydroxyl groups on the surface of the flat pores increased the ability of the flat pores to store quaternary ammonium cations, thus increasing the electric capacity of the supercapacitor. In the acetonitrile solvent system, the critical size of the desolvated quaternary ammonium cation increased with a decreasing radius. The desolvated quaternary ammonium cation lost electrons when embedded in the hydroxyl-flat pores, while the hydroxyl-flat pores exhibited enhanced metallicity and conductivity after gaining electrons.In this experiment, we studied the desolvation behavior of quaternary ammonium cationic complexes in AA- and AB-stacked flat pores and hydroxyl-flat pores with acetonitrile as a solvent using the first-principles calculation method. The complete desolvation flat pore size of [TEA(AN)]


Activating transcription factor 6α (ATF6α) is an endoplasmic reticulum protein known to participate in unfolded protein response (UPR) during ER stress in mammals. Herein, we show that in mouse C2C12 myoblasts induced to differentiate, ATF6α is the only pathway of the UPR activated. ATF6α stimulation is p38 MAPK-dependent, as revealed by the use of the inhibitor SB203580, which halts myotube formation and, at the same time, impairs trafficking of ATF6α, which accumulates at the cis-Golgi without being processed in the p50 transcriptional active form. To further evaluate the role of ATF6α, we knocked out the ATF6α gene, thus inhibiting the C2C12 myoblast from undergoing myogenesis, and this occurred independently from p38 MAPK activity. The expression of exogenous ATF6α in knocked-out ATF6α cells recover myogenesis, whereas the expression of an ATF6α mutant in the p38 MAPK phosphorylation site (T166) was not able to regain myogenesis. Genetic ablation of ATF6α also prevents the exit from the cell cycle, which is essential for muscle differentiation. Furthermore, when we inhibited differentiation by the use of dexamethasone in C2C12 cells, we found inactivation of p38 MAPK and, consequently, loss of ATF6α activity. All these findings suggest that the p-p38 MAPK/ATF6α axis, in pathophysiological conditions, regulates myogenesis by promoting the exit from the cell cycle, an essential step to start myoblasts differentiation. Here, specialized domains are devoted to the control of Catraction ,2. Aparttraction ,4,5. Thetraction . In thistraction ,8,9. In traction ,11,12,13traction .Myogenesis is a multistep process, in which myoblasts arrest cell division and then start to elongate and fuse forming multinucleated myotubes . These eA number of investigations indicate that the p38 MAPK signaling presides over myogenic differentiation or cross-talks with the UPR ,23. IndeAlthough ER stress activates simultaneous with the UPR pathways ,14, diffAn inverse relationship between the PERK activity and muscle differentiation is quite common. Indeed, an increased expression of CHOP, a downstream target of PERK, inhibits myogenic differentiation by repressing the transcription factor MyoD . In otheConcerning IRE1, a number of reports suggest that this branch of the UPR could give opposite results in myoblasts differentiation. Indeed, while on the one hand both IRE1 and XBP1 knockdown suppress C2C12 myoblasts differentiation , XBP1 ovTo date, few studies have explored the role of ATF6α in promoting myogenesis, and little is known about the involvement of ATF6α in the development of skeletal muscles ,37,38. I2. Myotube formation was induced by growing C2C12 myoblasts in a differentiation medium (DM) containing 2% horse serum and 1% penicillin/streptomycin for 24, 48 and 72 h. Cells were incubated for 5 h with tunicamycin (TN)  2 μg/mL to induce ER stress [Mouse C2C12 myoblasts were purchased from the American Type Culture Collection  . Cells wR stress . TreatmeR stress . For DexATF6α knockout was performed using the lentiCRISPRv2 plasmid to knockout ATF6α. Short guide RNAs (sg RNAs) against ATF6α [® Golden Gate Assembly Kit E1602S, New England Biolabs, Ipswich, MA, USA). Stable transfectants were selected in a growth medium containing 4 μg/mL Puromycin . Sub-confluent cells were transfected 24 h after plating by using TRANSIT . Empty lentiCRISPRv2 vectors were used to generate clones as an un-edited control (EV), while un-transfected C2C12 cells were used as a basal control. Stable transfectants were isolated by collecting individual