Z = 6 in space group P21/m.The title compound crystallizes with  7H14N3+·Br−, has been synthesized and structurally characterized using single-crystal X-ray diffraction and NMR. The compound crystallizes in the monoclinic space group P21/m with the non-hydrogen atoms of one cation lying on general positions and the others lying on a mirror plane. One bromide ion also lies on the mirror. The extended structure exhibits only weak intermolecular interactions between heterocyclic C—H groups and Br− ions.An ionic compound consisting of a triazolium cation and bromide anion, C All the atoms of the N4 cation and Br2 occupy general positions. The bond lengths in the triazolium rings indicate aromaticity with C—N bonds exhibiting distances in the range of 1.305 (2)–1.366 (2) Å and N—N bond distances near 1.365 Å; the N—C—N bond angles in the triazolium ring range from 106.93 (18) to 111.35 (18)°. The C1—N2—C5—C6 torsion angle of the ethyl side chain in the N1 cation is constrained to be 0° by symmetry and the corresponding C8—N5—C12—C13 torsion angle in the N4 cation is 24.4 (2)°.The molecular structure of the title compound is shown in Fig. 1The crystal packing of the title compound is displayed in Fig. 23 on a 400 MHz Varian spectrometer and referenced to the residual solvent peak (δ in p.p.m.).1-Ethyl triazole was purchased from AmBeed. All other compounds used in the syntheses of the title compound were obtained from Sigma-Aldrich. All materials in the synthesis were used as received. The synthesis was performed under nitrogen using reagent grade solvents, which were used as received without further purification. NMR spectra were recorded at room temperature in CDCl2Cl2 solution. Yield: 1.04 g (23%). 1H NMR: CDCl3, δ (p.p.m.) 11.99 , 8.85 , 5.13 2), 4.63 , 1.74 2), 1.65 . 13C NMR: δ (p.p.m.) 142.27 (N—CH—N), 141.84 (N—CH—N), 53.15 [CH(CH3)2], 48.36 (N—CH2), 23.14 [CH(CH3)2], 14.22 (N—CH2CH3).1-Ethyl-1,2,4-triazole  and isopropyl bromide  were added to toluene (20 ml) and the mixture was refluxed for 48 h. Once cooled, the liquid was decanted, the white solid product that formed was washed with ether, filtered, and dried. The title compound crystallized as clear needles by slow diffusion of pentane into a CHCrystal data, data collection, and structure refinement details are summarized in Table 210.1107/S2414314623007848/hb4448sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623007848/hb4448Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623007848/hb4448Isup3.cmlSupporting information file. DOI: 2293675CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Among these subsets, human T-IELs are predominantly TCRαβ+ (over 80%), whereas those in mice are mostly TCRγδ+ (~60%). Of note, the majority of the TCRγδ+ subset expresses CD8αα in both species. Although T-IELs have been extensively studied in humans and mice, their profiles in cattle have not been well examined. Our study is the first to characterize bovine T-IELs using flow cytometry, where we identified several distinct features. The percentage of TCRγδ+ was comparable to that of TCRαβ+ T-IELs (both ~50% of CD3+), and the majority of bovine TCRγδ+ T-IELs did not express CD8 (CD8−) (above 60%). Furthermore, about 20% of TCRαβ+ T-IELs were CD4+CD8αβ+, and the remaining TCRαβ+ T-IELs were evenly distributed between CD4+ and CD8αβ+ (~40% of TCRαβ+ T-IELs each) with no TCRαβ+CD8αα+ identified. Despite these unique properties, bovine T-IELs, similar to those in humans and mice, expressed a high level of CD69, an activation and tissue-retention marker, and a low level of CD62L, a lymphoid adhesion marker. Moreover, bovine T-IELs produced low levels of inflammatory cytokines such as IFNγ and IL17A, and secreted small amounts of the immune regulatory cytokine TGFβ1. Hence, bovine T-IELs’ composition largely differs from that of human and mouse, with the dominance of the CD8− population among TCRγδ+ T-IELs, the substantial presence of TCRαβ+CD4+CD8αβ+ cells, and the absence of TCRαβ+CD8αα+ T-IELs. These results provide the groundwork for conducting future studies to examine how bovine T-IELs respond to intestinal pathogens and maintain the integrity of the gut epithelial barrier in animals.Intraepithelial T lymphocytes (T-IELs), which constitute over 50% of the total T lymphocytes in the animal, patrol the mucosal epithelial lining to defend against pathogen invasion while maintaining gut homeostasis. In addition to expressing T cell markers such as CD4 and CD8, T-IELs display T cell receptors (TCR), including either TCRαβ or TCRγδ. Both humans and mice share similar T-IEL subsets: TCRγδ Gastrointestinal (GI) disorders, ranging from acute infections to chronic inflammatory diseases, present considerable economic and health implications in humans as well as in cattle. Intraepithelial T lymphocytes (T-IELs), which make up about 90% of all intestinal intraepithelial lymphocytes and 50–60% of total T lymphocytes in humans, play a critical role in pathogen clearance and gut homeostasis, and could be a central target for developing intervention strategies against GI diseases ,4,5,6,7.In contrast to the peripheral T cells, T-IELs are found in the antigen-abundant mucosal environment and possess unique homing molecules, activation states, and effector functions ,31,32,33+CD8αα+ and TCRγδ+ subsets, both of which exhibit innate-like features [+CD8αα+ subset remains a topic of debate, it has been generally accepted that they originate in the thymus [+CD8αα+ subset expresses natural killer (NK) receptors such as NKG2A/CD94 and the Ly49 family, which modulate immune responses by detecting altered MHC class I molecules on compromised cells and by producing immune regulatory cytokines like IL-10 and TGFβ1 [+ cells depend on IEC-intrinsic MyD88 signaling, which induces TCRγδ+ T-IELs to produce antibacterial lectin RegIIIγ along with cytokines like IFNγ and IL-13, facilitating the clearance of pathogens such as Salmonella, Toxoplasma, Listeria, and Nippostrongylus [+CD4+ and TCRαβ+CD8αβ+ subsets, which originate from antigen-stimulated peripheral T cells [+CD4+ T-IELs contribute to anti-inflammatory responses by producing immune regulatory cytokines, such as IL-10 [+CD8αβ+ subset initiates cytotoxic effector memory responses against pathogens such as simian immunodeficiency virus, lymphocytic choriomeningitis virus, Toxoplasma, and Giardia through TCR [+CD8αβ+ T-IELs can also respond in an antigen non-specific manner by expressing NK receptors in conditions like celiac disease whereas, in mice, it is the TCRαβ+CD8αα+ subset that performs this function [T-IELs are categorized into two groups based on their origin: natural and induced . Naturalfeatures ,84,85,86features ,88,89,90e thymus ,80,87,91e thymus ,93,94,95nd TGFβ1 ,96,97. Orongylus ,106,107. T cells ,79 and fas IL-10 ,108,109,ough TCR ,113,114.function ,115. Des+ population constitutes approximately 60 percent of the total peripheral blood mononuclear cells (PBMC) in young cattle, a proportion significantly higher compared to the 2–15% found in humans and mice [+ T cells in blood and lymph nodes mount immune responses that differ partially from those of humans and mice [+ and CD8+ cells, proliferate in response to various pathogens such as Cryptosporidium parvum and Escherichia coli [+ and CD8+ T-IEL population correlates with increased disease severity [+ and TCRαβ+ T-IEL subsets, the presence of distinct subsets such as