Limited progress in terms of an effective treatment for spinal cord injury (SCI) emphasizes the urgent need for novel therapies. As a vital central nervous system component, the resident astrocytes play crucial roles in regulating recovery after SCI. In this study, recovery after SCI was compared following the transplantation of either A1 or A2 astrocytes. A1 astrocytes are harmful as they upregulate the neurotoxic classical complement cascade genes. Conversely, A2 astrocytes are characterized as neuroprotective as they upregulate the production of many neurotrophic factors.We used different supernatant obtained from microglia stimulated with lipopolysaccharide or interleukin-4 to generate A1 and A2 astrocytes. We detected the influence of astrocytes on neurons by co-culturing A1 and A2 astrocytes with neurons. We transplanted astrocytes into the lesion site of the spinal cord and assessed lesion progression, neural restoration, glia formation and locomotor recovery.Astrocytes were polarized into A1 and A2 phenotypes following culture in the supernatant obtained from microglia stimulated with lipopolysaccharide or interleukin-4, respectively. Furthermore, co-culturing A2 astrocytes with neurons significantly suppressed glutamate-induced neuronal apoptosis and promoted the degree of neuron arborization. Transplantation of these A2 astrocytes into the lesion site of the spinal cord of mice significantly improved motor function recovery, preserved spared supraspinal pathways, decreased glia scar deposition, and increased neurofilament formation at the site of injury compared to the transplantation of A1 astrocytes. Additionally, enhanced A2 astrocytes with potentially beneficial A2-like genes were also detected in the A2 group. Moreover, luxol fast blue staining and electron microscopy indicated increased preservation of myelin with organized structure after transplantation of A2 astrocytes than of A1 astrocytes.A2 astrocyte transplantation could be a promising potential therapy for SCI.Video abstractThe online version contains supplementary material available at 10.1186/s12964-022-01036-6. Spinal cord injury (SCI) is a devastating disease that can impair neurons and axons, resulting in sensory and motor deficits and an increased risk of paralysis , 2. The SCI lesions comprise three diverse lesion-associated tissue elements: (1) central non-neural lesion core; (2) astrocyte scar boundary; and (3) spared but reactive neural tissue. During the formation of the second element, astrocytes form a border restricting neuroinflammation to protect the adjacent viable neural tissue . During Recently, two types of reactive astrocytes induced by neuroinflammation and ischemia, defined as A1 and A2 astrocytes, have been found to play different roles in the central nervous system (CNS) recovery and repair . A1 typel-lysine-treated wells. For microglia and astrocyte isolation, a 100 μm cell sieve  was used to filter the cell suspension; the strained cells were then cultured with 10% DMEM/F12 at 37 °C under 5% CO2. After 14 days of culture, microglial cells were shaken down, following which the upper layer contained microglia, whereas the lower layer contained astrocytes. Microglia were stimulated with LPS at 40 ng/mL  or IL-4 (interleukin -4) at 40 ng/mL  for 3 days. The cellular supernatants termed LPS-activated DMEM or IL-4-activated DMEM from microglia after stimulation were used to culture astrocytes for 3 days. To explore the direct influence of differentiated astrocytes on neurons, we established a co-culture model by using Transwell chamber inserts with 0.4-μm filters . Neurons were incubated in the lower chamber with 500 μL neurobasal medium for 24 h and polarized astrocytes were cultured in the upper chamber filled with 500 μL 10% DMEM. A 100 μM glutamate (GLU) solution was used to induce excitotoxicity.The Ethics Committee of Nanjing Medical University approved the study for scientific research (IACUC-2108026). C57BL6/J and neonatal mice were acquired from the Experimental Animal Center of Nanjing Medical University. Neonatal mice were then sacrificed and immersed in 75% alcohol for 5 min. The dura mater from the head was removed, and the cerebral cortex from the brain tissue was stripped and placed in cold phosphate-buffered saline . Under an anatomical microscope, the pia meninges and blood vessels on the cerebral cortex surface were removed using micro-anatomical tweezers and washed with cold phosphate-buffered saline  3 times. The enzyme papain was added  for tissue digestion at 37 °C for 30 min, and the digestion was terminated with 10% Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 . For neuronal extraction, neurobasal medium containing B27 and glutamine (Gibco) was used to resuspend the cell mix, which was cultured for 4 h. Then, the medium was replaced to remove suspended cells and, neurons were allowed to adhere to poly-4 cells/well and incubated for 24 h. Cells were incubated in the CCK-8 test reagent  for 4 h, and the absorbance of each well was measured at 450 nm.The suitably treated astrocytes were cultured in a 96-well plate  at 1 × 105 cells/mL in serum-free medium, and the lower chamber was filled with 500 μL 10% DMEM. After 24 h culture, unmigrated astrocytes on the upper chamber of the membrane were eliminated and migrated astrocytes on the lower surface were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 20 min. The scratch wound assay was performed by culturing the treated astrocytes in 2-well inserts  in 24-well plates . The cells migrating into the gap were counted at 24 h.Astrocytes, after stimulation, were cultured in Transwell chamber inserts with 8-μm filters . The upper chamber was filled with 2 × 10The neuronal apoptotic rate was examined using the Annexin V- fluorescein isothiocyanate/PI apoptosis kit  according to the manufacturer’s instructions. The pretreated neurons were incubated with Annexin V and propidium iodide at room temperature in the dark for 5 min. F4/80  and ACSA-2  were used to detect the purity of Primary cell. Acquired data were analyzed using FlowJo software.Protein expression of the A1 astrocyte marker C3 (complement 3) and A2 astrocyte marker S100A10 was determined by western blot. Total protein was extracted from astrocytes using the whole cell lysis kit , and protein concentration was determined using the BCA assay . Equal amounts of proteins per sample were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes . The blots were then incubated with primary antibodies against C3 , S100A10 or glyceraldehyde 3-phosphate dehydrogenase .After blocking the unbound sites with bovine serum albumin, the microglial, astrocyte or spinal cord sections were incubated with the primary antibodies against glial fibrillary acidic protein , S100A10 , ionized calcium-binding adapter molecule 1 , NF 200 (1:200), C3 (1:50), microtubule-associated protein 2  and neuronal nuclear protein  overnight at 4 °C. Then, the samples were incubated with secondary antibodies Alexa 488 and Alexa 594  for 1 h at room temperature, and the nuclei were stained with 4′,6-diamidino-2-phenylindole fluoromount-G . Cells or areas positive for the presence of IBA-1, GFAP, NF-200, C3, and S100A10 were quantified by ImageJ software ImageJ .A Sholl analysis of neurons was performed to evaluate the branching complexity according to previous research . BrieflyFbln5, forward, 5′- CTTCAGATGCAAGCAACAA-3′ and reverse, 5′- AGGCAGTGTCAGAGGCCTTA-3′; Serping1, forward, 5′- ACAGCCCCCTCTGAATTCTT-3′ and reverse, 5′- GGATGCTCTCCAAGTTGCTC-3′; Srgn, forward, 5′- GCAAGGTTATCCTGCTCGGA-3′ and reverse, 5′- TGGGAGGGCCGATGTTATTG-3′; Clcf1, forward, 5′-CTTCAATCCTCCTCGACTGG-3′ and reverse, 5′-TACGTCGGAGTTCAGCTGTG-3′; Tgm1, forward, 5′-CTGTTGGTCCCGTCCCAAA-3′ and reverse, 5′-GGACCTTCCATTGTGCCTGG-3′; Emp1, forward, 5′- GAGACACTGGCCAGAAAAGC-3′ and reverse, 5′- TAAAAGGCAAGGGAATGCAC-3′; GAPDH, forward, 5′- AAGAGGGATGCTGCCCTTAC-3′ and reverse, 5′- TACGGCCAAATCCGTTCACA-3′. GAPDH expression was employed as an internal control. The 2ΔΔCt method was employed to analyze the relative expression.Total RNA was extracted from astrocytes using Trizol in accordance with the manufacturer’s instructions , and reverse transcription was conducted using Prime Script TM Master Mix . Real-time PCR was performed using SYBR Green Mix  and the primers were obtained from Genscript . The following primers were used: 5 A1 astrocytes transplanted immediately after SCI; (4) A2 group, in which the mice were treated with 5 μL of hydrogel containing 1 × 105 A2 astrocytes transplanted instantly following SCI.Female C57BL/6 J mice aged 6–8 weeks and weighing 20–25 g were anesthetized with ketamine and xylazine via intraperitoneal injection. First, the skin was disinfected and incised, and laminectomy was performed at the T10 level. The T10 spinal cord of the mice underwent moderate contusion injury with an impactor  (10 g × 20 mm). The mice were randomly assigned into four groups in accordance with the treatment methods: (1) the control group, in which the mice underwent laminectomy without contusion; (2) the injury group, in which the mice underwent laminectomy with contusion and were injected with hydrogel  alone; (3) A1 group, in which the mice were administrated 5 μL of hydrogel containing approximately 1 × 10A footprint analysis was conducted as previously described . The forThe electromyography (EMG) signals of gastrocnemius muscle were detected by installing stimulating electrodes in the motor cortex according to a previous study at 6 weeks post SCI . The recLongitudinal hematoxylin and eosin (H & E) staining was conducted to visualize the damaged area 7 days after SCI and the histologic score  