Therefore, new treatment options in radiotherapy need to be developed to inhibit MGMT and increase radiotherapy response. Methods: Lomeguatrib, a highly specific MGMT inhibitor, was used to inactivate MGMT protein in vitro. Radiosensitivity of established human glioblastoma multiforme cell lines in combination with lomeguatrib was investigated using the clonogenic survival assay. Inhibition of MGMT was analyzed using Western Blot. Cell cycle distribution and apoptosis were investigated to determine the effects of lomeguatrib alone as well as in combination with ionizing radiation. Results: Lomeguatrib significantly decreased MGMT protein and reduced radiation-induced G2/M arrest. A radiosensitizing effect of lomeguatrib was observed when administered at 1 µM and increased radioresistance at 20 µM. Conclusion: Low concentrations of lomeguatrib elicit radiosensitization, while high concentrations mediate a radioprotective effect.Background: Treatment resistance of glioblastoma multiforme to chemo- and radiotherapy remains a challenge yet to overcome. In particular, the O Glioblastoma multiforme (GBM) is still one of the most devastating diagnoses. Despite developments in alternative treatment options including novel chemotherapeutic agents, inhibitors, and targeted miRNA delivery, as well as extensive research in radio- and chemoresistance, improvement of patient survival is still poor. With a five-year survival of only 0.05–4.7% after diagnosis , mortaliMagnetic resonance imaging (MRI) serves as the first tool of diagnosis, however, a biopsy and pathological examination are required to confirm GBM and determine the subtype. First in line for the treatment is the maximal safe surgical resection of the tumor to relieve symptoms caused by the increased intracranial pressure such as headaches, nausea, vomiting, somnolence, and visual impairments . Due to 2 for five consecutive days for six weeks [2 for maintenance [Since 2005, the chemotherapeutic agent temozolomide (TMZ) is orally administered with a daily dose of 75 mg per mntenance . After antenance .3 position and two positions of the base guanine are N7 (70%) and O6 (5%) [6-methylguanine (O6-MG), is assumed to have cytotoxic and mutagenic potential [6-MG creates a wobble base pair with thymine, which is recognized by the mismatch repair (MMR) pathway. MMR excises the mismatched thymine but will subsequently replace it with another thymine. These futile circles of thymine deletion and insertion will eventually lead to a depletion of deoxythymidine triphosphates (dTTP) resulting in lack of DNA synthesis and ultimately cell death by apoptosis [6-methylguanine-DNA-methyltransferase (MGMT) specifically removes these methyl adducts from the O6-MG preventing futile thymine deletion and insertion circles [TMZ is an alkylating agent methylating several sites within the DNA: one site in the base adenine is the N O6 (5%) ,11. Onlyotential ,13. Duripoptosis . The O6- circles . The met circles . The res circles . MGMT is circles . The pro circles . However6 position with heterocyclic moieties they synthesized guanine derivatives compatible with the stereochemical requirements at the MGMT’s active site. First characterization experiments in vivo were performed by Middleton et al. [−1 lomeguatrib [O(6)-(4-bromothenyl)guanine also known as PaTrin-2, lomeguatrib, O6BTG, or 4BTG is a potent MGMT inhibitor first synthesized by McElhinney et al. . By modin et al. . They obeguatrib .Therefore, this work aimed to investigate the effects of lomeguatrib on radiosensitivity of GBM cell lines with an unmethylated MGMT promoter region. Other cellular processes such as apoptosis, cell cycle distribution, and DNA repair were investigated as well.Colony-forming assay (CFA) was performed in order to determine the radiosensitivity of established human glioblastoma multiforme cell lines. Cells were pre-plated in 12-well plates 24 h prior to 0 Gy, 1 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy ionizing radiation.50 of 3.30 Gy, while LN18 was intermediate radiosensitive with a D50 value of 2.26 Gy, while U118 was the most radiosensitive amongst the three tested cell lines with a D50 of 1.66 Gy. Significant differences in the survival curves were detected between LN18 and T98G (p < 0.0001), between LN18 and U118 (p < 0.0001), and between T98G and U118 (p < 0.0001) cell lines  were exposed to different lomeguatrib concentrations for 4, 6, 8, 24, or 48 h and lysates were subjected to Western Blot analysis .Already 4 h after lomeguatrib treatment a decrease in MGMT protein was observed in T98G and U118 cell lines at all tested concentrations. In LN18 cells MGMT inhibition became visible after 24 h of lomeguatrib treatment at all concentrations. Inhibition of MGMT was still detectable after 48 h. Since MGMT inhibition was visible after 24 h in all cell lines, we decided to use this treatment duration (24 h) for further experiments.p = 0.0197) was detected at the highest concentration of 20 µM as well as a trend towards an increased G1 fraction (p = 0.0562) at 20 µM lomeguatrib, compared to the untreated sample (p = 0.0411) was detected in the U118 cell line at 20 µM lomeguatrib, compared to the untreated sample  and T98G (p = 0.0150) upon treatment with 1 µM lomeguatrib, while in U118 cells only a non-significant increased radiosensitivity was observed (p = 0.1468). Radioresistance was increased in T98G (p < 0.0001) and U118 (p = 0.0008) upon 20 µM lomeguatrib treatment, and a trend towards an increased radioresistance was observed in LN18 cells (p = 0.0954). Differences between 0 µM and 1 µM as well as 0 µM and 20 µM for each radiation dose were calculated using Student’s t-test and are indicated in Two-Way ANOVA was used to calculate differences between the treatment groups. Increased radiosensitivity was observed in LN18  to 3.05 ± 0.04 Gy upon 20 µM lomeguatrib (p = 0.045) but did not change upon 1 µM lomeguatrib  and decreased to 2.54 ± 0.18 Gy (p = 0.030) in the presence of 1 µM lomeguatrib  upon 20 µM lomeguatrib in U118 cells and remained unchanged upon 1 µM lomeguatrib treatment , while 20 µM lomeguatrib treatment resulted in a significant decrease in SER in all cell lines .Deguatrib . For theeguatrib . D50 valreatment . The SenNext, we investigated the combined effect of lomeguatrib and radiation on cell cycle distribution. Cells were treated with lomeguatrib for 24 h before irradiation and were fixed 24 h later.p < 0.0001; T98G: p = 0.0003; U118: p < 0.0001). Treatment with 1 µM without irradiation did not change cell cycle distribution in any of the tested cell lines (p = 0.0140), while in the other cell lines no difference was detected. 8 Gy irradiation combined with 20 µM lomeguatrib decreased G2/M phase in LN18 cells (p = 0.0085) and increases G1 phase accordingly (p = 0.0332) with a trend towards a decreased S phase (p = 0.0687) compared to the 8 Gy irradiated sample without lomeguatrib. An increased G1 phase (p = 0.0342) and a trend towards a decreased G2/M phase (p = 0.0511) in the 8 Gy irradiated and 20 µM lomeguatrib treated sample was observed in T98G as well, compared to the 8 Gy irradiated sample. In U118 cells, no significant difference was detected comparing the 8 Gy irradiated sample to the 8 Gy and 20 µM lomeguatrib treated sample, with only a trend towards an increased G1 phase (p = 0.0809).8 Gy ionizing radiation alone significantly enhanced G2/M cell cycle fraction , as well as in combination with 1 µM lomeguatrib  increased apoptotic cell fraction in all cell lines. 20 µM lomeguatrib combined with 8 Gy radiation did not change apoptotic cell fraction in LN18 and U118 cells, however, an increase was observed in T98G cells (p = 0.0125).The effects of lomeguatrib in combination with radiation on apoptosis were measured 48 h after irradiation via caspase-3/7 FACS analysis. 1 µM, as well as 20 µM alone, decreased apoptotic cells in the LN18 cell line . Lomeguatrib showed a radiosensitizing effect at lower concentrations and an increased radioresistant effect at higher concentrations. Further, higher concentrations of 20 µM reduced the G2/M cell population in combination with 8 Gy ionizing radiation and increased the G1 and S phase cell fraction accordingly. The underlying mechanism has yet to be investigated and we propose an interaction between lomeguatrib and the key regulators of the G1-to-S or G2-to-M transition point. As DNA damage plays a crucial role in the efficacy of radiotherapy the effect of lomeguatrib on DNA double-strand breaks, as well as subsequent cell death needs further investigation; here we propose a strong dose-dependent effect of lomeguatrib.This study presents new insights on the strong dose-dependent effects of lomeguatrib in vitro, which could help to minimize myelosuppression and hematologic side effects observed with other MGMT inhibitors, such as O6-benzylguanine ,22. We a−1 penicillin, and 100 U mL−1 streptomycin . All cell lines were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Cells were seeded 24 h before lomeguatrib treatment i.e., 48 h before irradiation treatment.Established human glioblastoma multiforme cell lines were obtained from the University Hospital of Heidelberg  or purchased from the American Type Culture Collection . Cells were regularly checked for the absence of mycoplasma and cell line authentication was performed by Eurofins Genomics. LN18 and U118 were cultured in high glucose DMEM  and T98G in low glucose DMEM  supplemented with 10% FCS, 100 U mLLomeguatrib was purchased from MedChemExpress LLC  and dissolved in DMSO . The stock solution of 6.13 mM was stored at −80 °C and diluted at 1:10 or 1:100 in medium immediately before use. Final DMSO concentrations did not exceed 0.3%. Cells were seeded 24 h before lomeguatrib treatment for 24 h before irradiation.−1 at 15 mA and 200 kV with a 0.5 mm copper filter and a distance to the x-ray tube of 15 cm.X-ray irradiation was performed at an RS225A irradiation device  at a dose rate of 0.9 Gy minRadiosensitivity of all cell lines was determined using the clonogenic survival assay. Cells were pre-plated at appropriate cell numbers per dose per 12-well plate and treated with 0 µM, 1 µM, or 20 µM lomeguatrib 24 h prior to 0 Gy, 1 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy irradiation. Followed by an 8-day (T98G and U118) respectively 12-day (LN18) incubation period, colonies were fixed with 100% −20 °C cold methanol and stained using 0.1% crystal violet. Colonies consisting of at least 50 cells were manually counted as one colony. Plating efficiencies, as well as survival fractions, were calculated in order to plot survival curves fitted to the linear-quadratic model:D10 and D50 values were calculated using the following formulas:α and ß values were derived from the linear-quadratic model and Cell proliferation was measured using the alamarBlue™ Cell Viability Reagent  in a 96-well format. Following lomeguatrib treatment for 24, 48, and 72 h, 10% of the alamarBlue Reagent was added to the cells and incubated for 4 h at 37 °C. Absorbance at 570 nm and 630 nm was determined on a Microplate Reader EL808 . Doubling times were calculated using the following formula:48 h after lomeguatrib, i.e., 24 h after radiation treatment, cells were lysed in RIPA buffer  on ice for 30 min with vortexing every two minutes. After centrifugation at 12,000 rpm for 10 min at 4 °C protein lysates were collected from the supernatants and stored at −80 °C for further use. Protein concentration was determined using the Pierce™ BCA™ Protein-Assay . Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Incubation with the primary antibodies anti-MGMT  and anti-ß-actin  was done overnight at 4 °C. Secondary antibody incubation using the anti-mouse IgG (H+L) AP conjugate  was done for 2 h at room temperature. Proteins were detected using the Novex™ AP Chromogenes Substrate (BCIP/NBT) .−1 PI , 0.15% Triton X-100 and 0.2 mg mL−1 DNase-free RNase A) was added to the samples and incubated for 30 min in the dark at room temperature. Acquisition was performed in the BD FACSCalibur™ . Cell cycle distribution was analyzed using ModFit LT™ software .24 h after irradiation, i.e., 48 h after lomeguatrib addition, cells were fixed in −20 °C cold 70% for at least two hours. Propidium iodide (PI) staining solution  according to the manufacturer’s instructions. 48 h after radiation treatment, i.e., 72 h after lomeguatrib treatment samples were collected and incubated with the Caspase-3/7 Green Detection Reagent for 40 min at room temperature. Staining of the dead cells was done using the SYTOX AADvanced dead cell stain solution for 5 min at 37 °C. Immediately, FACS analysis was performed in the BD FACSCalibur™  and quantification of apoptotic cells was analyzed in the BD CellQuest™ software.t-test. Two-Way ANOVA was used to evaluate significant differences between cell lines in the colony-forming assay using GraphPad Prism. Probability values of p < 0.05 were regarded as statistically significant. In order to ensure reproducibility of the results, each experiment was repeated at least three times.Mean values were calculated and are presented as ± standard error of the mean (SEM). Differences in mean values between groups were compared using Student’s


A Disintegrin And Metalloproteinase with ThromboSpondin Motifs 16 (ADAMTS16), an extracellular protease that was previously reported to be essential for optic fissure fusion in zebrafish eye development. To test the functional relevance of this observation, we targeted dpy19l1 or dpy19l3 in embryos of the Japanese rice fish medaka (Oryzias latipes) by CRISPR–Cas9. We observed that targeting of dpy19l3 partially caused defects in optic fissure fusion, called coloboma. We further showed in a cellular model that DPY19L1 and DPY19L3 mediate C-mannosylation of a recombinantly expressed thrombospondin type 1 repeat of ADAMTS16 and thereby support its secretion. Taken together, our findings imply that DPY19L3-mediated C-mannosylation is involved in eye development by assisting secretion of the extracellular protease ADAMTS16.C-mannosylation is a modification of tryptophan residues with a single mannose and can affect protein folding, secretion, and/or function. To date, only a few proteins have been demonstrated to be C-mannosylated, and studies that globally assess protein C-mannosylation are scarce. To interrogate the C-mannosylome of human induced pluripotent stem cells, we compared the secretomes of CRISPR–Cas9 mutants lacking either the C-mannosyltransferase DPY19L1 or DPY19L3 to WT human induced pluripotent stem cells using MS-based quantitative proteomics. The secretion of numerous proteins was reduced in these mutants, including that of   •TSR1 of ADAMTS16 can be C-mannosylated.•Deletion of DPY19L1 or DPY19L3 in hiPSCs caused reduced secretion of ADAMTS16.•dpy19l3 in medaka occasionally led to coloboma.Targeting of  We identified ADAMTS16 as a target protein for C-mannosylation and showed that this modification is needed for proper secretion of ADAMTS16. Targeting a distinct C-mannosyltransferase by CRISPR–Cas9 in medaka fish embryos caused defects in eye development. We conclude that these developmental defects are caused by reduced secretion of ADAMTS16 when C-mannosylation is missing. The combined analysis of our cellular and organismal studies uncovered the importance of C-mannosylation for secretion and function of ADAMTS16.Acknowledging the importance of C-mannosylation for protein secretion and motivated by an interest in the functional role of C-mannosylation for early developmental processes, we quantitatively compared the levels of secreted proteins from cell culture supernatants of human induced pluripotent stem cells (hiPSCs) and derived KOs of the C-mannosyltransferases DPY19L1 or DPY19L3 by MS. In a complementary study, we applied targeted genome editing on 2, and 85% relative humidity in cell culture vessels purchased from Greiner Bio-One. Cell culture reagents were purchased from Thermo Fisher Scientific unless otherwise stated. All stem cell–based experiments were performed as described previously  and genTGG and 5’-GCTGATGGCTAGGCTATAGGAGG (DPY19L1); 5’-GATAATCTAGGTACAATTGGTGG and 5’-GAGCTGTGACATAGATCGCCTGG (DPY19L3). sgRNA templates were individually cloned into a plasmid coding for a SpCas9 endonuclease and enhanced GFP (eGFP) (6 cells were cotransfected with 5 μg of each of the two sgRNA coding plasmids using a NEON transfection device (Thermo Fisher Scientific) with 1200 V for 2 × 20 ms. Cells were reseeded in mTeSR1 medium supplemented with 10 μM Rho-associated coiled-coil containing protein kinase inhibitor , and eGFP-positive cells were sorted after 3 days applying fluorescence-activated cell sorting. About 5 × 103 sorted cells were seeded on a murine embryonic fibroblast–coated 10 cm dish in stem cell medium supplemented with 10 μM RI and cultured for 10 days until separate single-cell colonies became visible. Colonies were picked manually by using 200 μl pipette tips and under a light microscope at sterile conditions. Picked colonies were transferred into murine embryonic fibroblast–coated 48-well plates in stem cell medium supplemented with 10 μM RI. After 4 days, single-cell clones were passaged, and cells were extracted for direct cell lysis and genotyping by PCR analysis (B).For genome editing in stem cells, we applied the CRISPR–Cas9 system each with two different single-guide RNAs (sgRNAs) to mediate macrodeletions in DPY19L1 and DPY19L3, respectively. The CCTop algorithm  was applP (eGFP) . hiPSCs analysis B.For total RNA extraction, NucleoSpin RNA Kit (Macherey–Nagel) was applied. Samples were sequenced on an Ion Torrent S5 XL Instrument (Thermo Fisher Scientific). Read mapping was carried out with STAR, version 2.6.0 with default parameters and the GRCh37 release 87 reference genome . HTSeq, myc epitope, and polyhistidine tag. This resulted in the pCAG-SecTagB vector. Protein coding sequences were amplified from hiPSC-derived complementary DNA and cloned into KpnI and NotI sites of the pCAG-SecTagB vector (7 hiPSCs were transfected with 50 μg of plasmid (split into four transfections with each 2.5 × 106 cells and 12.5 μg plasmid) using a NEON transfection device (Thermo Fisher Scientific) with 1200 V for 2 × 20 ms. Cells were reseeded in four T25 cell culture flasks on Matrigel in TeSR–E8 medium supplemented with RI. For protein expression in HEK 293T and CHO-K1 cells, 3 × 106 cells were seeded into a T75 cell culture flask in 20 ml DMEM/Ham's F-12 + 10% FCS or 5% FCS, respectively. For protein expression in KO cells, two approaches were pooled in the end. Cells were transfected after 24 h with 20 μg plasmid using 100 μg polyethyleneimine in 2 ml Opti-minimum essential medium followed by medium exchange after additional 6 h. For coexpression with a construct encoding myc-tagged epidermal growth factor repeats 9–14 of mouse Notch1, cells were cotransfected with additional 5 μg of the respective plasmid using 125 μg polyethyleneimine. Secreted proteins were purified from the medium after 3 days by applying nickel affinity chromatography and SDS-PAGE as described  was excised (XhoI and BglII) and replaced by a section of pSecTagB (Invitrogen) comprising the signal peptide for the secretory pathways, multiple cloning site, c-B vector . For proFragment Ion Calculator (Institute for Systems Biology) and annotated manually in detected MS/MS spectra. In addition, MS spectra were automatically searched with ProteinLynx V2.2.5 software (Waters) against custom-made databases containing sequences of individual proteins using preconfigured settings except “peptide tolerance” was set to 0.3 Da and “validation of results” was switched off. Oxidation (M), C-mannosylation (W), and modification by fucose or fucose–glucose  were chosen as variable modifications and carbamidomethyl (C) as fixed modification.Proteolytically digested proteins were analyzed using an electrospray ionization (ESI) quadrupole-TOF (Q-TOF) Ultima mass spectrometer (Waters) coupled with a nanoACQUITY UPLC System with an analytical C18 column . The MS analysis was performed as described previously . LC was g for 5 min to remove cell debris. Proteins were precipitated applying the trichloroacetic acid/sodium lauroyl sarcosinate method  Orbitrap-Velos mass spectrometer coupled to a reversed-phase LC system as described previously . ReverseLFQ min. ratio count set to 1 and Fast LFQ switched off. The MaxQuant output file “proteinGroups.txt” is provided in MaxQuant software suite, version 1.6.11.0 , with thhttp://annotations.perseus-framework.org) was used for filtering and statistical analysis . Levels of secreted proteins in different cell lines were compared by pairwise t tests. Proteins containing consensus sites for C-mannosylation were annotated by applying a custom-made R script searching all UniProt ID–related protein sequences of a protein group for the presence of WxxW or WxxC motifs. Fisher exact tests were applied to identify protein domains enriched within the group of proteins with altered abundance in the KO secretomes. Results were visualized by GraphPad Prism software (version 5).Perseus software, version 1.6.2.1 , with imt tests . p = 0.05) were applied to identify enrichment of protein families (according to the Pfam database) among the 25 proteins that were significantly affected in the DPY19L1 KO or DPY19L3 KO groups and that contain at least one consensus site (WxxW/WxxC) for C-mannosylation, referred to the frequency within all 398 