TCRαβ+CD4+CD8αβ+ and TCRγδ+CD8−, and the absence of TCRαβ+CD8αα+ cells. Despite these variations, bovine T-IELs, similar to their human and mouse counterparts, continue to express T-IEL markers and produce various cytokines in the gut.As ruminants, cattle possess a four-chambered stomach and a significantly longer intestine, differing from humans and mice not only in terms of gut environment but also in variations in the immune system ,120,121.and mice ,123,124.and mice ,118. Desand mice ,126. Forhia coli ,19,127. Since 1958, the Wye Research and Education Center, University of Maryland Experimental Station  ,128, has3 fragments and subjected to mechanical disruption and digestion in 5 mL of RP10 medium supplemented with 400 U/mL of V Collagenase, 0.1 mg/mL of DNase, and 2.5 U/mL of hyaluronidase at a temperature of 37 °C for a duration of 2 h [g-force (G) for 30 min. Following centrifugation, the buffy coat at the interface was carefully collected into a new 15 mL tube and re-suspended in 8 mL of 1× phosphate-buffered saline (PBS) . Then, 5 mL of lymphocyte separation medium (LSM) with a density of 1.077 g/mL  was added, followed by 30 min of centrifugation at 900G with break off. The interface’s second buffy coat was collected and cleaned twice with PBS. The cell pellet was resuspended in 5 mL Allos medium following the final rinsing, and a small aliquot was used for cell counting. Allos media was RPMI-1640 supplemented with FCS (10%), HEPES (10 mM), MEM non-essential amino acid (1×), sodium pyruvate (1 mM), penicillin and streptomycin (100 U/mL), L-glutamine (2 mM), and 2-mercaptoethanol (50 μM) .The inguinal lymph nodes (IGLN) were cut into 2–3 mmn of 2 h ,21. The n of 2 h ,131,132.2 pieces. These tissue pieces were then incubated in 50 mL of CMF Hanks with 2% FBS at 200 rpm at 37 °C for 30 min . After each incubation, the supernatant was collected, and the procedure was repeated twice using fresh CMF Hanks with 2% FBS. The accumulated supernatants were pooled together into a 200 mL beaker, allowing the epithelial cells to settle for 10 min. Without disturbing the sediment, the supernatants were decanted into new 50 mL conical tubes  and then strained using 70 µm cell strainers . The tubes were then centrifuged at 500 G for 15 min at 22 °C. The resulting pellets were resuspended in 40% isotonic Percoll  in RPMI supplemented with 5% FBS. These suspensions were carefully layered over 80% isotonic Percoll and centrifuged at 600 G for 30 min at 22 °C without break. Cells from the 40%/80% interface were collected into 15 mL conical tubes. They were then washed with RPMI1640 containing 5% FBS, followed by centrifugation. The final cell pellet was resuspended for counting and subsequent experiments.The T-IEL isolation protocol for the abomasum was adapted from , and forAll the antibodies used in this study are listed in the following tables: 6 cells were allocated to FACS tubes  for surface staining. These cells were sequentially stained with primary antibodies  to achieve a final concentration of 1× in the medium to activate T cells while ensuring retention of the cytokines they produce within the cells [2 for 4 h to allow stimulation. Surface staining was performed first, which was followed by permeabilization using P/W for 15 min at 4 °C. The subsequent intracellular staining followed the same protocol as the previously described surface staining process, with all antibodies incubating for 25 min at 4 °C. After each antibody incubation, the cells were washed with P/W. After the final P/W wash, cells were rinsed with SB and then resuspended in 100 μL SB. Isotype controls were stained using isotype antibodies, and an unstained control was included following the same protocol. Flow cytometry was performed, acquiring a minimum of 20,000 events. Data analysis was conducted using FlowJo version 10 .For the intracellular staining, 10he cells ,134. Thihe cells ,136. Thep < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.Statistical analyses were performed with Prism 8 ; specific details thereof are provided in the figure legends. Overall, all data have passed the Anderson–Darling normality test. All data were analyzed using one-way ANOVA with Tukey’s Multiple Comparisons Test. Asterisks indicate statistical significance. * 6 in the ileum mucosa (ILM) and the jejunum mucosa (JJM) from a 10 cm segment of each, which dropped by about 90% in abomasum mucosa (ABM) (~100 g) , but comparable to mice [Tissues from the blood and inguinal lymph nodes were collected from finished steers as in our previous report . The pro(~100 g) B. The lo and ILM D, which  and ILM C. The frand IGLN E. This e to mice .+ T-IELs make up a small population of T-IELs in humans (<15%), but a major proportion in mice (40–60%) [+ T-IELs were approximately 40% of T-IELs (CD3+) (+ T-IELs are CD8αα+ (>75%); bovine TCRγδ+ T-IELs were predominantly (>60%) CD8-negative (+CD8+ T-IELs expressed both CD8 subunits (CD8αβ+) or only the CD8α+ subunit (CD8αα+)  . In aduls (CD3+) E, comparnegative A,B, and (CD8αα+) B as defiwith age . In addiunctions ,143,144.+ T-IELs constitute a major portion of T-IELs in humans (>80%) and a significant portion in mice (>30%) [+ T-IELs expressing either CD4 and/or CD8α made up more than 50% of the total T-IELs (CD3+) across all the tissues , and collectively reached the number of TCRγδ+ cells in T-IELs (CD3+) (~40%) (+ in T-IELs was lower in JJM than in PBMC and IGLN (+CD8α+ (double positive) TCRαβ+ T-IELs in the mucosa, which was almost absent in the blood and IGLN  . Indeed, tissues A,B. Furt) (~40%) B. The frand IGLN B. Notabland IGLN B. These y CD8αβ+ C, so then humans . The TCRe future ,146,147. tissues , suggest+, TCRαβ+CD8α+, and TCRαβ+CD4+ T-IELs compared to those in PBMC and IGLN (+CD4+ T-IELs exhibited significantly less CD69 expression than the TCRγδ+ and TCRαβ+CD8α+ T-IELs (+CD8+ and TCRαβ+CD4+ cells had lower levels of CD62L than TCRγδ+ (+ T cells in response to TCR stimulation in vitro [+ and TCRγδ+ T cells from the blood, but is virtually absent in CD8+ T cells [+CD4+ and TCRγδ+ had modest but significantly higher levels of CD25 than TCRαβ+CD8+ (CD69 is a marker for T-IEL activation  and is aand IGLN A,B, indi+ T-IELs B. Consisn TCRγδ+ C. The suin vitro , and is  T cells . To dete T cells , among TRαβ+CD8+ , followiRαβ+CD8+ , indicat+CD4+ T-IELs producing less than their PBMC counterparts (+ T cells within PBMCs but induced no response in the TCRαβ+ T cells within PBMCs or in any T-IEL subtypes (+CD4+ T-IELs were rarely stained positive for Foxp3, a Treg marker (+CD4+ T-IELs are not Tregs. These data confirm that different subsets of bovine T-IELs differ in their functions from their counterparts in the blood and lymphoid tissues, as demonstrated by cytokine production differences.Cytokines play an important role in regulating immune responses. To test whether T-IELs could be induced to produce cytokines, these cells were stimulated with an activation cocktail as described in our previous reports ,121, usiterparts A–C. IFNγterparts A, suggesterparts E, suggessubtypes . TGFβ1-pg marker D, implyi+ and TCRαβ+ T-IEL subsets. Uniquely, subsets such as TCRαβ+CD4+CD8αβ+ and TCRγδ+CD8− are present in bovine T-IELs, while the TCRαβ+CD8αα+ T-IELs are notably absent. Despite these distinct features, bovine T-IELs continue to demonstrate key immunological functions analogous to those in humans and mice, specifically in their