clones, which were further amplified. Single clones and bulk culture (pool) were analyzed by Western blotting to verify gene inactivation. Clone 8 was chosen for further characterization , which revealed seven putative p38 MAPK phosphorylation sites in the ATF6 protein sequence: S13; S16; S94; S130; T166; and S548. Phosphorylation sites T166 and S130, not yet characterized, were chosen for the experimental procedures. Mutagenesis of plasmid p3xFlag-ATF6α, expressing full-length wild type human ATF6α , was obtained by using the In-Fusion Cloning Kit . Two different pairs of Inverse primers  were used to amplify the p3xFlag-ATF6α vector and to obtain serine/alanine (S130A) and threonine/alanine (T166A) substitution, respectively. For the In-Fusion Cloning reaction, the linear DNA was re-circularized at the site of the 15 bp overlap and mutagenic changes were included. Finally, constructs were validated by Sanger sequencing .Plasmid expressing p3xFlagATF6α  was used as a template for mutagenesis to obtain mutants T166A  and S130http://rsb.info.nih.gov/ij/, accessed on 10 November 2022).Whole-cell lysate was prepared by lysing cells in a solution containing 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1% Igepal, 1× protease inhibitor and a phosphatase inhibitor cocktail, as described previously ,46. Prot−ΔΔCT (cycle threshold) method was used to calculate the results, and mRNA expression levels were determined as fold-induction relative to the Ctrl cells, set as 1 [https://www.ncbi.nlm.nih.gov/nucleotide (accessed on 4 October 2021) and used as input, in order to use the mRNA sequence for primer design. The primer melting temperature was set to be 60 °C. The “Exon junction span” parameter was selected as “primer must span an exon-exon junction” and “Intron inclusion” parameter was flagged. Finally, “Primer specificity stringency” was set as “Primer must have at least 3 total mismatches to unintended targets, including at least 3 mismatches within the last 5 bps at the 3′ end”.Total RNA was purified by using an ultrapure TRizol reagent  according to the manufacturer’s instructions. The concentration and purity of RNA were determined spectrophotometrically by reading the absorbance at 260 and 280 nm. Aliquots of total RNA were subjected to DNase I digestion  and reverse transcribed using EasyScript Plus cDNA Synthesis Kit  according to the manufacturer’s protocol. Real-time PCR was carried out using the PowerUP Syber green master mix , the Quant Studio 7 Flex instrument and the fast gene-expression method with the following conditions: a first denaturation step at 95 °C for 20 s followed by 40 cycles at 95 °C for 1 s, 60 °C for 30 s, and then melting curve analysis was performed, raising the temperature from 60 °C until 95 °C with a 0.5 °C/s increase. Reactions were carried out in triplicate, and the 18S gene was used as an internal control to normalize the variability in expression levels. The 2set as 1 . The prihttp://rsb.info.nih.gov/ij/, accessed on 10 November 2022).RT-PCR and XBPI Splicing Assay: Total RNA was retro-transcribed with the EasyScript Plus cDNA Synthesis Kit  according to manufacturer instructions. To amplify the mouse XBP1 mRNA, PCR was carried out for 30 cycles  using the following primers forward, 5′-CCTTGTGGTTGAGAACCACC-3′; and reverse, 5′-CTAGAGGCTTGGTGTATA-3′, as previously described . Un-splix-axis projection using stack images [To perform immunofluorescence analysis, undifferentiated and differentiated cells were washed in phosphate-buffered saline (PBS), fixed in PBS-4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 5 min . Thereafk images . Co-locak images . The co-5 cells/well) were seeded into 12-well plates; at the end of each treatment, cell suspensions were centrifuged, and pellets were resuspended in a hypotonic lysis solution containing 50 µg/mL propidium iodide (PI). After incubation at 4 °C for 30 min, cells were analyzed by a Becton Dickinson FACScan flow cytometer, using the Cell Quest software version 4 [To determine the number of apoptotic nuclei and cell cycle phases analysis, cells .Statistical significance was determined by one-way analysis of variance (ANOVA), followed by Bonferroni test. Each value represents the mean ± SD of at least three independent experiments performed in triplicate  supplemented with 2% horse serum . Phase-contrast microscopy revealed