was measAreas 1 mm above and below the lesion area of the spinal cord were isolated and embedded in paraffin dividing into 3 μm thick sections. We isolated areas 1 mm above and below the lesion area of the spinal cord and embedded them in paraffin dividing them into 3 μm thick sections. The sections were stained with luxol fast blue (LFB) and sealed with neutral resin. The images were observed under microscopy . Six of 30 serial paraffin sections were analyzed in each analysis. Electron microscopy was carried out on spinal cord sections of SCI mice at 28 dpi. Briefly, spinal cords were perfused with 3% paraformaldehyde and 1% glutaraldehyde and samples were observed at 70 nm by transmission electron microscope . For G-ratio analysis, at least 100 fibers of each mouse were measured [t-test and analyzed the differences among three or more groups via one-way ANOVA followed by Tukey’s post hoc test. SPSS Statistics for Windows, version 20.0 software  was employed to conduct statistical analyses. A p < 0.05 was considered statistically significant.All data are presented as mean ± SD. We compared two groups by two-tailed unpaired Student’s Mixed cells from the cerebral cortex were obtained from newborn mice. After culture for 14 days in DMEM, microglia and astrocyte cells showed proliferation along with stratification Fig. : Fig. S2Fbln5, Serping1, and Srgn) and three A2-specific gene markers . Results demonstrated that in the LPS-activated DMEM group, significant upregulation of Fbln5, Serping1, and Srgn expression but a marked reduction of Clcf1 and Emp1 expression than in the control group was observed  or the S100 protein family member S100A10 an A2 marker). Significantly higher C3 protein was detected in astrocytes cultured with LPS-activated DMEM than in those cultured with IL-4-activated DMEM and control groups. In contrast, astrocytes stimulated with IL-4-activated DMEM displayed significantly increased S100A10 expression than those stimulated with LPS-activated DMEM and control groups , microglia (IBA-1 positive), and neurofilaments (NF-200-positive) were performed to assess tissue repair at 28 dpi. Microglia and astrocyte contribute to glial scars that impede neurofilament regeneration after SCI. Astrocyte proliferation was significantly elevated in the A1 astrocyte-transplanted mice than in A2 astrocyte-transplanted mice after SCI Fig. A. QuantiFbln5, Serping1, and Srgn, were remarkably increased in the A1 group as opposed to the A2 group and injury group   FG–I.Fig. To examine the effect of transplanting A1 or A2 astrocytes on myelination following SCI, tissue samples were stained with LFB and myelination was analyzed in the lesion site. Lesions of the mice treated with A2 astrocytes displayed significantly increased LFB staining compared to those of the mice transplanted with A1 astrocytes Fig. A, B. TheAstrocytes, as an abundant resident cell type in the CNS, play crucial roles in managing homeostasis by supplying neurotrophic support and sustaining synaptic integrity and function –35. AstrIn recent years, cell transplantation has been identified as one of the most promising therapies, aiming to replace dead cells and create a suitable environment for neural repair after SCI. Different cell types, including microglia, neural progenitors, and stem cells, have been transplanted; some of these studies are being applicated to phase I clinical trials . ConsideClcf1, Tgm1, and Emp1.Systemic intravenous injection of transplantation cells into the tail has its limitations. First, the transplanted cells need to pass through the blood–brain barrier to arrive at the lesion site of SCI, and second, injection may have side effects on other tissues and organs, resulting in a higher cell loss. Moreover, according to recent research, although direct transplantation causes more damage to the spinal cord than does intravenous administration, orthotopic injection is more efficient due to the requirement for a lower number of cells to be injected . Thus, cRemyelination is another key recovery process following CNS injury –53. MyelOur study had some limitations. First, primary astrocytes with no immunogenicity were difficult to obtain. Second, the mechanisms underlying the potentially beneficial therapeutic effects of astrocytes are unknown. However, with the development of gene-editing technologies such as the CRISPR-Cas system –63, otheIn summary, the findings of this study suggest A2 astrocytes can protect neurons and transplantation of A2 astrocytes increases neurofilament formation and improves motor function recovery of mice after SCI. Our data open avenues for the possible use of astrocyte transplantation in the treatment of SCI and identifies transplanting A2 astrocytes as an effective therapy.Additional file 1: Fig. S1. Analysis of microglial and astrocyte population after separation. A Flow cytometry assay results of cells in the lower layer. B Flow cytometry assay results of cells in the upper layer.Additional file 2: Fig. S2. Immunofluorescent staining for GFAP and C3 after directly stimulated by LPS. Scale bars = 200 μm.Additional file 3: Fig. S3. Gene Expression of Astrocytes after Treated with LPS-activated DMEM or IL-4-activated DMEM. A–C RT-PCR analysis of relative expression of A1-like genes: Fbln5, Serping1 and Srgn relative expression and D–F A2-like genes: Clcf1, Tgm1, and Emp1 relative expression in primary astrocytes after 3 days in the control, LPS-activated DMEM and IL-4-activated DMEM groups. Error bars showed means ± SD (n = 6 in each group). #p < 0.05, **p < 0.01, ##p < 0.01 compared to control group.Additional file 4: Fig. S4. Identification of optimal MOI of lentiviruses with GFP.Additional file 5: Fig. S5. Cell survival of astrocytes in hydrogel detected by CCK-8 test reagent.Additional file 6: Fig. S6. Analysis of GFP-positive astrocytes transplanted to the SCI mice. A Representative images of axial section of spinal cord on day 3, 7, 14, or 28 after SCI with A1 or A2 astrocytes transplantation. Scale bars = 100 μm. B Quantification of (A): number of GFP-positive cells. Error bars showed means ± SD.Additional file 7: Fig. S7. Hindlimb EMG response at 6 weeks after SCI in different groups. A Schematic diagram indicates EMG potentials of contralateral gastrocnemius muscle was recorded after electrical stimulation in the motor cortex of mice at 6 weeks after SCI. B Representative examples of EMG potentials were recorded by motor-cortex stimulation in the control, injury, A1 astrocyte and A2 astrocyte treatment groups after SCI.Additional file 8: Fig. S8. Morphological analysis of spinal cord on day 28 after SCI in different groups. A Representative images of spinal cord on day 28 post SCI with A1 or A2 astrocytes transplantation. B Quantification of (A): injured cord area. Error bars showed means ± SD. **p < 0.01, compared to A1 group.


BHLHA9 gene. The disease inherits with variable expressivity and significant incomplete penetrance as high as 50%.Split-hand/ foot malformation with long bone deficiency 3 (SHFLD3) is an extremely rare condition associated with duplications located on 17p13.3, which invariably encompasses the BHLHA9 entire coding sequence was unremarkable for both families.We have detected 17p13.3 locus one-allele triplication in a male proband from family 1 (F1.1), and duplication in a male proband from family 2 (F2.1) applying array comparative genomic hybridization (array CGH). The rearrangements mapped to the following chromosomal regions–arr[GRCh38] 17p13.3(960254–1291856)×4 in F1.1 and arr[GRCh38] 17p13.3(1227482–1302716)×3 in F2.1. The targeted quantitative PCR revealed that the 17p13.3 locus was also duplicated in the second affected member from family 2 . In the next step, we performed segregation studies using quantitative PCR and revealed that F1.1 inherited the triplication from his healthy father—F1.2, whereas the locus was unremarkable in the mother of F2.1 & F2.2 and the healthy son of F2.1. However, the duplication was present in a healthy daughter of F2.2, an asymptomatic carrier. The breakpoint analysis allowed to define the exact size and span of the duplicated region in Family 2, i.e., 78,948 bp chr17:1225063–1304010 (HG38). Interestingly, all symptomatic carriers from both families presented with variable SHFLD3 phenotype. The involvement of secondary modifying locus could not be excluded, however, the Sanger sequencing screening of We have shed light on the one-allele CNV triplication occurrence that should be considered when a higher probe (over duplication range) signal is noted. Second, all SHFLD3 patients were accurately described regarding infrequent limb phenotypes, which were highly variable even when familial. Of note, all symptomatic individuals were males. SHFLD3 still remains a mysterious ultra-rare disease and our findings do not answer crucial questions regarding the disease low penetrance, variable expression and heterogeneity. However, we have presented some clinical and molecular aspects that may be helpful in daily diagnostic routine, both dysmorphological and molecular assessment, of patients affected with SHFLD3.The online version contains supplementary material available at 10.1186/s13023-022-02480-w. Isolated split-hand/ foot malformation (SHFM), i.e., ectrodactyly, is a heterogeneous group of limb anomalies that manifest either as mild single phalanx hypoplasia or a complete aplasia of phalanges and metatarsals accompanied by additional peripheral skeleton defects, in its most severe form . FollowiBHLHA9 gene (Fingerin) encoding a transcription factor involved in embryonic limb development  17p13.3(960254–1291856)×4 in F.1.1 and arr[GRCh38] 17p13.3(1227482–1302716)×3 in F2.2. The