pre-activated states and with the production of immune regulatory as well as inflammatory cytokines, suggesting similar functions in maintaining gut homeostasis and pathogen control. This is the first time that T-IELs in the bovine gut have been characterized, furthering our understanding of bovine mucosal immunity and offering insights into the development of innovative drugs and vaccines against mucosal diseases.Bovine T-IELs, while demonstrating certain similarities, evidently differ from their counterparts in humans and mice. These variations are particularly noticeable in the distribution of the TCRγδ+ T-IELs in the ileum immediately post-birth and during early life and the presence of higher proportions of CD4+ and CD8+ T-IELs in weaned calves compared to those in suckling calves [+ and TCRαβ+ T-IELs. As bovine peripheral TCRγδ+ T cells inhibit the proliferation of TCRαβ+ T cells by producing IL-10 and TGFβ1 and the depletion of TCRγδ+ T cells results in enhanced proliferation of antigen-specific TCRαβ+ T cells in cattle infected with Mycobacterium bovis [+ T-IEL in young calves could assist in tolerating non-harmful antigens. In contrast, adult cattle may host a larger proportion of induced T-IELs in the gut due to increased antigen exposure over time. Additionally, induced T-IELs constitute a large proportion of the total T-IELs in humans, while mice maintain a significant proportion of natural T-IELs [Variations in the number and proportions of T-IELs might stem from the diverse GI microenvironments and age-related differences across species . While dg calves ,173,174.um bovis ,142,143,l T-IELs . It coul+CD8− subset in bovines potentially plays a crucial role in pathogen defense and immune homeostasis. Bovine TCRγδ+ T-IELs are predominantly CD8-negative, with only a small fraction expressing either CD8αβ and/or CD8αα. In contrast, mouse TCRγδ+ T-IELs mainly express CD8αα [+ is associated with cytotoxicity, while CD8αα is linked to immune suppression and cell survival [+ T-IELs exhibit cytotoxic responses and can enhance T cell sensitivity to a cognate antigen by 100-fold compared to their CD8αα-expressing counterparts [+ T cells or excessive cytolytic responses, thus serving as an immunosuppressive and survival-aiding molecule in mice [+ T-IELs expressing CD8αβ likely plays a dominant role in defending against pathogens, whereas those expressing CD8αα might promote immune homeostasis. Moreover, the TCRγδ+CD8− population is also observed in the blood, peripheral lymph nodes, skin, and spleen marginal zones in cattle [+CD8− T cell subset being known to display inflammatory functions in response to various infectious agents such as Anaplasma marginale and Mycobacterium bovis in the bovine periphery [+ subpopulation in T-IELs and in blood expressed low levels of TGFβ1. However, the function of the TCRγδ+ T-IELs may differ from their counterparts in the blood, as further stimulation enhanced TGFβ1 production only in those from the blood, not those from T-IELs. We predict that TCRγδ+ T-IELs play a crucial role in maintaining intestinal immune tolerance and also mediate certain immune responses. Additionally, the absence of the TCRαβ+CD8αα+ subset in cattle suggests a potential divergence in their immunological profile compared to humans and mice, implying the possibility of compensation through alternative subsets. Under normal conditions, TCRαβ+CD8αα+ T-IELs constitute a significant portion of T-IELs in mice and perform immunomodulatory functions, supported by a wide array of NK receptor expressions, including NKG2A/CD94 and the Ly49 members [+CD8αβ+ T-IELs in mice are geared towards immune responses and cytolytic functions, demonstrated by their substantial capacity to produce cytokines such as IFNγ and express high levels of granzyme B [+CD8αα+ T-IELs, while the TCRαβ+CD8αβ+ subset exhibits the dual capacity for immunomodulation and cytotoxicity by expressing NK receptors and displaying the potential to produce IFNγ and granzyme B [+CD8αβ+ T-IELs in cattle. Given these findings, it is worth considering that other bovine T-IEL subsets such as TCRγδ+CD8−, TCRγδ+CD8αα+, and/or TCRαβ+CD4+ T-IELs might compensate for the immune regulatory role of the TCRαβ+CD8αα+ T-IELs found in mice. However, further research is required to generate confirmatory results.The unique TCRγδss CD8αα ,116,175.survival ,176,177.terparts ,176. Con in mice ,176,177.n cattle . Despiteeriphery ,144, theeriphery ,142,143. members ,79. In canzyme B ,79. Inte+CD4+CD8αβ+ T-IELs in cattle may possess the combined characteristics of both CD4+ and CD8αβ+ T-IELs. In mice, various GI microenvironment factors such as food components like retinoic acid and gluten, as well as cytokines like TGFβ1 and IFNγ, can downregulate the CD4 lineage transcription factor ThPOK and upregulate the CD8 lineage transcription factor Runx3, which leads to the expression of CD8αα in CD4+ T-IELs [+CD4+CD8αα+ and TCRαβ+CD4+ T-IELs contribute to immune tolerance [+CD4+ T-IELs has been well established [+ T cell will generally not express CD8αβ, and vice versa [+CD4+CD8αβ+ T-IEL subset in cattle might possess both the immunoregulatory functions of CD4+ and the cytotoxicity of CD8αβ+ T-IELs. Nonetheless, this concept warrants further studies.The significant presence of distinct TCRαβ+ T-IELs ,181,182.olerance ,179,183.ablished ,185, it ce versa . At pres+ and TCRαβ+CD8αα+ T-IELs express substantially lower levels of CD62L compared to their PBMC and IGLN counterparts (Bovine T-IELs exhibit an activated-yet-resting state in the gut, similar to their human and mouse counterparts, but with potential differences. CD62L is essential for T cell adhesion, facilitating lymphocyte homing to secondary lymphatic organs. In contrast, CD69 serves a dual role: it counters the sphingosine-1-phosphate receptor, which typically signals to T cells to move into the bloodstream, and acts as a T cell activation marker ,188,189.terparts C. This v+ subpopulation, produced TGFβ1 under normal conditions, suggesting similar functions in cattle. Furthermore, stimulation enhanced TGFβ1 production only in TCRγδ+ T cells in the PBMC in cattle, indicating the regulatory role of this subset in systemic circulation during immune reactions as previously found [Bovine T-IELs, similar to those in humans and mice, secrete low amounts of the immune regulatory cytokine TGFβ1, which helps maintain immune tolerance under normal conditions ,198,199.ly found ,142,143.Our research shows that bovine T-IELs differ from those in humans and mice, making it of paramount importance to understand their roles in cattle. Although we characterized the bovine T-IELs by analyzing their surface and intracellular molecules, we have not yet tested their functions such as how they respond to different stimuli, which is currently an ongoing project in the lab.Our study reveals the unique composition and potential roles of T-IELs for the first time in cattle, highlighting functional conservation across species as well as specialized adaptations. These findings open avenues for further investigations into bovine T-IELs and their role in developing strategies against gastrointestinal disorders including both acute and chronic infections in cattle. Our next steps involve functional assays and molecular analyses to further understand the mechanisms by which bovine T-IELs contribute to maintaining homeostasis and immune responses in cattle.