that the formation of myotubes already appeared at 24 h, following cell incubation in DM. However, the higher number of multinucleated cells was obtained after 72 h in DM compared to the control cells kept in a normal growth medium (GM) A. The moActivation of the endogenous ATF6α was assessed by measuring the amount of the un-cleaved p90-ATF6α protein against the cleaved p50 form of ATF6α in the C2C12 myoblast, cultured in GM or in DM for the times indicated D. As sucInterestingly, in our experiments, we did not observe an activation of either PERK or IRE1 F,G. To eThe IRE1 endonuclease activity was established by assaying the expression of the spliced form of XBP1 in the C2C12 mRNAs extracted at the different times of myogenesis G. Resultp ≤ 0.001) in differentiating myoblasts with respect to the cells growing in GM, suggesting that C2C12 myoblasts, during myogenesis, are sustained by ATF6α. Finally, when we measured, by qPCR, the mRNA level of three markers of the ER stress, namely GRP78/BiP, Calnexin and Calreticulin H, we fouSince it has long been known that the p38 MAPK signaling pathway drives myogenesis , we inveSimilarly, we observed that either level of phosphorylated p38 MAPK (p-p38 MAPK) or MyoG expression were inhibited by the SB203580 at both the protein A and mRNThereafter, we investigated whether the inhibition of p38 MAPK by SB203580 could influence the endogenous ATF6α protein trafficking from the ER to the nucleus, via the Golgi complex, during C2C12 myoblasts differentiation. To this aim, C2C12 cells were cultured in GM or DM in the presence or absence of SB203580 and then subjected to confocal immune fluorescence analysis A. Co-locATF6α knockout (KO)   on the Chenotype . As we wmyotubes A,B againATF6α cells kept in GM or DM for 72 h and transfected with the wild-type 3xFlag-ATF6α expressing vector  after 72 h of differentiation time compared to the EV clone B clearlyAs we would expect, SB203580 kept myotube formation blocked B. These ATF6α cells, during cell differentiation. Moreover, T166A ATF6α showed a higher accumulation at the Golgi complex  to drive the myogenesis of C2C12 myoblasts. To this aim, we analyzed myotube formation in KO ATF6α C2C12 cells transfected with vectors expressing wild-type or mutant T166A ATF6α recombinant proteins or the recombinant S130A ATF6α protein A. Immunoonstruct C or the onstruct A, suggesonstruct A. FurtheWT ATF6α B. ATF6α cells transfected with WT ATF6  in the differentiated C2C12 cells treated with DEX and a significant reduction in the co-localization of the ATF6α fluorescent signal with the nuclear marker (p ≤ 0.01), compared to un-treated cells . As expected, DEX treatment kept cells in an undifferentiated state A, compared cells B. In lined cells C.In this study, we report that myotube formation in C2C12 myoblasts requires an unconventional UPR supported by ATF6α but not by IRE1 or PERK. This was shown by stimulating differentiation in C2C12 cells and observing that ATF6α is progressively processed, which suggested that this pathway of the UPR, to a certain extent, could play a role in supporting myogenesis. Following this observation, we set up a series of experiments in order to understand through which mechanism ATF6α takes place to the control of myogenesis. The steps of ATF6a activation have been mainly studied in the UPR. Mammals express two homologous ATF6 proteins, ATF6α (670 amino acids) and ATF6β (703 amino acids). ATF6α is a potent transcriptional activator, while ATF6β has a low capacity to act as a transcription factor ,57. The During myogenesis, we show, firstly, that, for ATF6α to be activated, it requires p38 MAPK phosphorylation. Our results showing that the p38 MAPK inhibitor SB203580 impaired myogenesis suggest that this modification is essential to promote not only ATF6α activation but also differentiation of myoblasts. Indeed, in the presence of SB203580, besides the loss of ATF6α activation, p38 MAPK inhibition has the effect of downregulating either the expression of the marker of differentiation MyoG or of the downstream targets of ATF6α . As a consequence of this, the expansion of the ER membrane network is greatly reduced, suggesting that ATF6α is required during myogenesis in order to ensure an efficient quality control of secretory proteins in the multinucleated myotubes and the full development of sarcoendoplasmic