targeted quantitative PCR revealed that the 17p13.3 locus was also duplicated in the second affected member from family 2 . In the next step, we had performed segregation studies using quantitative PCR and revealed that F1.1 inherited the triplication from his healthy father—F1.2, whereas the locus was unremarkable in the mother of F2.1 & F2.2 and a healthy son of F2.1. However, the duplication was present in a healthy daughter of F2.2, an asymptomatic carrier. We have also performed a series of quantitative PCRs to narrow down the triplicated or duplicated regions  or asymptomatic (F1.2 & F2.3) carriers of duplicated/ triplicated 17p13.3 locus and results of this analysis was unremarkable.Besides, we Sanger sequenced the BHLHA9  17p13.3(960254–1291856)×4 and arr[GRCh38] 17p13.3(1227482–1302716)×3, respectively. The chr17:960254–1291856 (HG38) triplication was also present in an unaffected individual F.1.2 (father of F1.1) .The lack of penetrance of reported here 17p13.3 triplication/ duplication is intriguing. However, lack of 17p13.3 duplication penetrance is a well known phenomenon that was described in all reported so far families , 17–19. We have shed light on the one-allele CNV triplication occurrence that should be considered when a higher probe (over duplication range) signal is noted. Second, all SHFLD3 patients were accurately described regarding infrequent limb phenotypes, which were highly variable even when familial. Importantly, we have noted the first bilateral monodactyly occurring in SHFLD3. Of note, all symptomatic individuals were males. SHFLD3 still remains a mysterious ultra-rare disease and our findings do not answer crucial questions regarding the disease low penetrance, variable expression and heterogeneity. However, we have presented some clinical and molecular aspects that may be helpful in daily diagnostic routine, both dysmorphological and molecular assessment, of patients affected with SHFLD3.® HF16 Automated Nucleic Acid Extractor (RBC Bioscience Corp.).Peripheral blood was collected from affected individuals and their healthy relatives, and next, gDNA was isolated via either the salting-out method or the MagCoreWe performed array CGH using Agilent Sure Print G3 Human CGH microarray 1 × 1 M kit. The hybridization signals were detected with SureScan Dx Microarray Scanner and visualized using Agilent CytoGenomics software  as described previously .−ΔΔCT method using control DNA as a calibrator. The results were normalised to albumin gene (ALB), whereas factor VIII (F8) was targeted in order to assure reliability of the assay (sex determination). Specific primers were designed using the Primer3 tool v. 0.4.0. For primer sequences, see Additional file We applied qPCR to determine the 17p13.3 locus dosage and narrow down its genomic coordinates using SYBR dye-based master mix . Reactions ran on the Viia7 cycler as described previously . In shorBHLHA9 gene in patients F1.1, F1.2, F2.1, F2.2 and F2.3 to establish the exact breakpoints of the duplication in members of Family 2. The PCR reactions and PCR products purification were carried out following standard protocols, whereas specific primers were designed via the Primer3 tool v. 0.4.0. Next, Sanger sequencing was performed on an automated sequencer Applied Biosystems Prism 3700 DNA Analyzer using dye-terminator chemistry kit v.3, ABI 3130XL [BHLHA9 coding region. The reaction conditions are available upon request. For primer sequences, see Additional file PCR followed by Sanger sequencing were applied to screen for pathogenic mutations in the I 3130XL . The anaAdditional file 1: Fig. S1 Results of copy number analysis in 17p13.3 locus in Family 1. Segregation studies in Family 1. We performed quantitative real-time PCR on DNA from all individuals from Family 1  and one unrelated control. DNA quantity from 17p13.3 locus and flanking regions was compared to 2 reference genes (ALB and F8) using the comparative 2−ΔΔCT quantification method (a). In addition, we narrowed down the 17p13.3 duplication and triplication regions for F1.1 (b). Error bars represent standard deviation. Ratios can be interpreted as follows: normal (0.9–1.1), one-allele duplication (1.4–1.6), one-allele triplication (1.8–2.4)Additional file 2: Fig. S2 Results of segregation studies in Family 2. We performed quantitative real-time PCR on DNA from all individuals from Family 2  and one unrelated control. DNA quantity from 17p13.3 locus and flanking regions was compared to 2 reference genes (ALB and F8) using the comparative 2−ΔΔCT quantification method. Error bars represent standard deviation. Ratios can be interpreted as normal (0.65–1.0) and one-allele duplication (1.4–1.5)Additional file 3: Table S1 Oligonucleotide primers used to perform copy number analysis of the 17p13.3 regionAdditional file 4: Table S2 Oligonucleotide primers used to perform PCR and Sanger sequencing


Herein, we exploit in situ X-ray absorptionspectroscopy powered by wavelet transform analysis and machine learning-assistedfitting to assess the impact of the zeolite composition on NH3-mobilized Cu-complexes formed during the reduction and oxidationhalf-cycles in NH3–SCR at 200 °C. Comparativelyanalyzing well-characterized Cu-CHA catalysts, we show that the Si/Alratio of the zeolite host affects the structure of mobile dicopper(II)complexes formed during the oxidation of the [CuI(NH3)2]+ complexes by O2. Al-richzeolites promote a planar coordination motif with longer Cu–Cuinteratomic distances, while at higher Si/Al values, a bent motifwith shorter internuclear separations is also observed. This is paralleledby a more efficient oxidation at a given volumetric Cu density atlower Si/Al, beneficial for the NOx conversionunder NH3–SCR conditions at 200 °C.Cu-exchanged chabaziteis the catalyst of choice for NO Nonetheless, previous studies16 of the catalysts investigatedherein point to a substantial agreement of Cu-speciation in the dehydratedstate with the theoretical compositional phase diagram by Paolucciet al.,12 based on a random Al distributionin the zeolite framework subject to the Löwenstein’srule.17After heating a Cu-CHA catalyst at temperatures above 400°Cand in the presence of O3-SCR reaction proceeds in a quasi-homogeneousfashion, over NH3-mobilized Cu-ions, yet electrostaticallytethered to their framework exchange sites.18 Spectroscopicstudies have been often performed by decoupling the reaction in thereduction and oxidation half-cycles, to facilitate the results interpretation.In the reduction half cycle exposure to a NO/NH3 mixture,results in the reduction of all Cu ions to form [CuI(NH3)2]+ complexes. In the oxidation half-cycle,these [CuI(NH3)2]+ complexesare exposed to O2, and the interaction of an O2 molecule with a pair of [CuI(NH3)2]+ leads to the formation of [Cu2(NH3)4O2]2+ complexes, which can bedescribed as a μ-η2,η2-peroxodiamino dicopper(II).21At 200 °C, the NH2 activation requires a [CuI(NH3)2]+ pair, leading to a quadratic dependencyof the NH3-SCR rate on Cu density at low Cu-loadings.6 Because the mobility of a [CuI(NH3)2]+ complex is limited to distancesbelow approximately 9 Å from their anchoring point in the zeolite,a low Cu density implies a lower propensity for [CuI(NH3)2]+ pair formation, and a larger fractionof unreacted [CuI(NH3)2]+. As the O2 activation implies a change in the oxidationstate of CuI to CuII, the fraction of unreacted[CuI(NH3)2]+ can be readilyquantifiable by Cu K-edge X-ray Absorption Spectroscopy (XAS).Paolucci et al. identified the volumetric Cudensity as the keydescriptor for the efficiency of the oxidation half cycle, validatingthat O3-SCR activity. However,the density and distribution of the framework Al atoms and Brønstedacid sites may influence the mobility of the [CuI(NH3)2]+ complexes, the stability of Cupairs, and thus the formation of the [Cu2(NH3)4O2]2+ complexes. The potentialimplications for low-temperature NH3-SCR performance throughsuch zeolite-host driven changes in [Cu2(NH3)4O2]2+ formation are still unexplored.In this scenario, the actual reaction takes place on mobile Cuspecies, and therefore, the stable position of framework-bound Cuions seems less relevant for the NH3-SCR reaction, we performed an in situ XASstudy empowered by wavelet transform (WT) analysis and machine learning(ML)-assisted fitting of the extended X-ray absorption fine structure(EXAFS) spectra in order to interrogate three Cu-CHA catalysts withvariable Si/Al ratios and Cu contents during the reduction and oxidationhalf-cycles in NH3-SCR at 200 °C. Cu content and Si/Alratio in the catalysts are balanced in such a way that the Cu densityin two of the three catalysts is equivalent, despite having differentSi/Al ratios. The catalysts used in this study have a Si/Al ratioof 5, 15 and 29, with a Cu/Al ratio of 0.1, 0.5, and 0.6 ,denoted hereafter as “0.1_5”, “0.5_15”,and “0.6_29”. These catalysts have been characterizedpreviously in depth after pretreatment in O2 at 400 °C.14 We use the 0.5_15 catalyst as a reference, becauseit was previously investigated under the same redox conditions.20 Additional details on the investigated catalysts,as well as on the experimental methods for in situ XAS data collection can be found in the SI, Section 1.To determine the influence of the zeolite properties on the NH2 at 400 °C and cooling down to 200 °C in the samegas feed, at the end of the reduction step in NO/NH3 andduring the exposure of the reduced catalyst to O2, alsocollected under isothermal conditions at 200 °C. In parallel, 2 at 400 °C 2]+ for all the Cu ions present. Forthe 0.1_5 composition, the reduction to [CuI(NH3)2]+ of the dominant Z2CuII species implies the formation of a new Brønsted acid site