The smart design intrinsically tunes the electronic structure, guarantees ion chemisorption–desorption equilibrium and increases the electroactive sites, which not only effectively accelerates charge transfer rate during reaction processes, but also endows more redox reactions, as verified by first-principle calculations. These merits can help the fabricated δ-MnOx2−−2.0 cathode to present a large specific capacity of 551.8 mAh g−1 at 0.5 A g−1, high-rate capability of 262.2 mAh g−1 at 10 A g−1 and an excellent cycle lifespan (83% of capacity retention after 1500 cycles), which is far superior to those of the other metal compound cathodes. In addition, the charge/discharge mechanism of the δ-MnOx2−−2.0 cathode has also been elaborated through ex situ techniques. This work opens up a new pathway for constructing the next-generation high-performance ZIBs cathode materials. In recent years, manganese-based oxides as an advanced class of cathode materials for zinc-ion batteries (ZIBs) have attracted a great deal of attentions from numerous researchers. However, their slow reaction kinetics, limited active sites and poor electrical conductivity inevitably give rise to the severe performance degradation. To solve these problems, herein, we introduce abundant oxygen vacancies into the flower-like The online version contains supplementary material available at 10.1007/s40820-023-01194-3. Based of δ-MnO2 .3, δ-MnOδ-MnO2, δ-MnOx2−−0.5, δ-MnOx2−−2.0 and δ-MnOx2−−5.0, as well as the adsorbed H+/Zn2+ at the above models, were optimized . The charge storage mechanism for the cathode materials can be unveiled through Eqs. .To further deeply understand the positive effects of vacancy modulation on improving the electrochemical performances, density functional theory (DFT) calculations were performed, and the corresponding analysis results are shown in Fig. ugh Eqs. –9 74], , δ-MnO2,ugh Eqs. :6\documeGH+/ΔGZn2+) of the redox reactionswere estimated, as shown in Fig. EH+ of δ-MnO2, δ-MnOx2−−0.5, δ-MnOx2−−2.0 and δ-MnOx2−−5.0 is −0.757, −0.989, −1.15 and −1.35 eV, respectively 6.4H2O and ZnMn2O4 formation reaction, as displayed in Fig. δ-MnOx2−−5.0 cathode possesses prominent electrochemical performances, whereas the δ-MnOx2−−2.0 cathode  verifies the highest specific capacity and rate capability in practice. It is because extremely robust H+/Zn2+ binding strength referring to δ-MnOx2−−5.0  may give rise to more difficulties in completing the charge reaction (left arrows) than that of the δ-MnOx2−−2.0, bringing about immensely inferior reversibility in δ-MnOx2−−5.0. Accordingly, δ-MnOx2−−2.0 provides relatively strong and moderate H+/Zn2+ absorption capability compared to the other obtained cathode materials. Therefore, the δ-MnOx2−−2.0 displays optimal H+/Zn2+ chemisorption–desorption equilibrium, which makes the reversible reactions easier to proceed, effectively accelerating the charge transfer rate during the redox reaction process and further demonstrating outstanding rate capability. Meanwhile, it greatly enhances the reaction activities, substantially guaranteeing the high capacity. Based on the above results, the introduced oxygen vacancies in the cathode materials own an optimal level rather than an infinitely improving vacancy concentration. Thus, our study offers a unique path for determining the optimal vacancy concentration, which is of extreme significance for constructing high-performance ZIBs cathode materials. In order to explore the bonding properties of the adsorbed H+/Zn2+ in the optimal δ-MnOx2−−2.0 and δ-MnO2, the corresponding electron density difference was calculated presented in Figs. +/Zn2+ at the δ-MnOx2−−2.0 in Fig. δ-MnO2 , which is quantitatively testified using the Bader charge analysis  of the δ-MnOx2−−2.0 and δ-MnO2 6.4H2O, JCPDS No. 44-0673) [–along with theconsumption of H+, which is consistent well with the previous studies [ex situ XRD patterns, presumably due to the H+ inserting into the interlayer of δ-MnO2 [2O4  phase labeled by red symbol at 59.1° and 66.3° can be detected during the discharge process (points IV and V) because of the Zn2+ intercalation. In the following charging process (points VI–IX), these new emerged diffraction peaks of the above produced ZSH and ZMO gradually vanish, and then, the intrinsic peaks of the δ-MnO2 can also recover well, indicating the superior reversibility of the δ-MnOx2−−2.0 cathode. Furthermore, the ex situ XPS tests were utilized to further demonstrate the phase transition mechanism during the discharge/charge process. In terms of the high-resolution Zn 2p spectra . When the cathode was charged to IX state again, the peak integral area of Mn3+and Mn4+ can be also significantly augmented owing to the extraction of Zn2+ from the cathode. Notably, the Mn2+ peak signal does not completely disappear, which can be mainly attributed to the captured Zn2+ in the cathode. Moreover, the O 2p spectra  , 76. The studies , 78. Notf δ-MnO2 . Except izations .δ-MnO2 nanostructures with moderate oxygen vacancies as the advanced ZIB cathodes. The created moderate oxygen vacancies in δ-MnO2 (δ-MnOx2−−2.0) not only effectively ensures H+/Zn2+ chemisorption–desorption equilibrium, making the redox reactions easier to proceed and further accelerating the electron transfer rate during the reactions, but also intrinsically enhances electrochemical activity and electrical conductivity, leading to faster reaction kinetics and more reversible redox reactions, as demonstrated by the series of the test techniques and the corresponding DFT calculations. Benefiting from the above advantages, the prepared δ-MnOx2−−2.0//Zn ZIBs can deliver a maximum specific capacity of 551.8 mAh g−1 at 0.5 A g−1, ultrahigh rate capability (262.2 mAh g−1 even at 10 A g−1) and a long-term cycle lifespan with a capacity retention ~ 90% after 1500 cycles. Meanwhile, the assembled ZIBs can also present a large energy density of 373.5 Wh kg−1 at 2.0 kW kg−1, and when the power density is increased to 747.5 kW kg−1, it still possesses the favorable energy density of 561.6 Wh kg−1. This work both provides a potential cathode candidate for next-generation aqueous ZIBs and unfolds an effective vacancy modulation strategy for constructing the other high-performance battery electrode materials.In summary, oxygen vacancies modulation strategies have been successfully employed to construct the unique flower-like Supplementary file1 (PDF 1762 kb)Below is the link to the electronic supplementary material.


Physalis as a source of anti-inflammatory constituents, the present study aimed at isolation for the first time some endophytic fungi from the medicinal plant Physalis pruinosa.Due to the extensive potential of previously studied endophytes in addition to plants belonging to genus P. pruinosa then purified and identified by both morphological and molecular methods. Comparative evaluation of the cytotoxic and ex vivo anti-inflammatory activity in addition to gene expression of the three pro-inflammatory indicators  was performed in WBCs treated with lipopolysaccharide (LPS) for the identified endophytes, isolated compounds and the standard anti-inflammatory drug (piroxicam). For prediction of the binding mode of the top-scoring constituents-targets complexes, the Schrödinger Maestro 11.8 package  was employed in the docking study.The endophytic fungi were isolated from the fresh leaves of P. pruinosa leaves. Selection of six representative isolates was performed for further bioactivity screening based on their morphological characters, which were then identified as Stemphylium simmonsii MN401378, Stemphylium sp. MT084051, Alternaria infectoria MT573465, Alternaria alternata MZ066724, Alternaria alternata MN615420 and Fusarium equiseti MK968015. It could be observed that A. alternata MN615420 extract was the most potent anti-inflammatory candidate with a significant downregulation of TNF-α. Moreover, six secondary metabolites, alternariol monomethyl ether (1), 3’-hydroxyalternariol monomethyl ether (2), alternariol (3), α-acetylorcinol (4), tenuazonic acid (5) and allo-tenuazonic acid (6) were isolated from the most potent candidate . Among the tested isolated compounds, 3’-hydroxyalternariol monomethyl ether showed the highest anti-inflammatory potential with the most considerable reductions in the level of INF-γ and IL-1β. Meanwhile, alternariol monomethyl ether was the most potent TNF-α inhibitor. The energy values for the protein –ligand interaction for the best conformation of the isolated compounds were estimated using molecular docking analysis.A total of 50 endophytic fungal isolates were separated from γ, IL-1β and INF-γ.The results obtained suggested alternariol derivatives may serve as naturally occurring potent anti-inflammatory candidates. This study opens new avenues for the design and development of innovative anti-inflammatory drugs that specifically target INF-The online version contains supplementary material available at 10.1186/s12906-023-03934-9. Physalis is a tropical plant, which belongs to family Solanaceae −.1HNMR : δH 2.68 , 3.79 , 6.76 , 6.99 , 7.28 , 12.51 . 13CNMR : δC 25.1 (6-CH3), 56.5 (5-OCH3), 100.1 (C-2), 100.3 (C-4), 104.8 (C-6), 111.4 (C-1), 117.5 (C-5), 128.5 (C-6), 131.5 (C-3), 138.4 (C-1), 142.2 (C-2’), 146.5 (C-4), 165.0 (C-3), 165.2 (C-7), 167.1 (C-5).Yellow crystals, ESIMS negative m/z 287.2 [M – H]−. 1HNMR : δH 2.71 , 6.38 , 6.65 , 6.72 , 7.26 , 11.70 . 13CNMR : δC 25.7 (6’-CH3), 97.8 (C-2), 101.3 (C-4), 102.1 (C-3′), 104.8 (C-6), 109.4 (C-1′), 118.0 (C-5′), 138.5 (C-1), 138.8 (C-6’), 153.1 (C-2′), 158.8 (C-4′), 164.5 (C-3), 165.1 (C-7), 165.9 (C-5).Colorless crystals, ESIMS negative m/z 257.0 [M – H]+. 