reticulum.Examining in deeper detail the molecular events underlying ATF6α activation, we show that phosphorylation by p38 MAPK of the threonine residues at position 166 of the ATF6α protein is essential for the proteolytic cleavage of p90 ATF6α at the Golgi complex and, as a consequence, for the import of p50 ATF6α inside the nucleus, where the truncated protein acts as transcriptional regulator. This event is very similar to that observed when inflammatory stimuli with IFN-γ-induce p38 MAPK activation, which in turn increases ATF6α phosphorylation and processing ,44. SimiATF6α gene keeps C2C12 myoblasts in an un-differentiated state. This confirms that the ATF6α pathway is essential for muscle cell differentiation.In order to establish the function of ATF6α in the development of myotubes, we performed gene inactivation experiments, which showed that ablation of the ATF6α KO cells, in which the ER-to-Golgi transport, cleavage and nuclear translocation of ATF6α regularly occurred and derived a differentiated phenotype. The treatment with SB203580, in the differentiation medium ATF6α KO cells transfected with 3xFlag-ATF6α WT vector led to a notable loss of differentiated morphology (ATF6α KO cells.Further validation of this can been seen by the rescue experiments, in which these effects were reverted by the 3xFlag-ATF6α WT expression in the rphology A, with tOn the other hand, p38 MAPK, through MyoD and MEF2, controls the expression of MyoG . Althougp38 MAPK may promote cell proliferation or induce cell cycle arrest, depending on the downstream pathways ,62. IntrMoreover, it has been shown that ATF6α signaling may play a role in developmental apoptosis and differentiation programs during muscle development ,65. HoweATF6α knockout blocks the exit from the cell cycle as shown by the evidence that the gene ablation reduces the percentage of cells in the inactive G1 phase and increases the number of cells in the active S phase, thus preventing the exit from the cell cycle and therefore preventing differentiation. Moreover, the transfection with the ATF6α WT construct, in the ATF6α KO cells, was able to promote the exit of the cell cycle, reverting the effect of the KO of ATF6α. Instead, we discovered, by genetic ablation experiments, that ATF6α contributes to cell cycle arrest, which is an essential prerequisite to start myoblasts differentiation. Therefore, since cells must exit the cell cycle, in order to differentiate a failure to exit the cell cycle would block all subsequent steps of differentiation. Therefore, it appears that the main mechanism through which ATF6 regulates myogenesis is the regulation of the exit from the cell cycle. This is supported by the finding that ATF6α KO cells transfected with the ATF6α WT. Our findings demonstrate that ATF6 influences the cell cycle, favoring the exit from the cell cycle by promoting myoblast cell differentiation.On the other hand, no effect was observed in the ATF6α KO transfected with the ATF6α T166A construct. A similar behavior was obtained after treatment with SB, the p38 MAPK inhibitor, in the Some studies demonstrate that ATF6α can regulate differentiation by inducing a selective apoptosis to eliminate myoblasts, which are incompetent to differentiate ,37,38,65The malfunction of p38 MAPK has already been counted among the potential causes of muscle atrophy ,67. As rIn this paper we showed that treatment with DEX, a major inducer of muscle cell atrophy, inhibits differentiation of C2C12 myoblasts and reduces the amount of phosphorylated p38, thus impairing ATF6α activation and in turn myogenesis, which suggests that the p38 MAPK/ATF6α could also play a role in the regulation of atrophic events. Our work represents a further example of how the UPR can be activated to obtain specific outcomes in different cell types and tissues by using the same machinery. In our case, p38 MAPK activates the ATF6α pathway of UPR, but p38 MAPK alone is not sufficient to sustain myogenesis, since p38 MAPK phosphorylation is active even in the absence of ATF6α. Our results strongly suggest that ATF6α, throughout p38 MAPK phosphorylation, can ensure important changes to achieve the differentiated state during myogenesis. The main important step is the cell cycle arrest, which occurs by ATF6α. Further work is required to demonstrate that all the other effects we see are independent of this.