extracellular proteins that remained upon filtering. For RNA-Seq analyses, normalization and differential expression calculations were carried out using the R package DESeq  and two individual clones of both, DPY19L1 KO and DPY19L3 KO, to minimize clonal effects caused by genome editing and clone selection. Two biological replicates for RNA-Seq and three biological replicates for secretomics were analyzed for WT and each KO clone. For secretomics, all biological replicates of independent cultures of WT (n = 3) and of both clones for DPY19L1 KO  and DPY19L3 KO (two clones with n = 3 each → n = 6) were grouped, and the WT group was compared separately to the DPY19L1 KO and DPY19L3 KO group by pairwise ge DESeq . RNA-Seqm/z range, of which two contained consensus sites for C-mannosylation, were calculated using Skyline, version 20.1 (via a NanoSpray III Source (both Sciex). Uncoated precut emitters  and a voltage of approximately 2.6 kV were applied for ESI.A THBS1 fragment comprising TSR1 to 3 was cloned directly into the HindIII and XbaI sites of pSecTagB .O. latipes) Cab strain used in this study were kept as closed stocks in accordance to Tierschutzgesetz §11, Abs. 1, Nr. 1 and with European Union animal welfare guidelines. Medaka experimentation was performed at the one-cell stage and analyzed at embryonic stages prior to stage 41, that is, prior to self-feeding and neither the loss of embryonic features and is thus not considered animal experimentation nor is an approval by an ethics committee required. Fish were maintained in a constant recirculating system at 28 °C on a 14 h light/10 h dark cycle .Medaka (in vitro transcription was performed as detailed previously (sgRNAs were designed with CCTop and standard conditions . The foleviously .O. latipes) Cab strain zygotes were microinjected into the cytoplasm with 150 ng/μl Cas9 mRNA, 15 ng/μl per sgRNA, and 10 ng/μl GFP or mCherry mRNA as injection tracer. Embryos were maintained in embryo-rearing medium  at 28 °C after injections and screened for GFP or mCherry expression 1 day post injection with a Nikon SMZ18 binocular. GFP- or mCherry-positive crispants were raised at 28 °C and phenotyped. Subsequently, PCR genotyping was performed on extracted DNA of pooled and single embryos: specimens were ground and DNA was extracted using 50 μl extraction buffer . Genotyping PCR was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs) with 98 °C at an initial denaturation for 2 min, followed by 30 cycles of 98 °C denaturation for 20 s, 67 °C annealing for 30 s, and 72 °C extension for 25 s. The following primers were used: dpy19l1_forward 5’-CCTCTAGTGTTTCTGTCCCCC, dpy19l1_reverse 5’-AGCTAGCACGCTAAAACCACT, dpy19l3_forward 5’-CATGCTGAACTCCAAACGCC, and dpy19l1_reverse 5’-GGGTTTGTGGCCTTTTTGCT. Amplicons were analyzed by gel electrophoresis, followed by gel extraction  and sequencing (MWG Eurofins).One-cell stage WT medaka . This strategy enabled simplified PCR-based screening of single cell–derived clones for genomic deletions (A and B). For both C-mannosyltransferases, two individual KO clones were obtained (C1 and C2), one bearing a biallelic macrodeletion (DPY19L1 KO C1 and DPY19L3 KO C1) and one containing a monoallelic macrodeletion combined with insertions and deletions (indels) on the second allele resulting in a frameshift (DPY19L1 KO C2 and DPY19L3 KO C2) (C and D).Two functional C-mannosyltransferases were reported for mammalian cells that are encoded by the genes  DPY19L3 . To stud DPY19L3  by apply DPY19L3  aiming tameshift A. This seletions , A and B3 KO C2) , C and DKlf4, Myc, Nanog, POU5F1, and Sox2 was similar. This suggests the absence of major genomic rearrangements or deleterious off-target effects that might have occurred during the procedure of gene editing (E).Whole genome expression analysis by RNA-Seq of WT and KO hiPSCs revealed that their overall gene expression pattern including the expression of the stem cell markers  editing E.DPY19L1 or DPY19L3 on C-mannosylation, a C-terminally myc-His6-tagged construct comprising TSRs, TSR2, and TSR3, of human THBS1 with a cleavable N-terminal secretion sequence was expressed in WT and KO hiPSCs (B). Its purification from cell culture supernatants by nickel affinity chromatography and separation by SDS-PAGE indicated that DPY19L1- and DPY19L3-deficient hiPSCs secreted less of the THBS1 fragment than WT cells (C). This is in accordance with our previous observation that C-mannosylation was required for proper secretion of UNC5A TSRs . Assignment of peaks in EICs was based on mass accuracy of the parental ion, peptide fragmentation spectra .To determine the effect of the genomic deletions in O hiPSCs B. Its puWT cells C. This iC5A TSRs . The THB as EICs D. Assign spectra  and RTs A) of which the majority (342 proteins) were identified in all cell lines. A statistical analysis revealed that, compared with the WT, in the secretomes of DPY19L1 KO and DPY19L3 KO clones, the abundance of 33 and 52 proteins was significantly altered (p ≤ 0.05), respectively, whereas reduction of transcription by more than twofold was only observed for five of these proteins . Affirming our approach, one of these candidates, THBS1, is already known as a target protein for C-Man . Based on this observation, we screened the entire list of 398 extracellular proteins for TSP1 domains in order to reveal more potential candidates that were not present in our initial screen. This analysis uncovered 11 proteins with TSP1 domains in total including several members of the ADAMTS family, of which several showed reduced abundance in the mutants' supernatants .Numerous studies have shown that C-mannosylation supports secretion of target proteins. This effect was used in this study to identify novel C-mannosylated proteins that are potentially involved in early developmental processes by quantitative comparison of protein levels in cell culture supernatants of WT and derived DPY19L1- or DPY19L3-deficient hiPSCs. The secretomes of each of three replicates of WT hiPSCs as well as of the KO cell lines DPY19L1–C1, DPY19L1–C2, DPY19L3–C1, and DPY19L3–C2 were analyzed by LC–MS/MS and processed with the MaxQuant and Perseus software tools , 35; sup; sup35; supplemental Fig. S3proteins . Screenior C-Man . As seveor C-Man , we perfor C-Man . This anajority 3 proteinsA). With the knowledge of the specific chromatographic RTs and the intensity patterns of fragment ions for the two peptides containing the WxxW sites for C-mannosylation from the recombinant THBS1, we now analyzed a native sample of hiPSC-derived proteins from cell culture supernatants by MRM. Indeed we detected the most intense fragment ions for the peptide DACPINGGWGPWSPW with one or two C-mannoses at the expected elution times and with the expected intensity profiles .Because of technical restrictions, no C-mannosylated peptides could be identified in the secretomics screen by MS. Thus, we set up a more sensitive targeted MRM approach  for deteFig. 3DPY19L1 or DPY19L3 in the medaka genome by zygotic microinjection of mRNA encoding Cas9 and two specific sgRNAs per gene at the one-cell stage (A). Targeting of dpy19l1 caused major shortening of the anterior–posterior axis and caudal bending in more than 80% of the injected embryos . In contrast, the vast majority of medaka embryos in which dpy19l3 was targeted showed normal embryonic development. However, in about 5% of the dpy19l3 crispants, the optic fissure did not close properly, a defect known as coloboma (B and C). Interestingly, a similar coloboma phenotype was previously observed in zebrafish upon KO of ADAMTS16 (C), we hypothesized that ADAMTS16 is a target for C-mannosylation and that C-mannosylation of ADAMTS16 is important for its proper secretion, thereby affecting its function.To assess the impact of defective C-mannosylation at an organismal level, we targeted the orthologs of human ll stage A. Targetcoloboma , B and CADAMTS16 . Since AADAMTS16 C, we hypA) was recombinantly expressed in CHO-K1 WT, DPY19L1 KO, and DPY19L3 KO cells (B). MS analysis of the secreted TSR1 of ADAMTS16 revealed that the WSDWSSWSPC motif of TSR1 of ADAMTS16 can be C-mannosylated at all three tryptophan residues. Deletion of DPY19L1 prevented C-mannosylation of the two first tryptophans, whereas C-mannosylation of the third tryptophan was not detected in the DPY19L3 mutant (C). Interestingly, the non–C-mannosylated TSR1 peptide of ADAMTS16 was not detected at all suggesting that it is not secreted without C-mannosylation.In order to analyze C-mannosylation of ADAMTS16, its TSR1 A was recKO cells . WesternKO cells B. MS ana3 mutant C. IntereMore than 2500 proteins harboring C-mannosylation sites were predicted by bioinformatics from the human genome , but to dpy19l1, in medaka embryos, major malformations appeared, suggesting a broad impact of dpy19l1-mediated C-mannosylation during ontogeny. Interestingly, dpy19l3 crispant medaka embryos partially developed ocular coloboma, that is a gap caused by incomplete closure of the optic fissure. A similar phenotype was recently shown in zebrafish upon morpholino-based KO of adamts16 (dpy19l3 crispant medaka embryos. Consequently, its levels in the extracellular milieu of the developing eye may not be sufficient to fulfill its functions. The low prevalence of the coloboma phenotype in our medaka model can be explained as a result of both, the mosaic organism generated upon injection of the CRISPR/Cas9 system and the non–cell autonomous effect of the secreted ADAMTS16 protease. In this study, we show that TSR1 of ADAMTS16 is C-mannosylated by DPY19L1 and DPY19L3. We also obtained strong evidence that secretion of ADAMTS16 depends on C-mannosylation as the secretion levels of the recombinantly expressed ADAMTS16 TSR1 as well as the endogenous ADAMTS16 were both reduced in C-mannosyltransferase–deficient CHO-K1 and hiPS cells, respectively.The quantitative secretomics analysis revealed for the majority of significantly affected proteins a reduced secretion in the C-mannosyltransferase–deficient hiPSC models, and identification of THBS1  can be cadamts16 . Accordiadamts16 , and wasadamts16 . Thus, Aadamts16 . ADAMTS1adamts16 . Concertadamts16 , 57. Conadamts16 , these fTaken together, we identified ADAMTS16 as a novel target protein for C-mannosylation and showed that C-mannosylation indirectly affects its function by affecting secretion of ADAMTS16.ProteomeXchange Consortium via the Panorama Public partner repository (via the PRIDE partner repository (MS Viewer repository with the search key p5vtjou6lz.All data are available in the main text or the supplemental files. The MRM data have been deposited to the pository  with thepository  with theThis article contains The authors declare no competing interests.