1HNMR : δH 2.07 , 3.45 , 6.04 , 6.09 . 13CNMR : δC 29.6 (C-9), 50.5 (C-7), 101.2 (C-4), 108.2 , 136.9 (C-1), 158.8 , 206.6 (C-8).Yellow crystals, ESIMS positive m/z 167.3 [M + H]−.1HNMR : δH 0.81, 0.82 , 1.16 , 1.35–1.42 , 1.90 , 2.35 , 3.61 , 3.78 . 13CNMR : δC 11.6 (C-10), 15.2 (C-11), 22.8 (C-9), 25.6 (C-7), 36.4 (C-8), 36.8 (C-8), 63.7 (C-5), 65.5 (C-5), 102.5 (C-3), 177.5 (C-2), 177.8 (C-2), 193.6 (C-6), 193.7 (C-6).Viscous, yellow oil, ESIMS negative m/z 196.1 [M – H]oC) for 10 min., then decantation of supernatant was performed and the tubes were permitted to drain. In 10 mL cold phosphate buffer saline , the pellets (WBCs) were suspended Followed by recentrifugation. The pellets were then resuspended using RPMI culture medium, which contained 2% L-glutamine and 10% fetal bovine. Then, dye exclusion method for assessment of WBCs viability and counting was utilized. Mixing of 50 µL cell suspension with the same volume of trypan blue staining solution (0.5%) was performed followed by hemocytometer loading. In each of the four corner quadrants , viable and nonviable cells were counted [Collection of a whole blood specimen was performed using a sterile heparin tube, then an aliquot (1 mL) of blood was transferred into a centrifuge tube (15 mL), which filled to capacity with fresh cold lysing solution. The tube was then inverted at room temperature for ~ 10 min. till the liquid turned into clear red. Centrifugation of specimens was done at 2000 rpm (4  counted .N / ml = Mean of WBCs counting x104× DN: Number of viable or nonviable cells.D: Sample dilution (1:1 with the trypan blue).2 incubator for six days. Seeding of WBCs was performed  in a 96 well cell culture plate followed by incubation in CO2 incubator (5% CO2 and 90% relative humidity at 37 °C).To use the cells for assays, at least 90% of them must be viable after incubating the culture in COAll experiments were performed in accordance with relevant guidelines and regulations.2 incubator for 72 h . Then MTT solution was added to each well after 72 h of incubation. 20 µL volume of plates were incubated in CO2 incubator for 3 h to enable the MTT to be reacted. Following incubation, the plates were centrifuged for 10 min. at 1650 rpm, then the medium was discarded. MTT byproduct, formazan crystals, was re-suspended using DMSO (100 µL) and then measurement of readings was performed using optima spectrophotometer at wavelength 570 nm for safe dose determination that cause 100% cell viability [200 µL of cultured medium ) were seeded with variable concentrations of the studied extracts/ isolated compounds  in RPMI medium without both fetal bovine serum and piroxicam (the standard anti-inflammatory drug) then incubation was performed in COiability .AT = Mean absorbances of cells treated with different concentration of each studied extract or isolated compound.AC = Mean absorbances of the control untreated cells (only culture medium).Ab = Mean absorbances of cells treated with vehicle of the studied extracts or isolated compounds .Using the percent viability derived from each studied extract/ isolated compound serial dilutions, the cytotoxicity assay of the drug was expressed as EC100 that calculated using the Graphpad Instat software .2 incubator. After 24 h, centrifugation of the plate (1650 rpm) was done for 5 min., afterward the supernatants were discarded. After that, 200 µL of crude extracts, isolated compounds or piroxicam were added using serial concentrations . Only cell culture medium was present in the control cells. Plates incubation occurred for extra 72 h in CO2 incubator. Following incubation, cell proliferations were assessed using MTT (as previously mentioned). Cell proliferations assessment was done using stimulation index (SI) [50 µL culture medium, which contained 100,000 of human WBCs, was dispensed per well in a 96 well plate. Inflammation induction was performed by adding 50 µL of LPS (20 µg/mL) to the plated cells followed by incubation in COdex (SI) .Stimulation index = (mean absorbance of LPS-stimulated cells or LPS-stimulated cells treated with different concentrations of the studied extracts or isolated compounds / absorbance of control untreated cells).Instate graph pad was used to determine the effective anti-inflammatory concentrations (EAICs) of the studied extracts and isolated compounds that could restore the aberrant proliferation of LPS-stimulated cells to the normal proliferation of control, untreated cells (SI = 1).Suspension of cell pellets was then made in solution R1 (50 µL), then it was mixed thoroughly for 30 s and incubated at room temperature (1 min.). 300 µL solution R2 was added, mixed thoroughly for 30 s and then centrifuged for 3–5 min. at 4ºC. In a spin column, the supernatant was transferred followed by centrifugation for 30 s at 14,000 rpm (4ºC). After removing the flow-through, working wash buffer (300 µL) was added into the spin column followed by centrifugation for 30 s (repeated twice). Recentrifugation of the spin column was performed at 10,000 rpm (1 min.) and then transferred to a sterile micro centrifuge tube (1.5 mL). Elution buffer (30 µL) was added to the central of the membrane, then incubated at room temperature (1 min.) and then centrifuged for 30 s at 14,000 rpm at 4ºC. Determination of the optical density (OD) of the extracted RNA was performed through measuring the absorbance (A260 nm) and purity (A280 nm) using spectrophotometer then kept in -80 °C till real time PCR .Total RNA or nuclease-free water (2 µg) in addition to oligo dT primer (1 µL) were mixed gently with nuclease-free water in PCR tubes in a total volume of 12 µL, centrifuged, incubated at 65ºC (5 min.) using PCR machine and then put back on ice instantly. 5X reaction buffer (4 µL), dNTPs mix (2 µL), RNase inhibitor (1 µL) and reverse transcriptase (1 µL) or nuclease-free water (1 µL) in place of reverse transcriptase for reverse transcriptase negative control were mixed gently with prior mixture, spin down and then incubated at 42ºC (60 min.) followed by heat inactivation at 70ºC (5 min.) in PCR machine .β-actin and 0.5 µL of 10 picomoles/mL for reverse primer of β-actin were added. The other tube was used as a non-template control (NTC), to assess for reagent contamination or primer dimers by adding 1 µL of nuclease-free water instead of template used. The tubes were gently mixed with 6.5 µL nuclease free water without creating bubbles (bubbles will interfere with the fluorescence detection) and spinned for few seconds. Samples were placed in the cycler and start the program as follows; 1 cycle of 95ºC for 10 min. , followed by 40 cycles of 95ºC for 15 s (denaturation), 60ºC for 30 s  and 72ºC for 30 s (extension) [In PCR tubes, a volume of 12.5 µL of 2 X SYBR green master mix was mixed with 5µL of cDNA 0.5 µL of 10 pico moles/mL forward primer and 0.5 µL of 10 pico moles/mL reverse primer for each primer. As for the reference tube, 0.5 µL of 10 pico moles/mL forward primer of tension) .Expressions fold levels of gene calculated by:ΔCt normal = Ct normal untreated cells – Ct reference.ΔCt tested sample = Ct tested sample-treated cells – Ct reference.ΔCt induced =- Ct LPS-exposed cells – Ct reference.In case of genes:ΔΔCT tested sample = ΔCt tested sample– ΔCt normal.ΔΔCT induced = ΔCt induced – ΔCt normal.In case of GAPDH:tested plant extract = ΔCt normal – ΔCt tested plant extract.ΔΔCTinduced = ΔCt normal – ΔCt induced.ΔΔCTFold change in gene expression = log (2−ΔΔCT)Where:Ct tested sample: Threshold cycle value of genes of extracted mRNA of the studied extracts/isolated compounds treated-LPS-stimulated WBCs which is the cycle number at which fluorescence produced during a reaction crosses the fluorescence threshold.Ct reference: Threshold cycle value of GAPDH which is utilized in normalization.Ct normal: Threshold cycle value of genes of extracted mRNA of untreated control WBCs.Ct induced: Threshold cycle value of gene of extracted mRNA of LPS-stimulated WBCs.The Schrödinger Maestro 11.8 package  was employed in order to predict the binding mode of the most potent bioactive compounds .https://pubchem.ncbi.nlm.nih.gov/) of the National Centre for Biotechnology Information in sdf file format. ChemDraw software  was used to draw the structures that weren’t included in PubChem database after searching through various literature. The chemical structure of each substance was imported into the Maestro 11.8 panel interface  once the dataset was generated. The 3D structure was created using the LigPrep 2.3 module , which was also used to search for various conformers. To geometrically optimize each ligand structure and compute partial atomic charges, the OPLS  force field was used.The two-dimensional structures of the isolated compounds along with the reference drug; piroxicam  were obtained from PubChem database (β (PDB ID 4DEP) [α (PDB ID 2AZ5) [γ (PDB ID 6E3K) [The crystal structures of human IL-1ID 4DEP) , TNF-α (ID 2AZ5)  and INF-ID 6E3K)  were obtoA around the centroid of the co-crystallized ligand and a grid spacing of approximately 0.375 oA was used to define the active site for docking on the prepared X-ray crystal structure of the targeted proteins. The targeted proteins crystal structure was subjected to binding interaction examination using this grid box size with the isolated compounds along with the reference drug.A grid box with dimensions of 15 × 15 × 15 The Glide 11.8 module  was used to perform flexible docking on the minimized and refined compounds while utilizing the Glide default parameters. The Glide-Dock program’s empirical