Results showed that Fe concentration in root and shoot increased by elevating the level of NPs from 100 mg L−1 to 500 mg L−1. Overall, the responses of maize seedlings to different sources of Fe oxide NPs were as follows: Fe3O4 > γ-Fe2O3 > α-Fe2O3 > bulk Fe3O4. The application of Fe at concentrations ranging from 100 mg L−1 to 500 mg L−1 had no significant effects on various growth parameters of maize, including biomass, chlorophyll content, and root length. Iron oxide NPs increased the plant biomass by 23–37% by root application, whereas it was 5–9% by foliar application. Chlorophyll contents were increased by 29–34% and 18–22% by foliar and root applications, respectively. The non-significant response of reactive oxygen species  suggested optimum maize performance for supplementing Fe oxide NPs. A confocal laser scanning microscope suggested that Fe oxide NPs entered through the epidermis and from the cortex to the endodermis. Our results provide a scientific basis that the root application of Fe3O4 at the rate of 100 mg L−1 is a promising approach to obtain higher maize performance and reduce the quantity of fertilizer used in agriculture to minimize environmental effects while improving crop productivity and quality. These findings demonstrated the tremendous potential of Fe NPs as an environmentally friendly and sustainable crop approach.Iron (Fe) oxide nanoparticles (NPs) improve crop growth. However, the comparative effect of root and foliar-applied different sources of Fe oxide NPs on plant performance at morphological and physiological levels still needs to be discovered. In this study, we characterized the growth and physiological responses of hydroponic-cultured maize seedlings to four sources of Fe (i.e., α-Fe It perff plants . Foliar-teosinte ,9. Howevo plants ,11. EDTAo plants .3O4), maghemite (γ-Fe2O3), and hematite (α-Fe2O3) remain a topic of interest for future studies [Nanotechnology is experiencing a growing trend in plant sciences, biomedicines, and environmental remediation due to its specific physicochemical properties and surface area . The fer studies . Bulk ma studies . Magneto studies . Magneti studies . The siz studies . Thus, d studies .Iron dynamic in soil-plant systems is well documented, and the uptake and translocation of Fe-based NPs vary widely with shape, size, concentration, and plant species ,21. The 2O3 NPs increases the root length, biomass, plant height, and chlorophyll contents of peanuts (Arachis hypogaea) [Glycine max L. Oxley) takes up Fe3O4 NPs, translocates it from root-to-shoot tissues, and improves chlorophyll contents and photosynthetic activity. Fe2O3 NPs application with a concentration of 200–400 mg L−1 and size range of 10–50 nm can reduce arsenic toxicity and improve the growth of mung bean [−1 α-Fe2O3 (hematite) application improves peanut growth [3O4) increases chlorophyll content, photosynthesis and the biomass of maize seedlings [3O4) and maghemite (γ-Fe2O3) with 50, 100, 200 mg L−1 increase Fe translocation and content in barley seedlings [Cucurbita mixta) shows higher root-shoot Fe3O4 NPs accumulation. These studies suggest that Fe NPs-based fertilizers can be promising in improving crop growth and agricultural productivity. However, the effect of Fe NPs can vary across different crop groups, NPs sources and application methods [The application of γ-Feypogaea) . Ref. [3ypogaea)  found thung bean . Similart growth . Foliar eedlings . Vibratieedlings . Hydropo methods . Thus, t2O3, γ-Fe2O3, Fe3O4) and application methods (root vs. foliar) in improving the performance of maize seedlings. Given different physiological characteristics, i.e., chlorophyll content, antioxidant enzyme activity, and Fe contents in root-shoot tissues, our findings will provide a scientific basis to better evaluate the source and application method of Fe oxide NPs in improving maize performance and agricultural productivity.Global maize production has increased in the past few decades, and maize is the leading cereal crop by production of 5.8 tons per ha on 197 million ha land worldwide . However2O3, γ-Fe2O3, and Fe3O4) and bulk Fe3O4 with sizes ranging from 10–30 nm and 142 nm, respectively, were procured from Macklin Inc. . The NPs were characterized based on their size and shape