Most importantly, Pt contents above 3 mol% removed up to 100 ppm methanol, isoprene and ethanol completely already at 40 °C and high relative humidity, while acetone was mostly preserved, as confirmed by mass spectrometry. When combined with an inexpensive, chemo-resistive sensor of flame-made Si/WO3, acetone was detected with high selectivity (≥225) over these interferants next to H2, CO, form-/acetaldehyde and 2-propanol. Such catalytic filters do not require additional heating anymore, and thus are attractive for integration into mobile health care devices to monitor, for instance, lifestyle changes in gyms, hospitals or at home.Catalytic packed bed filters ahead of gas sensors can drastically improve their selectivity, a key challenge in medical, food and environmental applications. Yet, such filters require high operation temperatures  impeding their integration into low-power  devices. Here, we reveal room-temperature catalytic filters that facilitate highly selective acetone sensing, a breath marker for body fat burn monitoring. Varying the Pt content between 0–10 mol% during flame spray pyrolysis resulted in Al Chemical gas sensors are promising for the next generation of handheld devices for air  or food 2/WO3 nanocrystals [2 that forms in the intestinal tract after food intake and can reach significantly higher concentrations (>50 ppm) than breath acetone . Al-ZnOexercise  and Si/Wrcise [3  are intercise [3 . Finallyanotubes  and Au vanotubes  are inteanotubes ).2. While Co3O4 and PdO nanocatalysts on In2O3 hollow spheres removed toluene, CO, H2 and NH3 quite effectively over acetone, their performance on ethanol has not been evaluated yet to assess possible interference [2O3 catalyst at 135 °C that featured unprecedented acetone selectivity (>250) over ethanol, H2, CO, isoprene, NH3, methanol, formaldehyde, acetaldehyde, toluene and m-xylene at 90% relative humidity (RH) [2O3 surfaces. In contrast, acetone remains intact since the Lewis acidic sites required for acetone conversion [Recently, a packed bed catalytic filter of flame-made ZnO nanoparticles at 260 °C was introduced that removed ethanol selectively  over acetone  by explorference . This waity (RH) , as provity (RH)  with intity (RH) ) by hydrnversion  are blocnversion  present nversion ), requirnversion .x clusters [2O3, while preserving selectivity [The reactivity of catalysts, and thus operational temperature, is influenced strongly by noble metals, as they lower the activation energy, for instance, by dissociatively adsorbing highly reactive oxygen . Specificlusters . Similarclusters  as was tclusters . Finallyectivity .2 and CO) selectively over acetone. This is achieved by flame spray pyrolysis (FSP) yielding highly dispersed Pt/PtOx clusters (0–10 mol%) on Al2O3 nanoparticles at controlled composition [2 adsorption. The catalytic performance towards methanol, ethanol (up to 100 ppm), isoprene and acetone was investigated at breath analysis-relevant 90% RH by proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS). To demonstrate the filters’ immediate practical impact, it was connected to a flame-made, chemoresistive Si/WO3 sensor [Here, room-temperature catalytic filters are introduced that remove alcohols, aldehydes and inorganics  to achieve a total metal content (Pt + Al) of 0.5 M and a Pt loading of 0, 1, 3, 5 and 10 mol%. This corresponds in the product (Pt on Al2O3) to 0, 3.7, 7.2, 16.8 and 29.8 wt%, respectively. The precursor was fed at 5 mL/min through a nozzle and dispersed by 5 L/min oxygen at a pressure drop of 1.7 bar. A ring-shaped premixed methane/oxygen flow  ignited the spray and sustained the flame. The powder was collected with a vacuum pump  on a water-cooled glass fiber filter  at 50 cm height above the burner. The particles were scraped off the filter with a spatula and thermally stabilized by annealing in air at 500 °C for 1 h in an oven .Pt/Ald by FSP  with a rd by FSP . Therefoθ = 15–70° with a scanning step and speed of 0.011° and 0.0057°/s using a Bruker AXS D8 Advance diffractometer operated at 40 kV and 30 mA. Crystal phases were identified by the software Bruker Diffrac.eva V3.1 by comparison to structural parameters of cubic γ-Al2O3 (PDF 10–0425), cubic Pt (PDF 01-1311) and tetragonal PtO (PDF 85-0714). To identify peak shifts, crystalline NiO  was admixed in a 1:1 w/w ratio as internal standard [D is the crystallite size in nm; K is the Scherrer constant ; λ is the wavelength of the CuKα X-ray source ; β is the full width at half maximum (FWHM) and θ is the peak position (both in radian). Bimodality of Pt was identified by subtracting first the pure Al2O3 pattern and subsequent peak deconvolution at 2θ = 39.7° using the software OriginPro 2018G (OriginLab Corporation). Al2O3 crystal sizes were determined with the Scherrer Equation at 2θ = 67.1°, while for 10 mol% Pt, the Al2O3 peak was first deconvoluted from the Pt peak.X-ray diffraction (XRD) patterns were recorded at 2standard  to align2 reduction, a high temperature cell was used . For this, the 3 mol% Pt/Al2O3 powder was filled into a 16 mm sampler holder featuring a 1 mm high edge . The particles were flushed with a constant flow of 50 mL/min H2/Ar and the temperature was increased stepwise with 0.5 °C/s from 30 °C to 150, 250 and 350 °C. The particles remained in isothermal conditions for 2 h at each temperature prior to analysis.For in situ XRD analysis during H2 adsorption was carried out at 77 K using a Tri-Star Micromeritics II Plus. All powders were degassed at 150 °C in N2 for 1 h prior to the analysis to remove humidity and other residues from the particle surfaces. The SSA was determined with the Brunauer-Emmett-Teller (BET) model. Particle sizes were calculated assuming separate Al2O3 and Pt spherical particles and an averaged density based on the relative amounts of Al2O3 (3.95 g/cm3) and Pt (21.45 g/cm3) while neglecting PtOx.N2O3 particles. The area-derived particle diameters were determined from HAADF-STEM images using the NanoDefine tool of ImageJ (version 1.53c) to measure the Pt particle/cluster area and assuming spherical particles. A lognormal fit was applied to derive the number count-based particle size distribution (PSD) and to identify the mean geometric diameter (dg) and standard deviation (σg).For electron microscopy imaging, the particles were dispersed in ethanol and deposited onto a copper grid-supported perforated carbon foil. High resolution transmission electron microscopy (HRTEM) images were acquired on a JEM-ARM300F  operated at 300 kV. Furthermore, a high-angle annular dark-field scanning transmission electron microscope  operated at 200 kV and equipped with an Energy-dispersive X-ray spectroscopy detector (EDXS) was used to image the Pt/AlnHm and NOx ≤ 100 ppb) at 150 mL/min, unless otherwise specified. Humidity was admixed by guiding synthetic air through a 125 mL glass bubbler  filled with ultrapure water  and adjusted to reach 0–90% RH that remained quite stable (±0.3% [The measurement setup comprised a gas mixing unit  that wase (±0.3% ) during 2/Ar (PanGas) at 350 °C for 2 h prior to measurements. The reactor was then placed inside an oven  and heated to the desired temperature . Effluent gas at the filter outlet was analyzed using a PTR-ToF-MS 1000  [3O+ as precursor ions at 600 V drift voltage, 60 °C drift temperature and 2.4 mbar drift pressure. Analyte concentrations were identified at m/z values of 33.03 (methanol [i) concentration at the inlet  and outlet  using the following formula:The catalyst consisted of 30 mg nanoparticles prepared as a packed bed inside a tailor-made quartz glass reactor (inner diameter = 4 mm) and fixated at both ends with quartz wool and quartz sand . Only whAustria)  with H3Omethanol ), 47.05 methanol ), 59.05 methanol ) and 69.methanol ). Prior 3 (Si/WO3) sensor [v/v) mixture of ethanol  and diethylene glycol monobutyl ether . For comparison, also a 0.5 mol% Pd/SnO2 sensor was prepared [2O3 particles. The Si/WO3 and Pd/SnO2 nanoparticles were directly deposited onto an Al2O3 substrate  featuring interdigitated electrodes and a Pt back-heater [Sensing tests were carried out with a flame-made 10 mol% Si-containing WO) sensor . The preprepared  with a pk-heater , situatek-heater  with a pk-heater . There, k-heater ) by pass2O3) and active (3 mol% Pt/Al2O3) catalytic filter (30 mg) that was fixated at both sides with quartz wool and quartz sand inside a compact glass tube . The filter was then connected downstream of the gas mixing unit and upstream of the sensor. Note that the filter was slightly heated , being the standard in breath analysis [2  and formaldehyde . The sensor response to each analyte was calculated as:airR and analyteR represent the film resistances in air or during analyte exposure, respectively. The acetone selectivity was defined as the ratio between the acetone response and that to a specific analyte following IUPAC guidelines [Sensing tests were performed both with an inactive  annealing [2O3 [x (not detectable by XRD) has been revealed with an extended X-ray absorption fine structure (EXAFS) on flame-made and similarly annealed Pt/Al2O3  before [First, the crystallinity and crystal sizes of flame-made and annealed (500 °C for 1 h) powders with 0–10 mol% Pt on Ale stars, a, in agrnnealing . Importannealing . While innealing  like Al2ing [2O3 , the pre) before .θ = 39.7° (3+ sites on the γ-Al2O3 (100) surfaces [2O3 before [Interestingly, the Pt peaks feature sharp tips and broader bases, indicative of bimodal crystal size distributions . In fact = 39.7°  reveals  = 39.7° b feature = 39.7° c for all = 39.7° b and aresurfaces , as had 3 before .2O3 crystal size from 8.1 to 6.7 nm  for 0–10 mol% Pt were 7.7–9.5 nm , suggesting some polycrystallinity.It is noteworthy that the addition of Pt decreased the γ-Alo 6.7 nm d, stars.analysis  with an ppm Pt4+ ) comparePt4+ [3+ ) at a co2O3. HRTEM reveals the presence of separate Al2O3 (brighter) and Pt (dark) particles/clusters  and standard deviation (σg) of 5 nm and 1.53, respectively, that agrees well with the average crystal size of the small Pt clusters  are obtained for all Pt contents  [To challenge the catalytic filter further, the 3 mol% Pt/Al ethanol  at 50% R 200 °C)  and ZnO  200 °C) , that ha2O3 at 40 °C were placed as packed bed filter ahead of a flame-made, chemoresistive Si/WO3 [2 (0.5) that can be present at orders of magnitude higher concentrations than acetone. The resulting selectivity at the same analyte concentrations range from 0.4–600 and are in fair agreement with earlier reports for ethanol  and isot 400 °C ). Howevet 400 °C  cycling t 400 °C .2O3 packed bed at 40 °C reduces these interferences (2O3 filter (at 135 °C); however, operated here at room temperature. Moreover, it outperforms state-of-the-art acetone sensors .3 sensor alone, when testing three identically prepared samples.End-tidal breath acetone levels are usually between 148–2744 ppb, as observed during weekly breath tests of 30 volunteers during 6 months . Therefo3 sensor alone that apparently compensates for the filter’s higher acetone loss  when altering Pt content, probably by stabilization on penta-coordinated Al3+ sites. Catalytic filters of such 3–10 mol% Pt/Al2O3 nanoparticles were highly reactive already at 40 °C, as confirmed by the complete conversion of up to 100 ppm ethanol with high robustness to 50–90% RH. We also suggested the presence of some PtOx, that was less reactive than metallic Pt but seemed beneficial for acetone selectivity.We demonstrated the systematic design of a room temperature catalytic filter for selective acetone sensing by optimizing the cluster size and loading of Pt/PtO2-based sensors  that is required for accurate breath monitoring of lipolysis . Due to the filter’s modular design, it can be combined flexibly also with other chemo-resistive sensors, like established  SnO2-bas Pd/SnO2 , Figure  Pd/SnO2  for brea Pd/SnO2 .