scoring functions was used to generate modeling scores. In order to investigate the most favorable binding modes of ligands, the Maestro interface, which displayed the 2D and 3D ligand-target protein interactions  was employed.oA . The re-docked complexes were superimposed onto the reference co-crystallized complexes with low root mean square deviation (RMSD) values .A pose selection approach was used to re-dock piroxicam ligand into the various binding sites of 4DEP, 2AZ5 and 6E3K in order to successfully validate the docking protocol. This was followed by estimating the root mean square deviation (RMSD) of the anticipated pose to the co-crystallized one piroxicam. The results showed high docking accuracy because they were less than 1 Each experiment was carried out in triplicate. All biological data were statistically analyzed using one-way analysis of variance (ANOVA), with p values less than 0.05 being regarded as significant. A neighbour-joining phylogenetic trees were constructed using MEGA X. Graphpad Instat  was used to calculate EC100 values and construct the dose response curves.P. pruinosa. Selection of six representative isolates was performed for further bioactivity screening based on their morphological characters , Alternaria infectoria MT573465 , Alternaria alternata MZ066724 , Stemphylium sp. MT084051 , Fusarium equiseti MK968015  and Alternaria alternata MN615420 . The isolated endophytic fungi were represented in the phylogenetic trees as shown in Figs. A total of 50 endophytic fungal isolates were separated from leaves of ers Fig. . IdentifP. pruinosa. Among the identified isolates, ethyl acetate extract prepared from A. alternata MN615420 strain exhibited the greatest anti-inflammatory activity. The obtained results suggested that A. alternata MN615420 strain holds bioactive compounds with potential anti-inflammatory activity. Thus, guided isolation of the bioactive compounds from A. alternata MN615420 strain was employed.Ethnopharmacologically important medicinal plants are considered as reliable sources for the isolation of endophytes which produce secondary metabolites with unique and therapeutically relevant bioactivities. Endophytes have already been investigated in a variety of plants, but this is the first report of the anti-inflammatory potential of the endophytic fungi associated with α-pyrone derivatives (1–3), one resorcinol derivative (4) and two tetramic acid derivatives (5 and 6) were isolated from A. alternata MN615420 ethyl acetate extract  [2) [3) [α-acetylorcinol (4) [5) and allo-tenuazonic acid (6) [Six secondary metabolites, including three dibenzo-act Fig. . The met ’ 3’-hydr. 1) [2) , alterna [2) [3) , α-acetyinol (4)  in additacid (6) , which wβ), which upregulates tumor necrosis factor-alpha (TNF-α) release that included in various inflammatory disorders. The etiology of autoimmune and chronic inflammatory illnesses has been linked to an increased release of gamma interferon (INF-γ). Using the MTT test, safety of the tested extracts and isolated compounds was evaluated and EC100 values that result in 100% cell viability were computed for the tested extracts  followed by extracts prepared from A. alternata MZ066724, A. infectoria MT573465, F. equiseti MK968015, Stemphylium sp. MT084051 and S. simmonsii MN401378 as they exhibited EC100 values of 20.38 ± 0.98 µg/mL, 18.99 ± 1.02 µg/mL, 9.76 ± 1.65 µg/mL, 7.42 ± 0.89 µg/mL and 5.97 ± 0.58 µg/mL, respectively. According to these findings, Alternaria species had a superior safety profile. Among the isolated compounds, compound 1  showed the highest EC100 value (77.0 ± 6.0 µM) followed by compound 3 . While the inseparable mixture (tenuazonic acid (5) and allo-tenuazonic acid (6)) was the most toxic as it possessed the lowest EC100 value of 8.0 ± 0.1 µM. This information came in accordance with a previous study which considered tenuazonic acid as the most toxic among Alternaria mycotoxins [It is well known that inflammatory triggers, including immune stimulatory lipopolysaccharides, cause macrophages to release a variety of pro-inflammatory mediators. The activated macrophages produce the pro-inflammatory cytokine interleukin-1beta  of the studied extracts and isolated compound was then estimated, which defined as the concentrations that could restore the aberrant proliferation of LPS-stimulated cells to the normal proliferation of control. The EAIC values of all extracts were evaluated and were all found to be lower than that of the positive control piroxicam (p < 0.05), thus indicating their efficacy. As concluded from Table α, IL-1β, INF-γ) was examined in order to determine the mechanism of anti-inflammatory activity of tested extracts and isolated compounds by real time polymerase chain reaction (PCR) in normal WBCs and the treated WBCs with lipopolysaccharide (LPS). The cell wall of gram-negative bacteria contains proteins known as lipopolysaccharides (LPS), which break down into O-antigen, lipid-A and core protein. Lipid-A is known to have strong immunogenic and pro-inflammatory effects. Pro-inflammatory cytokines such as TNF-α, IL-1β and INF-γ play crucial roles in immune system and inflammation process like vasodilatation and edema. In addition to their key role in chronic inflammation [A. alternata MN615420 extract was the most potent inhibitor of TNF-α with reducing the concentration to 0.20 ± 0.004 µg/mL. While A. infectoria MT573465 extract was the most potent INF-γ inhibitor with a reduction of its concentration to 0.15 ± 0.009 µg/mL. Stemphylium sp. MT084051 extract significantly reduced the elevated level of IL-1β to a value of 0.11 ± 0.007 µg/mL; compared to the value obtained with the positive control piroxicam, this value was significantly lower . Among the tested isolated compounds (Table γ (0.20 ± 0.07 µg/mL) and IL-1β (0.18 ± 0.004 µg/mL). While compound 1  was the most potent TNF-α inhibitor with a reduction of its concentration to 0.25 ± 0.02 µg/mL.Gene expression of the three pro-inflammatory indicators –ligand interaction for the best conformation of piroxicam (the standard anti-inflammatory drug) and the isolated compounds were estimated . A natural antagonist, the protein IL-1ra, binds to IL-1RI and prevents IL-1 from interacting with the receptor. IL-1β has a superior importance owing to its vital role in various immune responses in the body [2  followed by compound 3   was stabilized by a number of polar interactions with the following residues: Asn133, Asn135 and Asn136. All these interactions contributed significantly to the superior stability of compound 2  as a ligand. The docking scores and the experimental outcomes showed a very good match. As compound 2  showed the highest anti-inflammatory potential with the most considerable reduction in the level of IL-1β.Interleukine-1 is considered one of the most important families of cytokines, which consists of 11 members. This family is known as endogenous pyrogens and is implicated in pro-inflammatory reactions. The members of this family bind to a class of receptor known as IL-1R. There are two receptors for IL-1, IL-1 type 1 receptor (IL-1RI) and IL-1 type 2 receptor (IL-1RII). The two most significant IL-1 cytokine members are IL-1the body . In the e 2 Fig.  revealedα is a pro-inflammatory cytokine with a key role in apoptosis, inflammation, proliferation, differentiation and immune regulation. TNF-α plays a significant part in the inflammatory process, hence therapeutic research on drugs that block its action has gained a lot of attention recently [α with docking scores − 7.64 kcal/mol for compound 1 , − 6.41 kcal/mol for compound 3  and − 6.27 kcal/mol for compound 2  as illustrated in Table α. Thus, suggested that alternariol derivatives could be considered as direct inhibitors of the target protein as verified by the experimental results.TNF-recently . The isoγ is a well-known proinflammatory cytokine with a prominent function in inflammation and autoimmune diseases. The three isolated alternariol derivatives (compounds 1–3) were more active as compared to the reference anti-inflammatory drug, piroxicam  with compound 2  being the most active  as shown in Table 2 revealed four hydrogen bond contacts between the phenolic groups in ring A and PheA81, LysA43 and LysA88. Also, the ketonic group established hydrogen bond interaction with the side chain of LysA88 , one resorcinol derivative (4) and two tetramic acid derivatives (5 and 6) were isolated from A. alternata MN615420 ethyl acetate extract. It could be observed that A. alternata MN615420 extract was the most potent anti- inflammatory candidate with the most significant downregulation of TNF-α level. While A. infectoria MT573465 extract was the most potent INF-γ inhibitor. Moreover, Stemphylium sp. MT084051 extract significantly reduced the elevated level of IL-1β. Among the tested isolated compounds, compound 2  showed the highest anti-inflammatory potential with the most considerable reductions in the levels of INF-γ and IL-1β. While compound 1  was the most potent TNF-α inhibitor. The present study included a molecular docking analysis to investigate the conformational space of the ligand within the protein-binding site and predict the complex structure created by ligand-protein binding. The results obtained suggested alternariol derivatives as naturally occurring potent anti-inflammatory candidates. However, further extensive in vivo studies are required to verify the obtained results.In this work, isolation for the first time some endophytic fungi from the medicinal plant Below is the link to the electronic supplementary material.Additional file 1.