by using X-ray diffraction (XRD), transmitting electron microscopy (TEM), and selected area electron microscopy (SAED).Non-stoichiometric Iron oxide NPs  seeds were immersed in 10% H2O2 for 30 min and then washed with deionized water at least thrice. Seeds were spread on the petri plates with two layers of filter paper to keep the moisture. After 48 h, seedlings with 1 cm primary root length were wrapped using filter paper and grown in a standard growth chamber. After one week, consistent and uniform-sized seedlings were transferred into Hoagland’s nutrient solution with macro- and micronutrients as follows: 0.5 mmol L−1 Ca(NO3)2, 0.1875 mmol L−1 K2SO4, 0.025 mmol L−1 KCl, 0.0625 mmol L−1 KH2PO4, 0.1625 mmol L−1 MgSO4·7H2O, 0.25 × 10−3 μmol L−1 H3BO3, 0.25 × 10−3 μmol L−1 MnSO4·H2O, 0.25 × 10−3 μmol L−1 ZnSO4·7H2O, 0.25 × 10−4 μmol L−1 CuSO4·5H2O, and 1.25 × 10−6 μmol L−1 (NH4)6Mo7O24. The pH of the nutrient solution was maintained at around 6.8 with 1 mol L−1 NaOH. In case of root application treatment, the 100 mg L−1 and 500 mg L−1 of α-Fe2O3, γ-Fe2O3, Fe3O4 NPs, and bulk Fe3O4  were applied in nutrient solution after one week of acclimation, and later no Fe was supplemented in the control treatment. In this study, foliar spray treatments were conducted using NPs at two stages: once when the plant had four leaves and again when it had six leaves. The NPs were applied at the same concentration as in the root (100 mg L−1 and 500 mg L−1). No additional source of Fe was supplemented to the Hoagland nutrient solution. Amount of Fe 100 mg L−1 and 500 mg L−1 was calculated from different sources of Fe NPs. The given concentrations of NPs for root and foliar applications were selected based on previous studies [Maize  and root (0.4 g) were homogenized separately in 10 mL of 0.05 M pre-cooled phosphate buffer (pH 7.8). After centrifuging the mixture at 4000 rpm for 20 min, it was stored at 4 °C for further analysis. Analysis kits were procured from SolarBio®, Beijing, China.The activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were determined in the root and shoot tissues. Harvested root and shoot tissues were rinsed with distilled water to remove the surface contamination. Grinding was done in liquid N−1 methionine, 0.5 mL of 750 μmol L−1 NBT, 0.5 mL of 100 μmol L−1 EDTA-Na, 0.5 mL of 20 μmol L−1 lactochrome, and 3.5 mL of 0.05 mol L−1 phosphate buffer (pH 7.8). The entirely mixed sample was placed in light for 20 min, and distilled water was used to prepare the control sample and was placed in the dark. Finally, the absorbance was measured at 560 nm [The reaction mixture consists of 0.5 mL supernatant, 0.5 mL of 130 mmol Lt 560 nm ,42.−1 guaiacol, and 19 μL of 30% H2O2 were blended in 100 mmol L−1 phosphate buffer (pH 7.0). The prepared samples were run at 470 nm (A470) for every 30 s, and POD activity was calculated regarding absorbance change per minute (A470/min/g FW).The oxidative process of guaiacol, catalyzed by peroxidase, was used to estimate POD activity . To prep−1 phosphate buffer (pH 7.0) containing 0.01% H2O2. CAT activity was measured at 240 nm for 4.5 min after every 30 s [The CAT activity was calculated by mixing 0.5 mL of supernatant with 3 mL of 100 mmol Lery 30 s .Chlorophyll contents were determined with a UV-752 N spectrophotometer  using 80% acetone extracts as described . Fresh yDuring harvesting, fresh plant samples (leaves and roots) were collected for confocal microscopy LSM 800. Samples for confocal microscopy CLSM 800 imaging were prepared following a standard procedure described earlier . A confo3 to remove the adsorbed NPs on the surface [3 poured to digest the 0.3 g of the oven-dried samples. The next day, 2 mL of 30% H2O2 was added to the solution. Samples were digested for 10 min at 120 °C and then for 15 min at 180 °C in a microwave oven (MILESTONE Ethos D) controlled by a time and temperature regulator to get a clear solution. The Fe concentration in digested solution was determined using an inductively coupled plasma-optical emission spectrometer . The standard plant material  was used for quality control of the Fe analysis.After harvesting, the shoot and root tissues of maize seedlings were completely dipped in 0.01 M HNO surface . Then, sp ≤ 0.05) in the statistical package