tomaintain the charge balance in the zeolite. As NH3 is presentin the applied reduction conditions, this results in the formationof NH4+ ions 4O2]2+ complexes.20 However, the spectral shape reachedat the end of the oxidation step for the 0.1_5 catalyst differs fromthose observed for the 0.5_15 and 0.6_29 catalysts, especially inthe white-line peak region. This indicates a different coordinationmotif for [Cu2(NH3)4O2]2+ in the Al-rich 0.1_5 catalyst, and therefore, we undertooka more in-depth analysis of the in situ XAS data.During the oxidation step, thecharacteristic XANES features ofCuII after the oxidation step, from XANES linear combination fit (LCF)we found a residual amount of [CuI(NH3)2]+, determined by CHA Si/Al ratio, and rangingfrom 13% for Si/Al = 5, to 16% for Si/Al = 15 and 24% for Si/Al =29 (see pie charts in Section 2 for details).Although the majority of the Cu ions are reoxidized to Cux conversion in NH3-SCRfor the three catalysts as a function of temperature. At 200 °C,0.1_5 and 0.5_15 show almost equivalent conversion, which is insteadlower for 0.6_29. The inset of x conversionat 200 °C to a rate constant based on a first order kinetic model,corresponding to an integral reactor analysis,22 and accounting for Cu wt % in the catalysts . Considering the increase in residualCuI as Si/Al increases observed from XANES LCF, the TOFat 200 °C follows the opposite trend: it is higher in 0.1_5 andit decreases in an approximately linear way for the 0.5_15 and 0.6_29catalysts. This indicates that a more efficient oxidation of [CuI(NH3)2]+ by O2 activation is beneficial for the low-temperature NH3-SCRperformance.I fractionsis not entirely determined by the Cu density, but that the Si/Al ratioalso plays a role. I fractionfor the three catalysts investigated in this study and for the databy Paolucci et al.6 It is worth noticingthat the 0.1_5 and 0.6_29 catalysts show comparable Cu density, whilethe residual CuI fraction after oxidation is significantlylower for 0.1_5 than for 0.6_29. The data appear to branch based onthe Si/Al ratio, with a different dependence on the Cu density forlow and high Si/Al ratios 2]+, spectroscopically indistinguishable overthe compositional series.We note that the difference in residual Cu24 to unambiguously identify possible Cu–Cu scattering contributionspossessing a characteristic lobe centered at ca. 7 Å–1 along the k direction28 .To correlate quantitatively the structuraldifferences in the CHAzeolites with the observed trends in oxidation efficiency, we comparedthe EXAFS of the three catalysts at the end of both the reductionand oxidation steps 3. TogethI(NH3)2]+ complexes,without any detectable dependence on the Cu and Al contents in thecatalyst (3 ligands.EXAFS fits based on the [CuI(NH3)2]+ structure confirmed this picture .EXAFS confirms that afterthe reduction step all the Cu is presentas + (1) and for a bimodal distribution of the Cu–Cu distancesin μ–η2,η2-peroxo diaminodicopper(II). On the basis of the WT analysis, the latter is describedby combining contributions from the already proposed quasi-planarconfiguration (2/2′) and a “bent”motif (3) for the same complex, refined through ML-based optimizationof the Cu–O–Cu angle .To further understand the emerging structuralcomplexity, we employeda novel ML-assisted EXAFS analysis approach(NH3)2]+ 4a, 1 andguration 4a, 2/2′ t”motif 4a, 3 forI(NH3)2]+ agree wellwith those from XANES LCF, revealing the same trend in the oxidationefficiency as a function of the Si/Al ratio. The previously observedlack of fit at ca. 2.5 Å is now resolved for the whole set ofsamples.μ-η2,η2-peroxo diamino dicopper(II) coordination motifsare required to fully model the EXAFS of 0.5_15 and 0.6_29, whichis reflected in a different Cu–Cu distance for the bent (3.03–3.06Å) and planar complex (3.34–3.39 Å). Interestingly,for 0.1_5 the three-component fit restitutes Cu–Cu distancesof 3.38 and 3.39 Å, not distinguishable within the accuracy ofEXAFS analysis and both pointing to the same planar motif . The adopted approach allows refining, in principle,any structure starting from an initially guessed molecular complexwith significantly different interatomic distances and angles. Theseresults therefore highlight the potential of in situ XAS combined with integrated WT and ML-assisted EXAFS analysis.ML-EXAFS fitting ensures aquantitative understanding of the subtledifferences observed in the XANES spectral shape as well as in thehigh-R portion of WT maps, where the I(NH3)2]+ complexesby O2. Diffuse Reflectance UV–vis spectroscopy,qualitatively responsive to the structure of Cu-oxo species, furthersupports these results . Therefore, we suggest that the diverse electrostatic landscapeand the higher Brønsted acid site density in Al-rich zeolitescould trigger host–guest interactions promoting a longer internuclearseparation in the planar μ-η2,η2-peroxo diamino dicopper(II) motif. Plausibly, the NH3 ligands in the dicopper(II) complex could participate into H-bondinginteractions with ZH sites, as well as ZNH4 and ZNH4nNH3 associations, most likelyformed at the reduction step in the presence of NH3 at200 °C,32 triggering the observedelongation effect in Cu–Cu distances. Importantly, this isaccompanied by a more efficient oxidation at a given volumetric Cudensity, which is beneficial for the NOx conversion under NH3-SCR conditions at 200 °C. Withthis respect, Al-rich zeolites could favor dynamic Cu ion exchangebetween nearby sites, thus enhancing the mobility of [CuI(NH3)2]+ complexes beyond the limitdictated by electrostatic tethering to the initial exchange sites.A similar mechanism based on H+/H2O-aided diffusionof CuI was proposed in the context of continuous partialoxidation of methane to methanol over Cu-CHA, although in this caseZNH4 is suggested to hinder the exchange pathway.33 Overall, the findings obtained in this workprovide a robust experimental basis for further theoretical modelingof low-temperature NH3-SCR mechanism, aimed at validatingthese hypotheses within a quasi-homogeneous Cu-catalyzed oxidationhalf-cycle actively driven by the compositional characteristics ofthe zeolitic host.Summarizing, the XANES and EXAFS results for the Cu-CHA catalystsconsistently show, that the Si/Al ratio of the zeolite host affectsthe structure of mobile dicopper(II) complexes formed during the oxidationof the [Cu


Preexisting cell type–specific chromatin features represent an additional regulatory layer for interferon beta response and direct binding of STAT transcription factors and gene induction. The antiviral response induced by type I interferon (IFN) via the JAK-STAT signaling cascade activates hundreds of IFN-stimulated genes (ISGs) across human and mouse tissues but varies between cell types. However, the links between the underlying epigenetic features and the ISG profile are not well understood. We mapped ISGs, binding sites of the STAT1 and STAT2 transcription factors, chromatin accessibility, and histone H3 lysine modification by acetylation (ac) and mono-/tri-methylation  in mouse embryonic stem cells and fibroblasts before and after IFNβ treatment. A large fraction of ISGs and STAT-binding sites was cell type specific with promoter binding of a STAT1/2 complex being a key driver of ISGs. Furthermore, STAT1/2 binding to putative enhancers induced ISGs as inferred from a chromatin co-accessibility analysis. STAT1/2 binding was dependent on the chromatin context and positively correlated with preexisting H3K4me1 and H3K27ac marks in an open chromatin state, whereas the presence of H3K27me3 had an inhibitory effect. Thus, chromatin features present before stimulation represent an additional regulatory layer for the cell type–specific antiviral response.  Type I interferon (IFN) cytokines like IFNα and IFNβ are expressed across almost all tissues in human and mouse as a first line of defense against viral infections . They acHere, we dissected the cell type–specific IFNβ response by comparing mouse ESCs and MEFs. The comparison of ESCs to their differentiated counterparts is a well-established cellular system to distinguish DNA sequence versus epigenetically driven chromatin interactions of regulatory factors  derived by in vitro ESC differentiation were obtained from a 129/Ola mouse strain, as described previously  Figs 1A. We seleSequencing readouts and replicate number.Table S1 Cell type specific ISGs and STAT1/2-binding sites.Table S2 ISGs identified in ESCs, NPCs, and MEFs after IFNβ treatment.Table S3 We assessed by single-cell RNA sequencing (scRNA-seq) if the transcriptional response in ESCs and MEFs was homogeneous or if the observed up-regulation of ISGs arises from a subset of more strongly responding cells  and S1F.Ifit1 and Isg15 in Inspection of the UMAP plots showed no separate clustering of untreated (0 h) and 1 h IFNβ-treated ESCs, whereas they separated within the same clusters in MEFs. The differences are in line with the lower number of 57 and 115 ISGs detected by bulk RNA-seq after 1 h as compared with 188 and 452 genes after 6 h for ESCs and MEFs, respectively . After 6Next, we compared the transcriptional response with IFNβ in the three cell types in further detail.Irf9, Stat1, and Stat2 were already lowly expressed in unstimulated cells and showed a significant increase in expression after IFNβ treatment  or 727 (STAT1p721) was clearly increased after 1 h in MEFs as compared with ESCs and decayed to low levels at the 6 h time point. Furthermore, lower levels of active STAT1/2 protein complexes upon IFNβ induction are apparent from comparing the amounts of STAT1p701 and STAT1p727 between ESCs and MEFs. These differences in STAT1/2 protein levels under stimulated and unstimulated