N-(5-Acetyl-4-methylpyrimidin-2-yl)benzenesulfonamide was synthesized and structurally characterized. In the crystal, π–π interactions between the phenyl and pyrimidine groups of neighbouring molecules form molecular chains parallel to [010]. Adjacent chains are linked by N—H⋯N hydrogen-bonding interactions, resulting in a three-dimensional network. N-(5-Acetyl-4-methylpyrimidin-2-yl)benzenesulfonamide, C13H13N3O3S, was sythesized and characterized by single-crystal X-ray diffraction. In the crystal, π–π interactions between the phenyl and pyrimidine groups of neighbouring molecules form molecular chains parallel to [010]. Adjacent molecular chains are linked by N—H⋯N hydrogen-bonding interactions between the pyrimidine and amine groups of neighbouring molecules, resulting in a three-dimensional network. The asymmetric unit is shown in Fig. 1ar—C bond. Apart from a slight twist of the aldehyde group associated with the disorder, the 1-(2-amino-4-methylpyrimidin-5-yl)ethan-1-one segment of the molecule is essentially planar, the sulfonamide atom S1 being located only 0.423 (1) Å away from the plane of the pyrimidine group. The molecule exhibits a C7—N1—S1—C1 torsion angle of −79.0 (2)°, while the twist between the planes of the phenyl group and the pyrimidine ring comprises a dihedral angle of 63.07 (7)°.3.1 symmetry . The dihedral angle between the planes of the rings is 9.04 (10)° with a ring centroid-to-centroid distance of 3.769 (1) Å  and 1.58 Å . These π–π interactions form chains in the structure in which one AMBS molecule comprises the linker between two further molecules. The bent nature of the molecule results in a zigzag pattern of chains propagating parallel to [010].The packing of AMBS is shown in Fig. 2 Å Fig. 3. The slix, 1 − y, 1 − z). A pair of hydrogen bonds is formed between the pyrimidine and amine groups of the two molecules, resulting in a ca 2.7–3.4 Å help to consolidate the structure.The hydrogen-bonding interactions in the crystal are summarized in Table 1ry Fig. 2. The hyd4.et al., 2016CONQUEST benzenesulfonamide group gave 164 hits, i.e. too many for them all to be analysed in detail.A survey of the Cambridge Structural Database amino]pyrimidine (TPAP) benzenesulfonamide (PBS)  was added to a mixture of phenylsulfonyl guanidine (0.05 mol) and acetyl acetone (0.1 mol). The reaction mixture was then refluxed for 6 h. After cooling, the resulting precipitate was filtered and crystallized from ethanol.1H NMR : δ 2.49 , 2.52 , 7.57–7.66 , 8.00–8.02 , 8.93 , 12.34 . Analysis calculated for C13H13N3O3S (291.33): C, 53.60; H, 4.50; N, 14.42; S, 11.01. Found: C, 53.60; H, 4.49; N, 14.41; S, 11.00.Orange crystals; yield 45%; m.p. 469 K. 6.Uiso(H) = 1.2 or 1.5 Ueq(C). The acetaldehyde group of the molecule is disordered with two components related by a twist of 31.3 (1)° about the Car—C bond. In the refinement, the two components were restrained to have similar geometry (SAME in SHELXL) and atomic displacement parameters (SIMU and ISOR). The occupancies of the two components refined to 0.591 (11)/0.409 (11).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989023001871/yz2029sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023001871/yz2029Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023001871/yz2029Isup3.cmlSupporting information file. DOI: 2245275CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structures of four metal 3,5-lutidine pentaaqua sulfates  are presented and are shown to be isostructural. 4·H2O, CoSO4·7H2O, NiSO4·6H2O and ZnSO4·7H2O with 3,5-lutidine  yield crystals of pentaaquamanganese(II) sulfate, [Mn(C7H9N)(H2O)5]SO4, (1), pentaaquacobalt(II) sulfate, [Co(C7H9N)(H2O)5]SO4, (2), pentaaquanickel(II) sulfate, [Ni(C7H9N)(H2O)5]SO4, (3), and pentaaquazinc(II) sulfate, [Zn(C7H9N)(H2O)5]SO4, (4), which were characterized by single-crystal X-ray diffraction. The four crystals are isostructural, demonstrating near identical unit-cell parameters and atomic positions. The metal atoms are all octahedrally coordinated, with one lutidine ligand and five water ligands. The sulfate dianion hydrogen bonds with the coordinated water molecules of the dicationic metal complex salts, generating infinite three-dimensional networks.The reactions of MnSO The M—N distances (Table 1ii) with two metal-bound waters hydrogen bonding to each oxygen atom. There is also one slightly longer S—O bond (S1—O6) with four metal-bound waters hydrogen bonding to the oxygen. All S—O distances are listed in Table 1The s Table 1 observeds Table 1. The ful3.The ions in all of the compounds described are connected in an extended 3D network through hydrogen bonding. The major hydrogen bonds are between the metal–aqua complexes and the sulfate dianions Tables 2 ▸ ▸. T4.et al., 2020et al., 2010et al., 1991et al., 2011et al., 2012While there are many examples of metal–pyridine pentahydrate complexes, there is only one pyridine-based pentahydrate complex of a transition metal with a sulfate counter-ion, which is the dimer of zinc bridged by 1,2-bis(pyridin-3-ylmethylene)hydrazine  was dissolved in five drops of water and 2.5 mL of 3,5-lutidine. The resulting solution was heated to 338–343 K for twelve hours and allowed to cool slowly to room temperature producing single crystals suitable for X-ray diffraction. The manganese crystals formed as colorless blocks, the cobalt crystals formed as pink blocks, the nickel crystals formed as pale-green plates, and the zinc crystals formed as colorless blocks.A metal sulfate  Å. Isotopic displacement parameters were set to 1.5 Ueq of the parent oxygen atom. All other hydrogen atoms were placed in calculated positions . Isotropic displacement parameters were set to 1.2 Ueq of the parent aromatic carbon atoms and 1.5 Ueq of the parent methyl atoms.Crystal data, data collection and structure refinement details are summarized in Table 710.1107/S2056989023005261/zv2026sup1.cifCrystal structure: contains datablock(s) 1, 2, 3, 4. DOI: 10.1107/S2056989023005261/zv20261sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989023005261/zv20262sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989023005261/zv20263sup4.hklStructure factors: contains datablock(s) 3. DOI: 10.1107/S2056989023005261/zv20264sup5.hklStructure factors: contains datablock(s) 4. DOI: 2269315, 2269314, 2269313, 2269312CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


However, the role of three-dimensional (3D) chromatin structure in orchestrating changes in transcriptional regulation during direct cell reprogramming is not fully understood.Long-range interactions between promoters and Here, we performed integrated analyses of chromosomal architecture, epigenetics, and gene expression using Hi-C, promoter Capture Hi-C (PCHi-C), ChIP-seq, and RNA-seq during trans-differentiation of Pre-B cells into macrophages with a β-estradiol inducible C/EBPαER transgene. Within 1h of β-estradiol induction, C/EBPα translocated from the cytoplasm to the nucleus, binding to thousands of promoters and putative regulatory elements, resulting in the downregulation of Pre-B cell-specific genes and induction of macrophage-specific genes. Hi-C results were remarkably consistent throughout trans-differentiation, revealing only a small number of TAD boundary location changes, and A/B compartment switches despite significant changes in the expression of thousands of genes. PCHi-C revealed widespread changes in promoter-anchored loops with decreased interactions in parallel with decreased gene expression, and new and increased promoter-anchored interactions in parallel with increased expression of macrophage-specific genes.Overall, our data demonstrate that C/EBPα-induced trans-differentiation involves few changes in genome architecture at the level of TADs and A/B compartments, in contrast with widespread reorganization of thousands of promoter-anchored loops in association with changes in gene expression and cell identity.The online version