IBM SPSS 22.Each treatment contained three biological replicates, and the results are presented as mean ± SD (standard deviation). The statistical analysis of all collected data was carried out using three factor factorial ANOVA under CRD followed by Tukey’s HSD test  on the growth parameters  were analyzed. Results showed that the Fe oxide NPs significantly affected the shoot dry weight, shoot height, root dry weight, and root length (p < 0.05) and it was 87.6 cm, 85.8 cm, and 86.0 cm with Fe3O4, γ-Fe2O3, and α-Fe2O3 application, respectively. Moreover, root application of Fe oxide NPs significantly (p < 0.05) affected the root length and dry weight, and Fe3O4 NPs application increased 47% root length and 52% root dry weight, compared with the control treatment  and superoxide (O2) can lead to oxidative stress in plants. Plant produces antioxidants such as SOD, POD, and CAT to combat the toxicity of ROS [2O3, γ-Fe2O3, Fe3O4, and bulk Fe3O4) with 100 mg L−1 and 500 mg L−1. Three types of SOD, namely, Fe-SOD, Mn-SOD, and Cu, Zn-SOD can quickly convert O2 to H2O2 in plant cells. CAT is a well-known antioxidant enzyme that converts H2O2 to H2O and O2 [2O3 increased the CAT activity, which may be due to the degradation of H2O2. The no negative response of maize root suggested that oxidative stress did not occur in the root tissues. POD activity of root and shoot was not affected significantly with root and foliar application of Fe oxide NPs (3O4 NPs [Reactive oxygen species (ROS) such as hydrogen peroxide  was 200 and 330 mg kg−1 DW, respectively. These results suggested that maize roots and leaves efficiently absorbed Fe NPs on the surface due to smaller size, surface charge and magnetic characteristics by different Fe oxide sources . By comparing treatments with control in root application following the trend in root and shoot Fe concentration was observed Fe3O4 > γ-Fe2O3 > α-Fe2O3 > bulk Fe3O4  an average of 40.9 nm reduces its uptake efficiency in Arabidopsis [2O3 with a particle size of 14 nm efficiently utilized by barley [2O3 and Fe3O4 NPs also support the uptake of NPs. The size of aggregated NPs could be larger than the pores space of the cell wall, except for a few that freely translocate through the endodermis and cortex [Significant difference in Fe concentration in roots and shoots of maize seedlings was found by Fe NPs types, application methods, and application rate . In the lk Fe3O4 A. With fhen bulk B. Overalhen bulk . Iron cohen bulk C. We spean roots D, which bidopsis , and α-Fy barley . Microscy barley . Notablyd cortex . Fe NPs d cortex . As maizd cortex . Importad cortex . Thus, sd cortex . The epid cortex . Howeverd cortex .Finally, to gain better insight into Fe oxide NPs translocation in maize seedlings, root and leaves anatomy was observed using the confocal laser scanning microscopy (CLSM) . CLSM re2O3, γ-Fe2O3, Fe3O4, and bulk Fe3O4) on the growth and Fe nutrition of maize seedlings. We concluded that: (i) Fe oxide  has a positive impact on maize seedling growth and Fe nutrition; (ii) the application of Fe oxide NPs to the roots is more effective than foliar application in improving Fe concentration, root length, plant biomass, and chlorophyll content. This may be attributed to the translocation of Fe oxide NPs in the shoot, as confirmed by confocal microscopy and the longer exposure time of NPs to the roots; and (iii) overall, Fe3O4 NPs are the most efficient NPs among the four tested Fe sources, followed by γ-Fe2O3 and α-Fe2O3, when applied at a rate of 100 mg L−1. In a nutshell, the results of this study suggest that applying Fe3O4 directly to the roots at a concentration of 100 mg L−1 can serve as a promising Fe fertilizer to enhance maize yield. In future studies, the efficiency of α-Fe2O3, γ-Fe2O3, and Fe3O4 NPs regulated by site-specific covariates  should be investigated in detail and potential environmental risk with bioaccumulation of Fe NPs in food chain can be demonstrated in detail. The industrial application of Fe nanoparticles as absorbent of heavy metals from wastewater and drug delivery in nanomedicine is an interesting topic to be discussed in future studies.In this study, we investigated the comparative effects of root and foliar application of four Fe oxide NPs (α-Fe