conditions were not reflected in the RNA levels and suggest a reduced protein translation/degradation ratio of STAT1 and STAT2 in ESCs. We conclude that the globally attenuated response to IFNβ in ESCs involved epigenetic networks that lead to a reduced activity of key components of the JAK/STAT signaling pathway both on the RNA and protein level as compared with differentiated cells.The amount of STAT1 phosphorylated at residue 701  . Antibodies against STAT1ex.html) . Based oBinding sites of STAT1 and STAT2 in ESCs and MEFs.Table S4 n = 71 in ESCs; n = 112 in MEFs). In addition, ISG activation was provided for a smaller fraction of promoters by STAT2 in the absence of STAT1 . The latter finding is in line with the conclusion that the STAT2–IRF9 complex alone could provide some activation . For a remaining fraction of 105 (ESCs) and 306 (MEFs) ISGs, no STAT binding at the promoter was detected. Accordingly, these ISGs were either secondary target genes or become activated from non-promoter STAT-binding sites. Based on these findings, we focused on STAT1/2-binding sites as a proxy for the ISGF3 complex to further characterize the relation between non-promoter STAT1/2 binding and ISGs.To further dissect the activation mechanism, we analyzed the spatial relation between STAT-binding sites and ISGs. Almost half of the STAT1/2 peaks in ESCs and MEFs were located at promoters (defined as a window of ±1 kb around the transcription start site) with around 3/4 of them at the ISGs identified from the bulk RNA-seq analysis   . Thus, tin MEFs) , indicatin MEFs) . These oin MEFs)  and assiin MEFs)  and 4C. in MEFs)  to reveaOverview of scATAC-seq data.Table S6 Uba7 ISG. An IFNβ-induced co-accessible link between the STAT1/2 bound enhancer candidate and the promoter of the ISG Uba7 was detected. Another example of ISG regulation by STAT1/2 binding to distal putative enhancers is shown for the Ly6 gene cluster in MEFs . The 116 ESC-specific and 184 MEF-specific sites were predominantly at non-promoter loci (100/116 and 118/184) . We reasreadouts . These dustering . Five maustering : (i) “Acustering . (ii) “Austering . (iii) Tustering . (iv) Thustering . (v) “Reustering .Next, chromatin states at STAT1/2 sites in ESCs and MEFs and their changes were analyzed . The mosP-value and correlation coefficient R of a given mark with STAT1/2 binding are plotted in R = 0.42; MEF, R = 0.53), H3K4me1 , and H3K27ac  were the most strongly positively correlated marks, whereas H3K27me3  was anticorrelated with STAT1/2 binding. For the repressive H3K9me3 mark, the correlation was negative for ESCs (R = −0.26) and slightly positive for MEFs (R = 0.08) pointing to a more complex relation. We concluded that a preexisting active chromatin state  promoted STAT1/2 binding, whereas chromatin loci marked by the H3K27me3 impeded binding . STAT1 homodimers can also act as activators of type I IFN response  was added per 0.5 million cells in suspension. The mixes were incubated for 60 min on ice, spun down at maximum speed at 4°C for 30 min. The supernatant was transferred to a fresh tube and stored at −20°C. Gels were blotted on LF PVDF membranes using the trans-blot turbo transfer system (Bio-Rad) and blocked with 5% BSA in Tris-buffered saline supplemented with 0.1% Tween 20 detergent (TBST) at room temperature for 1 h. The primary antibodies were diluted according to manufacturer’s recommendations and incubated at 4°C overnight. On the following day, the membrane was washed three times with TBST buffer at room temperature for 5 min under agitation and incubated with secondary anti-HRP antibody  at room temperature for 1 h, washed three times with TBST, incubated with Clarity Western ECL substrate for 5 min, and imaged. The antibodies used are listed in Table S7.Antibodies used in this study.Table S7 Cells were seeded on a six-well plate. Two (ESCs and MEFs) or five (NPCs) days after plating, the cells were washed two times with PBS. Then, 500 µl LBP was added, and the cells were stored at −80°C. RNA was isolated with the NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s protocol. The elution step was done two times with 30 µl RNase-free water within the same tube. Concentrations were measured by Qubit RNA HS Assay Kit, and the quality of RNA was analyzed on an Agilent 2200 high sensitivity RNA screen tape system. Removal of rRNAs from isolated samples of IFNβ-stimulated ESCs and MEFs was done following the protocol of the Ribo-Zero rRNA Removal Kit (Illumina). An input of 5 µg total RNA was used, and the depleted RNAs were eluted in 30 µl RNase-free water supplemented with 1 µl RiboLock RNase Inhibitor (40 U/µl). Concentrations were measured by Qubit RNA HS assay kit. For NPCs, RNA samples were treated with DNase at 37°C for 30 min and purified by ethanol precipitation. Concentrations were measured by Qubit RNA HS assay kit, and 750 ng of DNase-treated RNA was used for rRNA depletion by NEB Next rRNA Depletion Kit (human/mouse/rat). The depletion was performed based on the manufacturer’s protocol. Samples were purified with RNA Clean XP beads (Beckman) with a 2.2× ratio and finally eluted in 8 µl nuclease-free water. Purified rRNA-depleted RNA samples of ESCs, MEFs, and NPCs were used to prepare NGS libraries based on the NEB Next Ultra II directional RNA library preparation kit from Illumina. As default, 50 ng of rRNA-depleted RNA was used as input. For less concentrated samples, 10 ng were used. The RIN value of all samples was above seven, and therefore the mixes were incubated at 94°C for 15 min. Furthermore, a fivefold NEB Next adaptor dilution was used as default at the adaptor ligation step. For lower concentrated samples, a 25-fold dilution was used. All samples were dual-barcoded with unique i5 and i7 primers. For 50 ng samples, a total of nine cycles and for 10 ng samples 11 cycles were performed during the PCR enrichment of the adaptor ligation DNA step. Samples were measured with Qubit dsDNA HS assay kit, and the fragment size was determined with a Tapestation D5000. In total, four replicates of ESCs, two replicates of MEFs, and four replicates of NPCs treated with IFNβ for 0, 1, and 6 h, respectively, were acquired. The corresponding RNA-seq libraries were 50-bp single-end sequenced on a HiSeq 4000 System (Illumina) with at least 50 million reads per sample. Sequencing of RNA, and that of all other sequencing readouts, was done at the DKFZ Genomics and Proteomics Core Facility.6 cells per sample were used as input for the ChIPs. After formaldehyde fixation, chromatin fragmentation was done with the EpiShear Probe Sonicator (Active Motif) at 4°C with 50% amplitude and 6–10 “on” and “off” cycles of 30 s duration to yield an average fragment size of around 150 bp. The immunoprecipitation was conducted with 10 µg of chromatin in a total volume of 500 µl and addition of antibodies (Table S7). The sequencing libraries were prepared using the NEB Next Ultra II DNA library preparation kit for Illumina with 40 µl ChIP sample and added 10 µl 10 mM Tris–HCl, pH 8.0. For the input reaction, a 1:10 dilution was made and from this dilution 4 µg chromatin were used and filled up with 1 × 10 mM Tris–HCl, pH 8.0, to a total volume of 50 µl. Concentrations were measured by Qubit dsDNA HS assay kit, and fragment distribution was analyzed on a Tapestation D5000. The libraries were sequenced as described above for RNA-seq.STAT1p701 and STAT2 ChIPs were performed with the ChIP enzymatic chromatin IP kit from Cell Signaling Technology according to the manufacturer’s protocol. Around 4 × 102, 10 mM KCl, 0.1% NP-40, 1 mM DTT, and 0.5 mM PMSF). A 10 min incubation step on ice and a centrifugation step at 400g for 5 min at 4°C were performed. 4 × 107 cells were resuspended in 100 µl MNase buffer and 40 U MNase was added. After an incubation step at 37°C for 15 min, 100 µl of 10× sonication buffer and 800 µl water were added. Samples were incubated on ice for 5 min, transferred into 12 × 24-mm tubes, and sonicated for 15 min  on a Covaris sonicator. A centrifugation step was performed at 16,200g and 4°C for 15 min. The supernatant was transferred into fresh tubes, and chromatin was snap frozen with liquid nitrogen and stored at −80°C. A quality check of reverse cross-linked samples was performed and yielded a fragment size of around 150 bp for the sheared chromatin. Pre-equilibrated 25 µl protein G beads were used per sample at room temperature for 10 min in sonication buffer . A sample precleaning step was performed by adding 25 µl protein G beads with 4 µg IgG antibody (rabbit or mouse) to chromatin and incubated, rotating at 4°C for 2 h. Beads were pelleted, and supernatant was transferred to fresh tubes. Antibodies were added to chromatin samples and incubated at 4°C for 2 h (Table S7). Then, 25 µl of pre-equilibrated beads were added to the samples and incubated rotating at 4°C overnight. The beads were washed by rotating at 4°C for 5 min with high-salt buffer , lithium buffer , and 2× with TE-buffer . Each sample was eluted two times with 250 µl elution buffer  at 37°C for 15 min on a shaker. Reverse cross-linking was performed by adding 20 µl 5 M NaCl and incubated at 65°C overnight. Subsequently, 10 µl EDTA (0.5 M), 0.5 µl RNase A (10 mg/ml), and 50 µl Tris  were added and incubated at 37°C for 30 min. Then, 2 µl proteinase K (20 mg/ml) was added and incubated at 55°C for 2 h. An isopropanol precipitation was performed to purify the DNA. Resuspended samples were measured with Qubit dsDNA HS assay kit and the fragment size was determined on a D5000 Tapestation (Agilent). Libraries were sequenced as described above for RNA-seq. In ESCs, two replicates for H3K4me1, H3K4me3, and H3K27ac and three replicates for H3K9ac, H3K9me3, and H3K27me3 were sequenced. In MEFs, two replicates of all modifications were sequenced.ESCs were cultured in 150 mm dishes and treated with IFNβ for 0, 1, or 6 h. Media was removed and cells were detached with Accutase, washed with PBS supplemented with PMSF at 0.5 mM concentration, and crosslinked with 1% formaldehyde  for 10 min at room temperature. 