We found downregulated expression of multiple myelin genes and decreased myelin thickness in the striatum of 16p11.2± mice versus wild type controls. Moreover, given that myelin is the main reservoir of brain lipids and that increasing evidence has linked dysregulation of lipid metabolism to ASD, we performed lipidomic analysis and discovered decreased levels of certain species of sphingomyelin, hexosyl ceramide and their common precursor, ceramide, in 16p11.2± striatum, all of which are major myelin components. We further identified lack of ceramide synthase 2 as the possible reason behind the decrease in these lipid species. Taken together, our data suggest a role for myelin and myelin lipids in ASD development.Myelin abnormalities have been observed in autism spectrum disorder (ASD). In this study, we seek to discover myelin-related changes in the striatum, a key brain region responsible for core ASD features, using the 16p11.2 deletion (16p11.2 Autism spectrum disorder (ASD) is a developmental disorder characterized by social communication difficulties, restricted interests, repetitive behaviors and intellectual disability . Signs oCyfip1 heterozygous rats  has revealed thinning of myelin sheaths in the corpus callosum together with decreased numbers of oligodendrocyte lineage cells including mature oligodendrocytes, the myelin-forming cells  mouse line, which is a CNV animal model of ASD with typical autistic phenotypes such as hyperactivity and impaired social ability  and maintained in B6/C57 background.Mice were maintained, with a maximum of 6 mice per cage, in pathogen-free temperature-controlled rooms (22 ± 1°C) under a 12 h light-dark cycle in an animal facility run by the Southern University of Science and Technology (SUST). All animal experiments were performed in accordance with the guidelines for care and use of experimental animals approved by the SUST Animal Care and Use Committee. The 16p11.2± mice (4 each) were used at postnatal day 60 (P60). Striatum tissues were dissected out from these mice under deep terminal anesthesia and immediately frozen in liquid nitrogen. RNA extraction and RNA Sequencing (RNAseq) analysis were performed by Applied Protein Technology . Briefly, total RNA was isolated using TRIzol  and 2 μg RNA per sample was used as input material to prepare a cDNA library (paired end 250bp) for high-throughput sequencing with the HiSeq 2000 sequencing system . After filtering out low quality and adapter sequences, reads were mapped to mouse genome using Hisat2 (Version 2.1.0)  . Read con 2.1.0)  was usedGapdh and the 2–ΔΔCT method was used for gene expression analysis  was carried out using a SYBR Green Master Mix  and gene specific primers  in PBS containing 2.5% glutaraldehyde under terminal anesthesia. Striatum tissues were collected and then post-fixed and kept in fresh Transmission Electron Microscopy (TEM) Fixative (Servicebio) at 4°C. The tissues were washed in 0.1 M phosphate buffer  3 times, each for 15 min, and then fixed with 1% OsO4 in 0.1 M PB for 2 h at room temperature (RT) in the dark. After washing in 0.1 M PB, the tissues were dehydrated at RT through a series of ethanol  and two changes of acetone (each for 15 min). The tissues were embedded in resin, followed by polymerization at 65°C for 48 h. The resin blocks were cut into 60–80 nm thin sections with an ultramicrotome (Leica EM UC7) and the sections were collected onto formvar coated 150 mesh copper grids for staining.The sections were first stained in 2% alcoholic uranyl acetate for 8 min in the dark, and then after rinsing with 70% ethanol (X 3) and ultra pure water (X 3), they were stained in 2.6% lead citrate for 8 min under CO2 free conditions. After further rinsing with pure water, the sections were dried overnight at RT. Images were taken in a transmission electron microscope and analyzed with Image J for measurement of inner diameters  and outer diameters of myelin sheaths. Myelin thickness was calculated as the difference between the outer and inner diameters. The myelin g-ratio  was also calculated.± mice (6 and 5 respectively) were used at P60 for lipidomic analysis. Striatum tissues were collected from these mice under terminal anesthesia and immediately frozen in liquid nitrogen. Lipid extraction and mass spectrometry (MS)-based lipid detection were performed by Applied Protein Technology. A small portion of each sample in each group was pooled together for use as a quality control sample. Samples were analyzed by liquid chromatography with tandem MS (LC-MS/MS). Lipid identification, peak extraction, peak alignment, and quantification were assessed with LipidSearch 4.1 (Thermo Fisher Scientific)  .Striatum tissues were lysed in RIPA Buffer  for protein extraction. After centrifugation at 12,000 rpm, 4°C for 15 min, the supernatant was collected and protein concentrations were determined with a PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated by SDS-PAGE and transferred to PVDF membranes . After blocking with 5% fat-free milk in 1XTBST , the blotted membranes were first incubated with primary antibodies  at 4°C oAnesthetized mice were transcardially perfused with 4% PFA. Brain tissues were collected, post-fixed in 4% PFA at 4°C overnight, cryoprotected with 30% sucrose for 2 days, embedded and frozen in Tissue-Tek OCT (Sakura Seiki), and stored at −80°C for cryosectioning. After incubation in blocking buffer (10% goat serum and 0.3% Triton X-100 in PBS), brain sections (30 μm thickness) were first incubated with primary antibodies  at 4°C ot tests were performed using GraphPad Prism to compare different groups and p < 0.05 was considered statistically significant. Data are presented as mean ± SEM.Unpaired Student’s ± mice and WT controls at P60. RNAseq data analysis revealed that the expression of multiple myelin-related genes , myelin oligodendrocyte glycoprotein , myelin basic protein , proteolipid protein 1  (yclic nucleotide phosphodiesterase (Cnpase) expression also showed a sign of reduction (p = 0.07) (platelet-derived growth factor receptor a (Pdgfra) and chondroitin sulfate proteoglycan 4 (Cspg4) was unchanged  in 16p11.2± striatum  . Althoug = 0.07) . In contnchanged . We furtstriatum . Taken t± striatum, we preformed TEM with striatum tissues from P60 16p11.2± mice and WT controls , decreased thickness of myelin sheaths  along with a higher g-ratio  was observed in 16p11.2± striatum , including diglyceride  in the makeup of myelin and the linkage between dysregulation of lipid metabolism and ASD , we askel lipids . Notablystriatum . No diff WT mice .± mice versus WT controls using our lipidomics data and identified the SM and CerG1 species showing significantly changed levels  in 16p11.2± striatum (p < 0.05) was observed in the levels of several long acyl-chain SM species in 16p11.2± striatum, including SM(d20:2/24:3), SM(d22:0/20:1), SM(d22:1/18:1), SM(d22:1/20:1), and SM(d22:2/20:1) (p < 0.05/p < 0.01) was found in the levels of 10 CerG1 species  in 16p11.2± striatum, including CerG1(d18:0/24:1), CerG1(d18:0/24:2), CerG1(d18:0/24:3), CerG1(d18:0/25:2), CerG1(d18:0/26:2), CerG1(d18:1/24:2), CerG1(d18:1/24:3), CerG1(d18:1/25:1), CerG1(d18:1/25:2), and CerG1(d18:2/24:0)  chain joined to a long chain sphingoid base  via an amide linkage . Given tstriatum . A drop :2/20:1) . Similar:2/24:0) .p < 0.05/p < 0.01) of certain species of Cer (the common precursor of SM and CerG1) in 16p11.2± striatum, including Cer(d18:0/23:0), Cer(d18:0/24:1), Cer(d18:1/24:2), Cer(d18:1/24:3) Cer(d18:1/25:1), Cer(d18:1/25:2) and Cer(d18:1/26:2) (ceramide synthase 2 (CerS2) expression was significantly lower (p < 0.05) in the striatum of 16p11.2± mice than in WT controls  . Cer pro:1/26:2)  employincontrols , whereasstriatum .CerS2 expression in the striatum of P60 16p11.2± mice versus WT controls and confirmed that CerS2 expression was markedly decreased (p < 0.01) in 16p11.2± striatum (p < 0.05) by Western blotting  knockout mouse model of ASD, MRI scans pointed to a reduction in myelin macromolecular content  in the internal capsule as well as other white matter regions at P60 (chromodomain-helicase-DNA-binding protein 8 (Chd8) conditional knockout mice where Chd8 is specifically ablated in oligodendrocyte lineage cells, autism-like behavioral changes appeared to correlate with myelin microstructural changes in the striatum  gene, located in the deleted DNA region of 16p11.2± mice, encodes a protein containing a TLC domain that is shared by CERS . Therefore, the possibility of Fam57b haploinsufficiency having an impact on Cer, CerG1 and SM levels in 16p11.2± striatum can be ruled out.In addition,  by CERS . In our https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE179056.The data generated in this manuscript can be found here The animal study was reviewed and approved by Southern University of Science and Technology Animal Care and Use Committee.NL, HLi, and YP designed the project, guided the project, and revised the manuscript. JJu, XY, and JJi did the experiments and wrote the draft. DW, YZ, XZ, XF, HLia, LZ, SL, S-TH, and LL helped to design the project, guided the project all through, and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Inefficient differentiation and insufficient maturation are barriers to the application of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) for research and therapy. Great strides have been made to the former, and multiple groups have reported cardiac differentiation protocol that can generate hPSC-CMs at high efficiency. Although many such protocols are based on the modulation of the WNT signaling pathway, they differ in their timing and in the WNT inhibitors used. Little is currently known about whether and how conditions of differentiation affect cardiac maturation. Here we adapted multiple cardiac differentiation protocols to improve cost-effectiveness and consistency, and compared the properties of the hPSC-CMs generated. Our results showed that the schedule of differentiation, but not the choice of WNT inhibitors, was a critical determinant not only of differentiation efficiency, which was expected, but also CM maturation. Among cultures with comparable purity, hPSC-CMs generated with different differentiation schedules vary in the expression of genes which are important for developmental maturation, and in their structural, metabolic, calcium transient and proliferative properties. In summary, we demonstrated that simple changes in the schedule of cardiac differentiation could promote maturation. To this end, we have optimized a cardiac differentiation protocol that can simultaneously achieve high differentiation efficiency and enhanced developmental maturation. Our findings would advance the production of hPSC-CMs for research and therapy. Howevert tissue . The devt tissue .et al. developed a cost-effective differentiation strategy employing a chemically defined platform to produce cardiac cultures containing up to 95% CMs in 11 hiPSC lines  with supplements (Thermo Fisher Scientific) and medium was refreshed every day. hPSCs were passaged at ∼70% confluency, usually around 3-4 days after seeding. On the day of passage, cells were first rinsed with DMEM/F12 (Thermo Fisher Scientific) and dissociated by incubating in accutase (1ml/well) (Thermo Fisher Scientific) at 37°C for 5 mins. Doubling time was calculated and used to determine initial seeding density for each cell