125 mM glycine was added to neutralize formaldehyde, and the cells were scratched from the plates on ice and collected in tubes. Afterward, the samples were washed three times with PBS/100 mM PMSF, and cell pellets were resuspended in 10 ml swelling buffer . A total of 50,000 cells were transferred into fresh tubes and centrifuged by 800g at 4°C for 5 min. For ESCs, the cell pellet was resuspended in 200 µl ATAC lysis buffer , incubated at room temperature for 2 min and centrifuged at 800g and 4°C for 5 min. The supernatant was discarded and pellets were resuspended in 20 µl ATAC reaction buffer containing 10 µl 2× transposase buffer and 2.5 µl Tn5 enzyme (Illumina). Samples were incubated at 37°C for 30 min. Reactions were stopped by adding 5 µl EDTA (100 mM) in Tris–HCl, pH 8.0, to a final concentration of 20 mM. For MEFs and NPCs, the cells were directly resuspended in 25 µl ATAC reaction buffer with digitonin  and incubated at 37°C for 30 min. The samples were purified with a MinElute PCR Purification Kit (QIAGEN) and eluted in 12 µl buffer. After PCR amplifications, sequencing libraries were purified with AMPure beads (Beckman). Concentration was measured with the Qubit dsDNA HS assay kit (Thermo Fisher Scientific) on a Qubit fluorometer, and size distribution of final library was checked on a D5000 Tapestation. Libraries were 50-bp paired-end sequenced on Illumina HiSeq 2000 or 4000 systems with at least 50 million reads per sample. Two replicates for ESCs and NPCs and four for MEFs were sequenced.ESCs were plated on six-well plates and treated for 0, 1, or 6 h with IFNβ at 500 U/ml. Cells were detached using Accutase, collected, and washed with MT-PBS buffer  and involved normalized strand coefficients and relative strand correlation values for each sample. Peak calling for STAT ChIP-seq was done with MACS2 . ESCs and MEFs were treated for 0, 1, or 6 h with IFNβ. The cDNA amplification was done by running 13 PCR cycles. The samples were eluted in 35 µl 10 mM Tris–HCl, pH 8.0. Concentrations of cDNA libraries were measured by Qubit dsDNA HS Assay Kit, and mean peak sizes of the samples were determined on a Tapestation D5000. Each of the final libraries were paired-end sequenced (26 bp and 74 bp) on one Illumina HiSeq 4000 lane following the manufacturing protocols. For scATAC-seq, ESCs and MEFs were treated with IFNβ for 0, 1 (only for MEFs), or 6 h, and libraries were prepared according to the Chromium Single-Cell ATAC v1.0 protocol (10× Genomics). Two (three for MEFs treated with IFNβ for 6 h) replicates per scATAC libraries were paired-end sequenced on an Illumina NovaSeq 6000 system, according to the manufacturer’s protocol.3.5 and 105, respectively), a TSS ratio above 4 and a ratio of fragments in blacklisted genomic regions to all fragments below 0.0225 (ESCs) and 0.016 (MEFs). Cell numbers and quality measures of scATAC-seq data are provided in Table S6.Sample demultiplexing and barcode processing of scRNA-seq data were conducted with the Cell Ranger pipeline from 10× Genomics. For ESCs, quality filtering was conducted by selecting only cells within a certain percentage of mitochondrial reads (2.5% < accepted cells < 7.5%) and number of detected genes , yielding 1,332 cells for time point 0 h, 2,085 cells for 1 h, and 4,825 for 6 h of IFNβ stimulation. For MEFs, quality filtering was conducted by selecting only cells within a certain percentage of mitochondrial reads (0.5% < accepted cells < 7.5%) and number of detected genes , yielding 9,771 cells for time point 0 h, 10,186 cells for 1 h, and 7,579 for 6 h of IFNβ stimulation. Further analysis including UMAP embedding was done using the R package Seurat . The scAhttps://github.com/RippeLab/RWire-IFN). For the co-accessibility analysis 2,700 cells per (sub-)type and treatment condition were selected that had a similar number of fragments per cell. The background co-accessibility signal was determined by randomly shuffling the accessibility values over cells and peaks . As an additional parameter, the percentage of accessible cells was calculated as the average of cells that had site 1 or site 2 accessible with the rational that correlations computed from a low number of cells are less reliable. Only co-accessible links of STAT1/2 bound sites were considered for further analyses. We observed no correlation between the Pearson correlation coefficients of co-accessible links and their percent accessible cells  and pseudo-bulk number of fragments .Regions of 2 kb size that were simultaneously open in the same cell within a 1 Mb window around STAT1/2 bound sites were identified from a co-accessibility analysis of the scATAC-seq data. Pearson correlation coefficients were calculated without aggregation of single cells for ESCs, epithelial-like and mesenchymal-like MEFs for each treatment condition based on our previously described RWire approach  implemennd peaks . As a thGSE160764 at Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). Software used for data analysis for the different sequencing readouts is listed in Table S8. The R-scripts for the co-accessibility analysis are available via Github at https://github.com/RippeLab/RWire-IFN.The data and computer code produced in this study are available from the following sources: All original sequencing and relevant processed data have been deposited under Data analysis software (Table S8 software .


The electrosynthesis mechanism has been systematically investigated, and the results show that the synthesis of the Cr2GeC MAX phase involves electro-separation and in situ alloying processes. The as-prepared Cr2GeC MAX phase with a typical layered structure shows the uniform morphology of nanoparticles. As a proof of concept, Cr2GeC nanoparticles are investigated as anode materials for lithium-ion batteries, which deliver a good capacity of 177.4 mAh g−1 at 0.2 C and excellent cycling performance. The lithium-storage mechanism of the Cr2GeC MAX phase has been discussed based on density functional theory (DFT) calculations. This study may provide important support and complement to the tailored electrosynthesis of MAX phases toward high-performance energy storage applications.The two-dimensional MAX phases with compositional diversity are promising functional materials for electrochemical energy storage. Herein, we report the facile preparation of the Cr Energy ge hosts . Xu et a0 cycles . Chen et with Li . Li et aproposed . In gene15Ge4) of Ge during the Li insertion/extraction process severely hampers its energy storage properties. Cr2GeC is one of the MAX phases, and the A-layer atom is Ge. The stable Cr2GeC MAX phase is expected to take advantage of the two-dimensional structural properties of the MAX phase and the Li-storage property of metallic Ge. If the typical alloying mechanism of Li15Ge4 in the Cr2GeC MAX phase is considered as the basis, the theoretical capacity can reach 535 mAh g–1 , GeO2 , graphite powders  with different molar ratios , and 10 wt% polyvinyl butyral  were mixed by ball-milling at 300 r/min for 5 h to prepare the powdered Cr2O3/GeO2/C precursor. About 0.5 g of the obtained mixed powders were pressed under 10 MPa to fabricate a Cr2O3/GeO2/C disc (10 mm in diameter). The Cr2O3/GeO2/C disc was wrapped by nickel foam and fixed on a Mo wire  Co., Ltd.) to form the Cr2O3/GeO2/C cathode system. A high-purity graphite rod  fixed with the Mo wire was used as the anode. CaCl2 and NaCl  were commonly baked at 300–400°C for 24–48 h and then used as electrolytes in a 1:1 M ratio. The electrodes and mixed salts were assembled in a corundum crucible to form an electrolytic cell, which was then placed in an electrolysis furnace sealed on one end. High-purity Ar gas was continuously introduced into the electrolytic furnace to create an inert atmosphere. The electrolysis furnace temperature was then ramped up to 700°C with a heating rate of 5°C/min. Pre-electrolysis was then performed between two graphite rods  at 2.0 V for 2–5 h to eliminate residual purities in molten salts. A constant voltage of 3.0 V was applied between the Cr2O3/GeO2/C cathode and the graphite anode for pre-set times. After electrolysis, the obtained electrolytic samples were washed with deionized water to remove solid salts and then, dried at 100°C in a vacuum drying oven for further characterization.Commercial Cr2GeC. In detail, a slurry made by mixing 80 wt% Cr2GeC as active materials, 10 wt% acetylene black , and 10 wt% polyvinylidene fluoride  in N-methyl pyrrolidone  was coated on a Cu foil; then, the obtained Cu foil coated with the slurry was dried under vacuum at 80°C for 10 h. The disc-shaped electrodes (12 mm in diameter) were cut off from the dried Cu foil. In addition, the lithium metal foil, 1.0 M LiPF6, and the polypropylene membrane  were used as the counter electrode, electrolyte, and separator, respectively. The coin cells were assembled in an argon glovebox.The two-electrode CR2032-type coin cell was fabricated to evaluate the lithium-storage performance of the as-prepared Cr–4 eV and a maximum force of 0.001 eV/Å. The 2 × 2 × 1 supercell of the Cr2GeC (010) slab contains 38 atoms, where the bottom three atomic layers were fixed. A vacuum layer of 15 Å was used to prevent the interaction between the near slabs.The phase composition of the samples was analyzed