line. Cells were seeded on Matrigel-coated 12-well plates in E8 medium supplemented with Y27632 inhibitor  to ensure cell survival. E8 medium was refreshed without Y27632 inhibitor within 24 h.Undifferentiated hESC (H7: WiCell) and hiPSCs [MD1-C16  and SCVIWe initiated cardiac differentiation when the confluency reached 70–90% 3-4 days after seeding. Cardiac differentiation was performed using the chemically defined medium (CDM3) as reported previously .In order to understand the effect of timing and WNT inhibitors on cardiac differentiation efficiency, three induction schedules and two combinations of WNT inhibitors were investigated in this study  to initiate differentiation on day (D)0. In the “1 + 2 + 2” differentiation protocol, CDM3 medium was refreshed the following day from D1 to D3, followed by the combination of IWP-2  with either IWR-1-endo  or XAV939  applied from D3 to D5. For the “2 + 2” differentiation protocol, a 2-day incubation in CHIR-99021 was followed by another 2-day incubation with either IWP-2 + IWP-1-endo or IWP-2 + XAV939. The “2 + 2 + 2” protocol started with CHIR-99021 treatment for 2 days, but with CDM3 medium refreshed from D2 to D4 before addition of either IWP-2 + IWP-1-endo or IWP-2 + XAV939 from D4 to D6. CDM3 medium were changed every 2 days until D15 to D18, when cells were dissociated for use of analysis by flow cytometry or cell passaging.On the day of analysis, cells were washed with Dulbecco’s phosphate buffered saline no calcium, no magnesium (DPBS-/-) (Thermo Fisher Scientific) and dissociated with Trypsin-EDTA (0.05%) (Thermo Fisher Scientific) at 37°C for 5–15 min (1ml/well). Cells were then neutralized with an equal volume of RPMI medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (HyClone) and centrifuged at 300 × g for 4 min at 4°C. Samples were prepared as previously described . An antiGlass cover slips were coated with Matrigel at least 1 h prior to plating. Cells were dissociated and plated onto the Matrigel-coated cover slips and cultured until the required time point. Cells were washed with phosphate-buffered saline (PBS), fixed in 4% PFA (prepared with PBS) for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Following blocking with 10% FBS in PBS for 30 min at room temperature, cells were incubated in primary antibodies prepared in 10% FBS in PBS at 4°C overnight. After washing with PBS, cells were incubated in secondary antibodies at 1:1,000 dilution for 1 h at room temperature, and washed in PBS for 3 times. The cover slips were then mounted on glass slides with ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). Cells were imaged using the Leica DM6B Motorized Upright Microscope or Nikon A1 Confocal Microscope.Cells were stained with antibodies against MLC2A (Synaptic Systems) and MLC2V (ProteinTech) as described above. 40X images were taken using the Leica DM6B Motorized Upright Microscope with 5-6 fields from each sample, >300 cells per sample. The proportion of MLC2A and MLC2V positive cells were counted using the ImageJ software.TRIzol Reagent (Thermo Fisher Scientific) was used to extract total RNA according to the manufacturer’s protocol. cDNA was synthesized with PrimeScript RT Master Mix kit (TaKaRa). Quantitative SYBR Green RT-PCR was then performed. Each sample was run using three technical replicates. RNA levels were normalized to that of B2M.  dye  and a fluorescently labelled palmitate analogue BODIPY FL C16  for 25 min. The cells were dissociated, washed and resuspended in Hank’s buffered saline solution (HBSS) containing 10 µM Y27632 inhibitor, 25 mM HEPES (Thermo Fisher Scientific), and 5% FBS. DAPI staining (2.5 µM) was also included to help gate out dead cells during flow cytometry. Cells were then loaded onto the Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed. BODIPY Fl C16 and MTDR staining were calculated as the median FITC-A and APC-A, respectively.Oxidative respiration was assessed with the mito-stress assay, which measures the oxygen consumption rate (OCR), using a Seahorse extracellular flux analyzer (XFe-96)  as per manufacturer’s protocols. CM cultures were seeded at approximately 1 × 104 cells/well on a Seahorse 96-well XF Cell Culture Microplate (Agilent). The mito-stress assay involved the sequential application of oligomycin (1 μM), FCCP (1 μM) and antimycin A/rotenone (0.5 μM) during which the OCR was measured. Basal and maximal respiration and spare respiratory capacity were calculated using the Wave software (Agilent).Human PSC-CMs were stained with anti-Ki67 antibody (Abcam) as described above. EdU incorporation assay was performed using the Click-iT™ Plus EdU Cell Proliferation Kit (Thermo Fisher Scientific) as per manufacturer’s instructions. The number of positive nuclei was quantified using the ImageJ software. >300 cells were counted per batch of cells.2 incubation. Individual cells from the videos were manually segmented and calcium transient traces were extracted with Leica LAS X software. The traces were analyzed with the software MATLAB R2019b using CalTrack code  and 0.02% Pluronic F-127 (Biotium) in CDM3 at 37°C for 30 mins, and washed in warm CDM3 media twice. Fluorescence videos of calcium transients were taken at 100 frames per second using Leica THUNDER imaging system with 37°C and 5% COack code .Two resources were used for miRNA-target prediction. mirTarBase is a database of miRNA target genes that is supported by experimental validation . Second,To identify target genes of cardiac TFs , we searched for binding sites conserved on human and mouse promoters using PWMSCAN . This met-test for comparisons between two groups. One-way ANOVA and Two-way ANOVA for one-factor and two-factor comparisons were used among three groups respectively. Significant differences were defined as *p < 0.05, **p < 0.01, and ***p < 0.005, respectively, and data are presented as mean ± S.E.M.Statistical analysis was performed using ratio et al., which is chemically-defined and is more cost-effective than the traditional B27 media used by Hamad et al. and Bhattacharya et al.  than “2 + 2” cultures , with the lowest CM yield in the “2 + 2 + 2” protocol   .+ cells , than “2 + 2” , but differences were not statistically significant  expression become predominant as hPSC-CMs mature (1 (KCNJ2) and If (HCN1), which are responsible for determining the resting membrane potential in mature CMs, and in automaticity in immature cells, respectively. KCNJ2 (9.74 ± 4.13 fold) was significantly upregulated, while HCN1 level was similar in “1 + 2 + 2” compared to “2 + 2” cultures. The levels of CACNA1C and ATP2A2, which encodes the L-type Calcium channel and SERCA (sarco/endoplasmic reticulum Ca2+-ATPase), were also significantly higher by 2.15 ± 0.40 and 2.18 ± 0.37 fold in “1 + 2 + 2” compared to “2 + 2” cultures  which is a surrogate measure of cell size, and showed that “1 + 2 + 2” cultures displayed a significantly greater FSC, and hence are larger, than the “2 + 2” cultures .We also sought to determine whether there was a difference in the mitochondrial functionality and metabolism in hPSC-CMs generated using different schedules. As adult CMs primarily rely on mitochondrial oxidative phosphorylation as a means to generate energy , a highe+ CMs compared to “2 + 2” cultures (8.28 ± 1.17 vs. 12.78 ± 1.44%)  . These w± 1.44%) . These r2+ transient properties of “1 + 2 + 2” and “2 + 2” hPSC-CMs using the Calbryte 590 calcium indicator (2+ release than those from the “2 + 2” protocol (22.8 ± 1.7 vs. 31.7 ± 2.2 beats/min), consistent with a more mature state. The amplitude of Ca2+ transients tended to increase, and this was accompanied by a modest increase in the rise time (188.3 ± 11.5 vs. 154.6 ± 9.2 ms) and similar decay time.We next evaluated the Candicator . H7-CMs We and others have shown that microRNAs (miRs) and cardiac transcription factors (TFs), such as miR-200c, and a set of GATA4/6, NKX2-5, TBX5, HEY2, and HAND1/2 can cooperatively regulate the expression of genes critical for cardiac development and function . To inveDuring the last decade, researchers have focused on promoting cardiac differentiation with high efficiency. As a result of such intense efforts, many groups have reported cardiac differentiation protocols that can efficiently generate CMs across many cell lines. Here we adapted and optimized protocols previously shown to generate highly pure hPSC-CM cultures, and compared the properties of the CMs generated. Two of the differentiation schedules tested generated CMs with high efficiency but the differentiated CMs differed in the expression of genes important for cardiac maturation, and in their functional properties. Thus temporal control of the WNT signaling pathway during early differentiation significantly affect not only cardiac differentiation efficiency, but also the maturation of hPSC-CMs.et al., Hamad et al. and Burridge et al., who each reported the generation of hPSC-CMs at >80% purity  Ki67+ CMs and altered gene expression. More recently, Prajapati et al. also compared differentiation protocols that differ widely in the use of small molecules and growth factors, proprietary differentiation media, co-culture with End-2 endodermal cells and in 2D vs. 3D culture (Many strategies have been devised to promote the maturation of hPSC-CMs , and thecultures . The reshPSC-CMs . Kerschebehavior . Convers culture . Single et al., Fan et al., and Quaife-Ryan et al. all showed that activation of the WNT signaling pathway can promote proliferation of hPSC-CMs (et al. evaluated the behavior and transcriptomic profile of hPSC-CMs treated with CHIR-99021, which could activate WNT signaling by inhibiting GSK-3β (Compared to the various reports comparing CMs generated using 2D- and 3D-methods, there have been few reports on the characterization of CMs generated using different 2D monolayer protocols. Here we defined the temporal control of the WNT signaling pathway to be a determinant not only of differentiation efficiency, but also of the maturation stage of the resultant cells. Specifically, we demonstrated differential structural, mitochondrial/metabolic and proliferative properties in hPSC-CMs generated using different differentiation schedules. It is well known that cardiac differentiation requires very precise control of the WNT signaling pathway, and that inappropriate activation/repression can reduce differentiation efficiency, as we also showed here with the “2 + 2 + 2” schedule. By contrast, the role of the WNT signaling pathway on cardiac maturation is less clear. Recent studies by Buikema hPSC-CMs . Since cg GSK-3β . TreatedMoreover, we performed bioinformatics analysis to show that select cardiac transcription factors and miR-200c may contribute to the mRNA differences observed between our “1 + 2 + 2” and “2 + 2” cultures. Our result demonstrates that the interplay of TFs and miR-200c could play a role in the differential regulation of genes important for differentiation and maturation among our cultures.In summary, we optimized a cardiac differentiation protocol that is robust, cost-effective, highly efficient, and promotes a more developmentally advanced phenotype. The successful use of hPSC-CMs for disease modeling and cardiotoxicity screening depends on their ability to recapitulate the properties of their adult counterparts. Our simple protocol will facilitate the production of highly pure, mature CMs for research and therapy.


We previously showed that zinc (Zn) deficiency affects the STAT3 signaling pathway in part through redox-regulated mechanisms. Given that STAT3 is central to the process of astrogliogenesis, this study investigated the consequences of maternal marginal Zn deficiency on the developmental timing and key mechanisms of STAT3 activation, and its consequences on astrogliogenesis in the offspring. This work characterized the temporal profile of cortical STAT3 activation from the mid embryonic stage up to young adulthood in the offspring from dams fed a marginal Zn deficient diet (MZD) throughout gestation and until postnatal day (P) 2. All rats were fed a Zn sufficient diet (control) from P2 until P56. Maternal zinc deficiency disrupted cortical STAT3 activation at E19 and P2. This was accompanied by altered activation of JAK2 kinase due to changes in PTP1B phosphatase activity. The underlying mechanisms mediating the adverse impact of a decreased Zn availability on STAT3 activation in the offspring brain include: (i) impaired PTP1B degradation via the ubiquitin/proteasome pathway; (ii) tubulin oxidation, associated decreased interactions with STAT3 and consequent impaired nuclear translocation; and (iii) decreased nuclear STAT3 acetylation. Zn deficiency-associated decreased STAT3 activation adversely impacted astrogliogenesis, leading to a lower astrocyte number in the early postnatal and adult brain cortex. Thus, a decreased availability of Zn during early development can have a major and irreversible adverse effect on astrogliogenesis, in part via multistep alterations in the STAT3 pathway.  •Maternal zinc deficiency disrupts offspring's brain cortical STAT3 activation.•Impaired PTP1B proteasomal degradation inhibits JAK/STAT3 activation.•Tubulin oxidation and decreased interactions with STAT3 can affect STAT3's nuclear localization.•Zinc deficiency impairs STAT3 acetylation.•Altered STAT3 activation leads to astrogliogenesis disruption which extends into adulthood. BMPbone morphogenetic proteinCcontrol (zinc sufficient diet)CTcortical tissueCT-1cardiotrophin-1Eembryonic daybFGFbasic fibroblast growth factorGAPDHglyceraldehyde 3-phosphate dehydrogenaseGFAPglial fibrillary acidic proteinhnRNPheterogeneous nuclear ribonucleoprotein A1JAK2Janus kinase 2LIFleukemia inhibitory factorMZDmarginal zinc dietNSCneural stem cellsPpostnatal dayPTP1Bprotein-tyrosine phosphatase 1BSHP2SH2 containing protein tyrosine phosphatase-2STATsignal transducer and activator of transcriptionSVZsubventricular zoneS100βS100 calcium-binding protein βVZventricular zoneZnzinc1Zinc (Zn) is essential for all living organisms and is one of the most abundant metals in the body. Zn plays a key role in the physiology of the nervous system. In this regard, Zn is a structural or functional component for a large number of proteins including enzymes, receptors, and transcription factors ,2, regulin vivo and in vitro [Given the critical role of Zn in the regulation of cell proliferation, differentiation, and survival, a decreased zinc availability during early development can have a major impact on brain structure and function . In thisin vitro . Dietaryin vitro  and affein vitro . The latin vitro and mouse models indicate that bone morphogenetic protein (BMP)-Smads [Almost half the cells in the adult human brain are glial cells , astrocyP)-Smads ,14, NotcP)-Smads ,15, and P)-Smads ,17.trans-activating activity [Our recent study showed that Zn deficiency impairs the activation of STAT1 and STAT3 in the developing rat brain and in neuroblastoma cells in culture, in part through oxidative stress-mediated mechanisms . We obseactivity . Thus, aBased on the above, this study investigated the effects of a marginal Zn nutrition imposed to rats from gestation day 0 until postnatal day (P) 2, on STAT3 activation, astrogliogenesis, and its consequences on corticocerebral astrocyte population in the young adult brain. The mechanisms of STAT3 activation were characterized at different developmental stages (embryonic day (E) 14, E19, P2, P56). A marginal decrease in fetal Zn availability caused alterations in STAT3 activation and astrogliogenesis leading to a lower number of cortical astrocytes in the young adult brain. These findings stress the relevance of a proper zinc availability during critical developmental periods for normal brain development and function.22.1Y705-STAT3), acetyl-lysine 685 STAT3 (ac-STAT3), total STAT3, phospho-tyrosine 1007/1008 Janus kinase 2 (JAK2) (pY1007/1008-JAK2), total JAK2 (JAK2), and ubiquitin were purchased from Cell Signaling Technologies . Primary antibodies for cardiotrophin-1 (CT-1) and glial fibrillary acidic protein (GFAP) were obtained from Abcam . The primary antibody for protein-tyrosine phosphatase 1B (PTP1B) was from Millipore . Polyvinylidene difluoride (PVDF) membranes were obtained from Bio-Rad . The ECL Plus Western blotting detection reagent was from Amersham Pharmacia Biotech Inc. . All other reagents were of the highest quality available and purchased from Sigma .Primary antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), leukemia inhibitory factor (LIF), SH2 containing protein tyrosine phosphatase-2 (SHP2), and S100 calcium-binding protein β (S100β), heterogeneous nuclear ribonucleoprotein A1 (hnRNP), β-actin, and Protein A/G PLUS-Agarose were obtained from Santa Cruz Biotechnology . Primary antibodies for phospho-tyrosine 705 STAT3 . At E14 and E19, dams were submitted to laparotomies, the gravid uterus was removed, and fetuses were collected, weighed and examined for gross structural malformations. Fetal rat brains were removed, weighed and either immediately processed for immunofluorescence or kept on ice to dissect cortical tissue (CT)-enriched regions which were subsequently frozen in liquid nitrogen and stored at −80 °C. In E14 embryos, CT comprised the cortical neuroepithelium with the subventricular zone (SVZ) and ventricular zone (VZ); in E19 embryos, CT comprised the cortical plate with the SVZ and VZ. P2 and P56 offspring brain/brain cortices were dissected after rat euthanasia, and processed as described above.2.34 containing inhibitors of proteases and phosphatases and 1% (v/v) Igepal). Samples were incubated at 4 °C for 20 min and then centrifuged at 10,000×g for 20 min. Supernatants were stored at −80 °C. Protein concentration was determined immediately before starting the assay [For preparation of total tissue extracts, CTs were manually homogenized . An equal amount of buffer A containing 0.2% (v/v) of Igepal was added and samples were incubated on ice for 10 min. After 15 min of centrifugation at 850×g, the supernatant (cytosolic fraction) was separated and stored at −80 °C. The pellet was rinsed with buffer A and centrifuged twice at 850×g for 2 min. The nuclear pellet was gently resuspended in 100 μl of buffer B (20 mM Tris (pH 8.0), 25% (v/v) glycerol, 1.5 mM MgCl2, 0.4 M NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.4 mM NaVaO4, 0.5 mM PMSF and inhibitors of proteases and phosphatases) and incubated for 15 min on ice. After centrifuging for 30 min at 10,000×g, the supernatant (nuclear fraction) was transferred into new tubes and stored at −80 °C.Nuclear and cytosolic fractions were isolated as previously described . Briefly2.4Cell extracts containing 20–35 μg protein were added with 4× Laemmli sample buffer and heated for 5 min at 95 °C. Proteins were separated by 8–12% (w/v) polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. Colored molecular weight  and biotinylated  standards were run simultaneously. Membranes were blocked for 1 h in 5% (w/v) nonfat milk and incubated at 4 °C overnight with the corresponding primary antibodies (1:1000–1:5000) in 5% (w/v) bovine albumin serum (BSA) in TBS-Tween. After incubation with the secondary antibodies , the conjugates were visualized using an ECL system in a Phosphoimager 840 .2.5Fetal rat brains were dissected out and fixed in a 4% (w/v) solution of paraformaldehyde in PBS overnight. Tissues were washed with PBS twice before cryoprotection by incubations in 15% (w/v) sucrose in PBS for 24 h, and subsequently in 30% (w/v) sucrose for 3 d. Brains were then submerged in Cryoplast , frozen, cut into 18-μm sections on a Leica CM 1850 cryotome  and mounted on positively charged microscope slides. Sections were blocked in 1% (v/v) donkey serum in 0.1% (v/v) Triton X-100 in PBS for 45 min and then incubated overnight at 4 °C with GFAP (1:700) and S100β (1:100) primary antibodies. Sections were washed once in 0.1% (v/v) Triton X-100 in PBS and once in 0.1 M phosphate buffer, pH 7.4, and later incubated for 2 h at room temperature with the corresponding Cy2 or Cy3-conjugated secondary antibodies (1:1000) . Cell nuclei were stained with Hoechst 33342.Sections were imaged using an Olympus FV 1000 laser scanning confocal microscope  or an Olympus BX50 epifluorescence microscope equiped with a Cool-Snap digital camera. Image Pro software  was used to merge and analyze the resulting micrographs. GFAP-positive cells were counted using ImageJ  and results were expressed as the number of positive cells per area. At least 300 cells were counted and analyzed in three independent experiments performed in triplicate on four animals per group. Colocalization of GFAP/S100β was analyzed by laser confocal microscopy, and optical sections (Z = 1 μm) of confocal epifluorescence images were obtained sequentially by means of a 60×  oil objective.2.6g for 10 min. Protein concentration was determined immediately before starting the assay. Protein lysates (400 μg100 μl)were immuno precipitated using STAT3 or PTP1B antibodies(4 μg)and immune complexes were collected on protein A/G agarose beads. The pellets were washed twice with lysis buffer Proteins were added with 4 x Laemmli sample buffer and heated for 5 min at 95 °C before Western blots. After SDS-PAGE and protein transfers, membranes for the STAT3 immunoprecipitates were probed with primary antibodies for PTP1B, SHP2, STAT3, pY705-STAT3, JAK2, β-actin and α-tubulin, while membranes for PTP1B immunoprecipitates were probed with primary antibodies for ubiquitin, JAK2 and PTP1B. After an overnight incubation at 4 °C, membranes were