by X-ray diffraction . The morphology, microstructure, and elemental distribution of the samples were characterized by scanning electron microscopy , transmission electron microscopy , and the affiliated energy dispersive X-ray spectrometer (EDS). A Bio-Logic HCP-803 electrochemical workstation was used to record the current–time curve of the electrolysis process and cyclic voltammetry curves of the fabricated coin cell. The charge–discharge tests of coin cells were carried out on a NEWARE CT-4000 battery test system. For computational details, all density functional theory (DFT) calculations were performed using the Vienna Ab-initio Simulation Package (VASP). The generalized gradient approximation (GGA) developed by Perdew, Burke, and Ernzerhof (PBE) was used as the exchange-correlation potential. The cutoff energy is set to 400 eV. A Monkhorst–Pack grid of 6 × 6 × 6 was used for bulk lattice optimization, and a Monkhorst–Pack grid of 3 × 3 × 1 was used for slabs. Electronic and ionic optimizations were performed using a self-consistent field (SCF) energy criterion of 102O3/GeO2/C used as a cathode was directly electrolyzed to prepare Cr2GeC in molten CaCl2–NaCl. Theoretical analyses of Cr2O3, GeO2, CaCl2, and NaCl were first performed based on Gibbs free energy, and the calculated temperature-dependent decomposition voltage plots are shown in 2O3 and GeO2) in the cathode at a wide temperature range below 1,000°C, in the case of avoiding the decomposition of chloride molten salts. Theoretically, oxygen ions ionized from cathodic Cr2O3 and GeO2 and discharged at the anode at the applied voltage. The in situ alloying reaction between the electrochemically reduced metals Cr, Ge, and C was expected to induce the formation of Cr2GeC. 2GeC MAX phase.Cr2GeC was first attempted using Cr2O3/GeO2/C with a molar ratio of 1:1:1 in molten CaCl2–NaCl at a relatively moderate temperature of 700°C. However, the XRD result  results in the generation of metallic Ge as the second phase in Cr2GeC. On this basis, the electrolysis of Cr2O3/GeO2/C with a molar ratio of 1:2:1 was further analyzed to understand the formation of the Cr2GeC MAX phase. 2O3/GeO2/C cathode (with a molar ratio of 1:2:1) at 700°C for different times. The results show that Ge, CaCr2O4, and Ca2GeO4 appeared in the cathode after 0.5 h of electrolysis. The generation of CaCr2O4 and Ca2GeO4 is caused by combination reactions between Cr2O3, GeO2, Ca2+, and O2−, which has been confirmed in previous works  in the nano state usually has a low melting point to enter the molten salt, and the latter (Ca2GeO4) has a certain solubility in molten salts , the products are Cr2GeC, Cr7C3, and Cr3C2 mixtures, thus showing a mixed irregular sintered morphology , sintered clusters appeared in the electrolytic samples due to the generation and growth of Ge from excessive GeO2, and the increase in the Ge content enables this phenomenon to become more apparent, as shown in As the synthesis conditions of Crtrolysis , irregularticles . The magarticles  shows thrphology . However2O3/GeO2/C at 3.0 V. The current value rose to 1.2 A at the brief initial electrolysis stage i and then, gradually decreased to about 0.2 A within 1 h (stage ii). Subsequently, electrolysis remained in a relatively stable current state until the end of electrolysis (stage iii). The current variation during electrolysis can be explained by the three-phase interlines (3PIs) mechanism  of this electrolysis process was calculated to be about 38.4% according to the following equation , as confirmed by the XRD results –(4) during electrolysis, wherein O2− is derived from the electrolysis of the oxides (GeO2 and Cr2O3) and the residual O2− in the molten salts. These oxides were also successively electrochemically reduced to metals Cr and Ge through reactions (2) and (5)–(7). As a result, the as-generated Cr and Ge can in situ react with C to form Cr2GeC by reaction (8). In general, the electrosynthesis of the Cr2GeC MAX phase involves the electro-separation of oxides and in situ alloying of Cr, Ge, and C.The electrochemical synthesis mechanism of the Cr results . In addi2GeC MAX phase was further investigated by TEM. 2GeC powder and the inset is its corresponding selected area electron diffraction (SAED) pattern. Evidently, Cr2GeC exhibits a nanoscale irregular shape with a particle size of about 100 nm. In addition, Cr2GeC nanoparticles show an interconnected morphology because of the sintering effect during the molten salt electrolysis process. The SAED pattern reveals the typical hexagonal property of the Cr2GeC MAX phase. From the high-resolution TEM image shown in 2GeC reveals the evident layers along the (0001) crystallographic direction. The regular lattice-resolved image commonly confirms periodic crystal structures  phase or the incompletely reversible intercalation of Li in the MAX phase structure  alloying reaction during (de) lithiation are believed to be responsible for the increase of the capacity during cycling  is shown in 2GeC nanoparticles.The DFT calculations were performed to preliminarily understand the Li-storage mechanism of Cr2GeC MAX phase with a typical two-dimensional layered structure has been electrochemically synthesized in molten salts. This electrosynthesis process consumes only electrons to directly convert Cr2O3/GeO2/C into Cr2GeC at a moderate temperature of 700°C. The synthesis mechanism mainly involves the electro-separation of oxygen ions from Cr2O3/GeO2 and in situ alloying of the as-generated Cr, Ge, and C. The as-prepared Cr2GeC MAX phase shows a uniform morphology of nanoparticles with a particle size of about 100 nm. Cr2GeC nanoparticles have been further investigated as anode materials for lithium-ion batteries, which showed attractive electrochemical performance with a specific capacity of 177.4 mAh g−1 at 0.2 C and excellent cycling performance. The possible lithium-storage mechanism of Cr2GeC has been discussed based on DFT calculations, whereby Ge atoms at edge sites of Cr2GeC nanoparticles undergo the (de) alloying reaction of Ge–Li.The Cr


Members of the carboxylesterase 2 (Ces2/CES2) family have been studied intensively with respect to their hydrolytic function on (pro)drugs, whereas their physiological role in lipid and energy metabolism has been realized only within the last few years. Humans have one CES2 gene which is highly expressed in liver, intestine, and kidney. Interestingly, eight homologous Ces2 (Ces2a to Ces2h) genes exist in mice and the individual roles of the corresponding proteins are incompletely understood. Mouse Ces2c (mCes2c) is suggested as potential ortholog of human CES2. Therefore, we aimed at its structural and biophysical characterization. Here, we present the first crystal structure of mCes2c to 2.12 Å resolution. The overall structure of mCes2c resembles that of the human CES1 (hCES1). The core domain adopts an α/β hydrolase-fold with S230, E347, and H459 forming a catalytic triad. Access to the active site is restricted by the cap, the flexible lid, and the regulatory domain. The conserved gate (M417) and switch (F418) residues might have a function in product release similar as suggested for hCES1. Biophysical characterization confirms that mCes2c is a monomer in solution. Thus, this study broadens our understanding of the mammalian carboxylesterase family and assists in delineating the similarities and differences of the different family members. Furdrolases ,10,11,12drolases . The humdrolases ,15,16. Tdrolases ,21,22,23Human carboxylesterase 2 (hCES2) is of special interest because of its prominent roles in metabolizing endogenous and exogenous chemicals including many important therapeutic medications, e.g., the anti-cancer chemotherapy drug CPT-11/irinotecan ,25. NotaIn this work, we take a structure-based approach to better characterize mCes2c and compare its experimental structure with the known structure of hCES1. Our data give detailed insights into the 3D structure of mCes2c determined by X-ray crystallography at high resolution. Despite overall similarities with hCES1, we also noted significant differences regarding binding pockets and the dynamics of the different modules. Furthermore, our in silico docking studies revealed a likely binding pose of the new COVID-19 drug molnupiravir in the substrate binding pocket of mCes2c. Thus, this study starts to fill the large gap in our knowledge of the different murine Ces2 family members and their physiologically relevant interrelationship to human carboxylesterases.TM cells. The resulting protein has 574 residues and a theoretical molecular mass of 64 kDa. The purified protein was active in hydrolyzing monoolein at a rate of 8900 nmol fatty acids*h−1*mg−1 in agreement to our recently published data [M) of 58.2 ± 0.5 °C for mCes2c. This high thermal stability was confirmed with CD-spectroscopy by following the changes in molar ellipticity at 222 nm TM,222~60.7 ± 0.1 °C , which are referred to as chains A–D, with the biological assembly being a monomer a,b. AminThe monomeric unit of mCes2c harbors an α/β-hydrolase fold with additional modules and insertions b,c. The The cap region  and hCES1 on the basis of binding of the anti-cancer drug CPT-11 in rCE and the fatty acid palmitate in hCES1 ,31. Two 2 = 1.4) to the experimentally determined crystal structure of mCes2c  showed a distinct peak at an elution volume of 15 mL. Based on our standard run, this corresponds to approx. 