incubated with the corresponding HRP-conjugated secondary antibodies and visualized using chemiluminescence detection in a Phosphoimager 840 .Fetal brain cortices were lysed in 1% (v/v) Nonidet P-40 buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) Nonidet P-40, 5% (v/v) glycerol, and proteases and phosphatase inhibitor cocktails). Lysates were cleared by centrifugation at 13,000×2.7PTP1B activity was measured as previously described with minor modifications ,23. Brie2.832P)-ATP using T4 polynucleotide kinase and purified using Chroma Spin-10 columns. Samples were incubated with labeled oligonucleotide  for 20 min at room temperature in 1× binding buffer [5× binding buffer: 50 mM Tris-HCl buffer, pH 7.5, containing 20% (v/v) glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl and 0.25 mg/ml poly(dI-dC). Products were separated by electrophoresis in a 6% (w/v) non-denaturing polyacrylamide gel using 0.5 × TBE  as the running buffer. Gels were dried and the radioactivity quantified in a Phosphoimager 840 .Oligonucleotides containing the consensus sequence for STAT3 (5′-GAT CCT TCT GGG AAT TCC TAG ATC-3′) were end-labeled with . A p-value < 0.05 was considered statistically significant. Values are given as means ± S.E.M. The litter was considered the statistical unit.Student unpaired 33.1Maternal MZD did not affect maternal and offspring outcomes at E14, E19, and P2. However, maternal MZD led to a higher  body weight of the female offspring at P56, and a trend  for higher body weight of the male offspring compared to controls . The rat3.2Y705-STAT3 by Western blot in the offspring CT at E14, E19, P2 and P56. Maternal MZD differentially affected STAT3 tyrosine phosphorylation depending on the developmental stage. Maternal MZD did not affect CT pY705-STAT3 levels at E14 and P56, while they were significantly lower  at E19 when compared to controls  in MZD than control CT at P2  did not vary between MZD and control groups at E14 and P56, while at E19 and P2 JAK2 phosphorylation levels were 92% lower and approximately 10-fold higher, respectively, compared to controls , kinases (JAK2) and phosphatases (PTP1B and SHP2). In agreement with the pattern of STAT3 activation, JAK2 phosphorylation levels at Tyr 1007/1008 , while at all other developmental stages they were similar, in MZD compared to controls H and I. During astrogliogenesis, STAT3 is activated by extracellular signals such as CT-1 and LIF ,17,24,253.3Data showed that the critical timing of initiation of cortical STAT3 activation is E19, which coincides with the beginning of active astrogliogenesis. At this time point, STAT3 was particularly susceptible to the deleterious effects of gestational MZD. Thus, this time was selected to further study potential molecules/mechanisms involved in maternal MZD-mediated impairment of fetal brain STAT3 activation.To examine whether zinc deficiency could affect the interactions of STAT3 with JAK2 and the phosphatases PTP1B and SHP2, we immunoprecipitated STAT3 from E19 CT and did immunoblotting for the interacting proteins. Compared to controls, maternal MZD caused a decrease  in the amount of JAK2 that co-immunoprecipitated with STAT3  A,C. In In terms of cytoskeletal proteins, the amount α-tubulin and β-actin that co-immunoprecipitated with STAT3 was 19% (p < 0.01) and 26% (p < 0.05) lower, respectively, in the E19 CT from the MZD group compared to controls D and E. 3.4We subsequently focused on PTP1B given that STAT3 and JAK2 are substrates of this enzyme, and that the amount of PTP1B co-immunoprecipitated with STAT3 was affected by developmental MZD. During astrocyte precursor differentiation, PTP1B down-regulation occurs via ubiquitin/proteasome-mediated degradation . Thus, w3.5Based on the reciprocal activation of STAT3 and JAK2 observed in E19 and P2 CT, we investigated if PTP1B varied accordingly. While PTP1B protein levels in CT at P2 was not significantly different, the enzymatic activity of PTP1B in MZD brain cortices was higher  at E19  A and lo3.6STAT3 transactivating activity requires STAT3 tyrosine phosphorylation, and its subsequent dimerization and transport into the nucleus. We next examined the effects of developmental MZD on STAT3-DNA binding activity in nuclear fractions. At E19 and P2, the ratio of nuclear/cytosolic STAT3-DNA binding was significantly lower  in MZD CT compared to controls A and B.FY705-STAT3/total STAT3 in MZD CT was lower at E19   C and D b < 0.05) E and F.STAT3 acetylation in the nucleus is another relevant mechanism leading to STAT3 activation. Acetylated (lysine-685) STAT3 levels in nuclear fractions were not detectable in both control and MZD CT at E19 C and D. 3.7We next investigated whether the developmental impairment of the STAT3 signaling pathway caused by zinc deficiency could affect the number of astrocytes in the offspring brain cortex. The astrocyte marker S100β was assessed by Western blot in the offspring CT at different developmental stages. S100β, a marker for mature astrocytes, was only detectable in the P56 brain cortex, and its levels were significantly lower  in the brain cortex of the MZD offspring than in controls A and B. 4This study shows that a decreased Zn availability during gestation and early postnatal stages affects cortical astrogliogenesis, in part as a consequence of a dysregulation of STAT3. The adverse effects of early developmental Zn deficiency on astrogliogenesis caused irreversible alterations in the astroglial population in the young adult rat brain. Maternal Zn deficiency adversely affects several steps in the JAK/STAT pathway, i.e. JAK2 and STA3 activation (phosphorylation), PTP1B degradation, STAT3-cytoskeleton interactions, and STAT3 acetylation.Mild maternal Zn restriction in rats does not affect general maternal and fetal outcomes . HoweverDuring brain development, STAT3 expression is region-specific . In the per se [Maternal marginal Zn deficiency increased the protein levels and activity of the STAT3 phosphatase PTP1B in the E19 CT. Given that PTP1B dephosphorylates both JAK2 and STAT3 , this phper se . Thus, Zper se ,50. The per se . Overallin vitro and in vivo [Zn deficiency causes oxidative stress and oxidative damage to lipids, proteins and DNA , being t in vivo . One con in vivo ,33,52,53 in vivo  and STAT in vivo . We did  in vivo . Thus, z in vivo ,56, the  in vivo ,58. TakeIn association with STAT3 dysregulation, a marginal zinc nutrition during gestation and until P2 impaired astrogliogenesis in the offspring brain, which extended into young adulthood. In mammals, astrogliogenesis is considered to be completed soon after birth and could be reactivated only in pathological situations. The astroglial population increases 6-8-fold in normal rats and mice during the first 3 postnatal weeks ,60, beinIn summary, our results show that marginal dietary Zn deficiency during pregnancy and until P2 causes a disruption in astrogliogenesis which extends into adulthood even after reinstatement of a diet with adequate Zn concentration shortly after birth. Given the mechanisms underlying astrocyte generation in the rodent CNS , it may The authors have no conflict of interest to declare.


Functional studies about IRAG1 exhibited that IRAG1 is specifically phosphorylated by the PKGIβ, regulating cGMP-mediated IP3-dependent Ca2+-release. IRAG1 is widely distributed in murine tissues, e.g., in large amounts in smooth muscle-containing tissues and platelets, but also in lower amounts, e.g., in the spleen. The NO/cGMP/PKGI signaling pathway is important in several organ systems. A loss of PKGI causes gastrointestinal disorders, anemia and splenomegaly. Due to the similar tissue distribution of the PKGIβ to IRAG1, we investigated the pathophysiological functions of IRAG1 in this context. Global IRAG1-KO mice developed gastrointestinal bleeding, anemia-associated splenomegaly and iron deficiency. Additionally, Irag1-deficiency altered the protein levels of some cGMP/PKGI signaling proteins—particularly a strong decrease in the PKGIβ—in the colon, spleen and stomach but did not change mRNA-expression of the corresponding genes. The present work showed that a loss of IRAG1 and the PKGIβ/IRAG1 signaling has a crucial function in the development of gastrointestinal disorders and anemia-associated splenomegaly. Furthermore, global Irag1-deficient mice are possible in vivo model to investigate PKGIβ protein functions.Inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1) is a substrate protein of the NO/cGMP-signaling pathway and forms a ternary complex with the cGMP-dependent protein kinase Iβ (PKGIβ) and the inositol triphosphate receptor I (IP The inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1), which is also named IRAG or as human homologue MRVI1, was first described in 2000 . . 3R-I) , followed by 30 min 5% K4[Fe(CN)6] in 3,7% HCl. Slides were washed (3 × 2 min) in ddH2O and counter-stained in 0.6% safranine O in 66% EtOH for 15 s and again washed (2 × 2 min) in ddH2O. Differentiation of the colors and dehydration of the slides was conducted by incubation in 80% EtOH for 2 min and 3 min 100% EtOH, followed by a final step of 5 min xylene and sections were embedded with DePeX . Pictures of the slides were taken with Axiovert 200 microscope  using the MosaiX module  and analyzed with AxioVision Software .Pieces of spleens were incubated overnight in 3% PFA-solution  overnight at 4 °C and then at −80 °C. RNA isolation was carried out with the PeqGOLD TriFastTM reagent , and cDNA-synthesis was performed with 2 µg RNA according to in-house standard protocol [® 480 system  was used as fluorescent dye SYBR green I . In separate runs for each tissue, the mRNA expression of Gucy1b1, Hamp, Irag1, Itpr1, Itpr3, Prkg1a, Prkg1b and Tfr1 was analyzed. Gapdh was used as a housekeeper. The sequences of specific primers are listed in Gapdh was calculated [Tissue samples were stored in RNAprotocol . For qRTlculated .t-test was used and calculated with “Graphpad Prism” and “Microsoft Excel.” Significant differences in the graphs are shown by (*) (p < 0.05), (**) (p < 0.01) and (***) (p < 0.001).All data are shown with mean ± SEM. For the statistical analysis, the Student’s