48 kDa, indicating that mCes2c is monomeric in solution a. An addric peak b. The mof mCes2c c,d. Toge2). However, a loop in the lid region (N100-L116) . This is in agreement with missing electron densities due to disorder for some of these residues in different chains of the lid. Furthermore, the RD shows higher values for the B-factors (average B-factor 46.1 Å2) which indicate flexibility and might enable breathing motions of mCes2c. Hence, entrance to the substrate binding pocket might be mediated by the flexible lid region and increased dynamics in the RD  pink,  exhibitsn the RD .The prodrug molnupiravir (EIDD-2801) was docked into mCes2c by using Maestro, Schrödinger. As a result, molnupiravir is positioned in the active site pocket of mCes2c with an extra precision (XP) glide score of −6.3 a. The maRecently, mCes2/hCES2 family members were investigated intensively with respect to their ability to hydrolyze lipids, and their possible role in lipid signaling pathways ,7,8. MamIn this work, we determined the crystal structure of mCes2c (561 amino acids) which is suggested to be the mouse ortholog of hCES2 . It is possible that flexibility in this region is responsible for the opening-closing mechanism of the substrate binding pocket of hCES1 together with the flexible regions of the RD domain of hCES1 . The entrance to the substrate binding pocket of mCes2c might be mediated by a flexible loop in the lid region (N100-L116) and dynamics in the RD  and is responsible for the closed entrance to the substrate binding pocket in hCES1 a,b. AddiIn hCES1 the substrate binding pocket opens on its top (between cap and RD), while in mCes2c the pocket opens on the side (between lid and RD), with closer proximity to the active site compared to hCES1. Whether different substrates enter via different entrances in carboxylesterases needs to be established in further structural studies with different substrates and different mCes/hCES family members.For hCES1, three features are described for the side door. Bencharit et al., 2006 describes the highly conserved gate residue M425 in hCES1 as the most important residue for regulating the release of products such as fatty acids. The highly conserved switch residue F426 might be a link between RD and the catalytic region, allowing a rotation of RD. The aromatic releasing valve residue F551 of hCES1 is the third feature and might be responsible for long-chain fatty acid release . SimilarBencharit et al. describe a regulatory mechanism for hCES1 that is dependent on its oligomeric state . As hexa®”. In vitro and in vivo studies of molnupiravir showed high efficacy to the replication machinery, high genetic barrier to resistance, and cytotoxicity. Molnupiravir is the isopropylester of the ribonucleoside analog β-D-N4-hydroxycytidine. It is administered orally and first needs to be hydrolyzed to N-hydroxycytidine, and then phosphorylated before it becomes incorporated in place of cytidine triphosphate or uridine triphosphate during RNA synthesis. Subsequently, these erroneous incorporations lead to numerous mutations in the whole viral genome, ultimately rendering the virus unable to replicate [Molnupiravir is a novel broad-spectrum antiviral agent that acts by introducing mutations into viral RNA via RNA-dependent RNA polymerase. Although initially developed against influenza, it obtained world-wide attention as treatment strategy for COVID-19 using the brand name “Lagevrioeplicate ,35,36,37eplicate ,38. Our In this manuscript, we use the suggested nomenclature of Holmes et al., 2010 when referring to human and mouse carboxylases, namely capitalized CES1-CES2 for proteins from humans and Ces2a-Ces2h for mouse proteins . For easChemicals were obtained from Sigma-Aldrich  or Carl Roth GmbH , if not stated otherwise; columns for protein purification were obtained from Cytiva . PierceTM Unstrained Protein MW Marker from Thermo Fisher Scientific was used as size marker for SDS-PAGE gels, unless otherwise noted. For protein production we used the mammalian Expi293F™ cells from Thermo Fisher Scientific.6-tag. For protein expression, the resulting vector was transfected into Expi293F™ cells using the ExpiFectamin 293 transfection kit. Seventy-two hours after transfection, we collected the medium containing the overexpressed and secreted protein, centrifuged, and then filtered it through a 0.45 µm filter to remove cell debris. Afterwards, we purified His6-tagged mCes2c protein by Ni-affinity chromatography followed by size exclusion chromatography (SEC) using a Superdex 200 Increase 10/300 GL column buffer A containing 20 mM Tris/HCl, pH 7.4, and 150 mM NaCl. The concentration of the purified protein was determined via absorption at 280 nm with the calculated extinction coefficient 73,130 M−1 cm−1; the purity was analyzed using SDS-PAGE.The cloning of mouse Ces2c  into a pFLAG CMV 5.1 expression vector was described previously . The finThe activity assay was performed using monoolein as substrate as described in Chalhoub et al. .g for 15 min at 4 °C prior the actual experiment. mCes2c was eluted from a Superdex 200 Increase 10/300 GL column  in a buffer B containing 20 mM potassium phosphate, pH 7.4 and 150 mM NaCl with a flow rate of 0.3 mL/min at 8 °C directly coupled to a MALS unit from Wyatt Technologies (TREOS II) equipped with three detection angles. Data were analyzed with the program ASTRA 7.1.2.5 (Wyatt Technologies). To determine the molecular weight based on the SEC, protein standard from Bio-Rad  was used .For characterization by SEC-MALS, purified mCes2c (1.5 mg/mL) was centrifuged at 12,851× M) was calculated from the first derivate of the fluorescence signal vs. the temperature. Experiments were performed as triplicates.For differential scanning fluorimetry, we mixed protein and dye solution in 96 well plates to a total volume of 20 µL per well. 4 µL mCes2c per well (1.3 mg/mL) were mixed with 14 µL buffer B, 2 µL of 50X SYPRO Orange (Thermo Fisher Scientific) per well were added to reach the final concentration of 5X for SYPRO Orange. The temperature was increased from 20 °C to 95 °C with increments of 0.5 °C every 30 s. The melting temperature  was performed at 222 nm every 0.5 °C/min with a JASCO J-1500 CD spectrophotometer using a 1 mm path length quartz cuvette. Spectra were measured at six accumulations collected at 100 nm/min scanning speed, data pitch of 0.2 nm, a digital integration time (DIT) of 4 s and a bandwidth of 2 nm.m/z, 1 Hz, source voltage 1.3 kV, dry gas flow 3 L/min at 180 °C. The protein mass spectra were deconvoluted by Bruker Data analysis software, using the MaxEnt2 algorithm. The following main parameters were applied: m/z range, min. 5000 to max. 100,000, instrument resolving power was set to 10,000. For peak detection, the Sum-peak method was used.Purified mCes2c was desalted using 10 kDa Amicon Ultra 0.5 mL centrifugal filter units . A final protein concentration of 200 ng/µL in 2% acetonitrile and 0.1% formic acid (in water) was obtained. Protein species were separated by nano-HPLC (Dionex Ultimate 3000) equipped with a Pepswift precolumn  and a ProSwift RP-4H column . Two µL of protein sample were injected and concentrated on the enrichment column for 3 min at a flow rate of 5 µL/min with 0.1% formic acid as isocratic solvent. Separation was carried out on the nanocolumn at a flow rate of 1 µL/min at 37 °C using the following gradient, where solvent A was 0.1% formic acid in water and solvent B was acetonitrile containing 0.1% formic acid: 0–2 min: 5% B; 2–17 min: 5–60% B; 17–20 min: 60% B; 20–20.1 min: 60–5% B; and 20.1–29 min: 5% B. The maXis II ETD mass spectrometer  was operated with the captive spray source in positive mode with following settings: mass range: 300–3000 Rg) from the scattering data. All the experiments were conducted with the following parameters: beam current = 200 mA, flux = 2.6 × 1012 photons/sec, wavelength = 1 Å, estimated beam size 1 mm × 100 µm, 10 frames (1 frame per second) were taken from each sample. All experiments were standardized to water  suite [SAXS measurements were performed at the BM29 beamline for Bio-SAXS at the European Synchrotron Radiation Facility . Three samples of purified mCes2c with a volume of 50 µL were prepared with three different concentrations . Samples and buffer A were shipped to ESRF Grenoble and the measurement was performed on site. The sample delivery and measurements were performed using a 1 mm thick quartz capillary, which is a part of BioSAXS automated sample changer unit. Before and after each sample measurement, the corresponding buffer was measured and averaged. Low-q regions of the buffer subtracted averaged frames were studied to identify aggregation. The Guinier approximation was used to extract the radius of gyration (y) suite .w/v PEG 8000 (ShotGun (SG1) Screen C5, Molecular Dimensions, Newmarket, UK). We obtained crystals of ~200 μm after 1 month of incubation at 20 °C. A complete diffraction dataset of mCes2c up to 2.12 Å, space group P21 21 21, resolution was collected at the DESY synchrotron facility PETRA III, P11 beamline in Hamburg, Germany. Data were processed using XDS and scaled using Aimless [The monomeric peak fractions from the final SEC run in buffer A were concentrated to 2.7 mg/mL and different standard crystallization screens were used for setting up the crystal plates. Crystallization experiments were performed with an ORYX 8 robot  using the sitting drop vapor-diffusion method in 96-well plates. The drops contained equal volumes (0.5 µL) of protein and reservoir solution and were equilibrated against 30 µL of reservoir solution. Crystallization condition containing 0.2 M ammonium sulfate, 0.1 M sodium cacodylate buffer pH 6.5 and 30%  Aimless ,41,42. T Aimless  and Isol Aimless  and refi Aimless . Waters  Aimless . The ref Aimless . The resMaestro (version 12.6.144), Schrödinger (2020-4) was used for the in silico docking analysis . The str