contains supplementary material available at 10.1186/s12915-023-01766-0. Cis-regulatory elements such as enhancers and promoters of genes they control can be separated by large genomic distances [istances . Howeveristances . Gene existances , 3. DeciGenome-wide contact frequencies determined by Hi-C have revealed higher-order chromatin structural features. Topologically associated domains (TADs) are large megabase-size genomic regions of increased self-interaction that are partially insulated from contacts with neighboring domains , 5. VariAnalysis of Hi-C heatmaps has revealed a “plaid pattern” of increased or decreased contact frequencies between domains which corresponds with their activity state. Active domains tend to contact other active domains, while inactive domains tend to contact other inactive domains, indicating that the nuclear genome is organized into two nuclear compartments referred to as A (active compartment) and B (inactive compartment) . Here agHi-C has been instrumental in discovering and defining the principles of the above aspects of higher-order genome organization. However in the absence of billions of read-pairs, Hi-C lacks the resolution to identify significant interactions between individual promoters and their long-range regulatory elements. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between individual promoters and their long-range regulatory elements in an unbiased manner –21. PCHiPre-B cells are precursors of B lymphocytes, which are adaptive immune cells of the lymphoid lineage , 28. MacIn this study, we used ChIP-seq, RNA-seq, Hi-C, and PCHi-C data to investigate higher-order chromatin organization and promoter interaction dynamics associated with gene expression changes during C/EBPαER-induced trans-differentiation of mouse Pre-B cells into macrophages over a 48-h period , 37. OurTo study the role of promoter-enhancer interactions during Pre-B cell trans-differentiation into macrophages, we employed direct lineage conversion of the C10 mouse Pre-B cell line which harbors an estradiol-inducible rat C/EBPα fused to the estrogen hormone receptor binding domain (C/EBPαER) , 38. UpoH3f3a and H3f3b) genes , macrophage scavenger receptor (Msr1), myeloid restricted serine protease C (Ctsc), and the myeloid cytokine-dependent chemokine 6 (Ccl6) were upregulated,  levels increased in the whole cell extracts through Pre-B trans-differentiation Fig. b. To ananes Fig. d. H3.3 ines Fig. d, consisnes Fig. e. The encis-contacts greater than 10Kb, approximately 15% cis-contacts < 10 Kb, and ~ 14% trans contacts, indicating very high-quality Hi-C libraries . GO analysis of coding genes (n = 6937) within TADs that show boundary changes shows enrichment for immune processes, such as response to chemokine, response to dsRNA, and response to pheromone . PC1 value of PCA shows a higher correlation between biological replicates than between different time points  libraries at each time point. PCHi-C data quality was checked using HiCUP analysis  did not change compartment status over the 48h period. A recent study that used C/EBPα to trans-differentiate a human B cell line to macrophage also shows conservation of TAD boundaries, and observed only a small percentage of dynamic compartments associated with gene expression changes . It is iOur results suggest that the rewiring of promoter-anchored interactions plays a major role in controlling differential gene expression and cell fate transition compared to the relatively minor role played by changes in higher-order organization of TADs and A/B compartments. We show that PIRs are significantly enriched in histone modifications associated with gene regulatory activity. Genes whose promoters are contacted by PIRs with the active enhancer mark, H3K27ac, are highly expressed, while promoters contacted by the repressive H3K27me3 modification are not. Thus, transcription factor-mediated differential loop formation between promoters and their distal regulatory elements is the major driver of the cell-type-specific, promoter interactomes  and is tOur integrated analyses of gene expression, histone modifications, transcription factor binding, and various levels of three-dimensional chromosome conformation during trans-differentiation have revealed that widespread changes in gene expression that characterize the transition from a Pre-B cell to a macrophage phenotype involve surprisingly few changes in TAD structure or A/B compartment status. Instead, we found reorganization of thousands of promoter-anchored loops, in conjunction with transcription factor binding and histone modification dynamics, occurring at thousands of differentially expressed genes suggesting that changes in chromatin interactions at the sub-TAD level are a major mechanism of differential regulation of gene expression that drives cell state transitions.6 C10 cells were plated in 10-cm cell culture dishes, followed by adding100 nM of β-estradiol , 10 nM IL-3 , and CSF-1 . The cell culture medium was replenished every 24h. The Pre-B cell trans-differentiation efficiency was measured by flow cytometry analysis and qPCR gene expression analysis of Pre-B cell-specific genes and macrophage-specific genes. For flow cytometry analysis, C10 cells at various differentiation stages were stained using PE anti-mouse/human CD11b  antibody and APC anti-mouse CD19 antibody . The percentage of Pre-B cells and macrophage population was analyzed on FACSCanto II analyzer (BD Biosciences). Flow cytometry data was analyzed by FlowJo  software. PE Rat IgG2b  and APC Rat IgG2a  antibodies were used as isotype controls to exclude the background fluorescence.C10 cells were cultured in RPMI 1640  medium supplemented with 10% heat-inactivated FBS , 1% Penicillin–Streptomycin , and 50 μM 2-Mercaptoethanol . For C10 cells trans-differentiating into macrophages, 5.0 × 10For western blots, cells were lysed by CelLytic buffer . The protein concentration was quantified by the Micro-BCA assay reagent . A 10 μg protein from each sample was loaded into 4–12% SDS–polyacrylamide  for electrophoresis. The following antibodies were used for Western blot experiments: Anti-C/EBPα , Anti-H3 , Anti-β-Tubulin . For immunofluorescence, C10 cells were first fixed in 4% PFA for 5 min, washed using cold PBS twice, followed by attaching cells to fibronectin and pre-coating the glass slides. Next, the cells were treated using blocking buffer  for an hour at room temperature, followed by primary antibody treatment overnight at 4 °C and secondary antibody treatment at room temperature for an hour. The primary and secondary antibodies were diluted in antibody dilution buffer (1% BSA and 0.3% Triton X-100 in 1X PBS), and DAPI  was used for nuclear staining. Anti-C/EBPα  and Goat anti-Mouse IgG (H + L) Alexa Fluor 488  antibodies were used for immunofluorescence staining. All the immunofluorescence images were taken by LSM 710 confocal microscope (Zeiss) and processed using Zen black (Zeiss).Gapdh using the 2−ΔΔCT method.RNA was prepared from C10 cells at various trans-differentiation stages using RNeasy mini kit  followed by on-column DNase I treatment. Total RNA (1 μg) was used for reverse transcription to generate cDNA by using iScript™ Reverse Transcription Supermix for RT-qPCR , followed by the real-time PCR amplification using Power SYBR Green PCR Master Mix . The real-time PCR was performed with QuantStudio 7 Flex Real-Time PCR System (Thermofisher SCIENTIFIC) for 40 cycles, 95°C for 15 sec, and 60°C for 1 min. Gene expression was normalized to the housekeeping gene For RNA-seq libraries preparation, 1μg of total RNA (RIN > 7) was used for rRNA depletion by using NEB rRNA Depletion Kit (E6310). Next, the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (E7770S) and NEBNext NEBNext® Multiplex Oligos for Illumina® (E7335) were used for library construction. RNA-seq library concentration was quantified by using the TapeStation and KAPA Library QANT Kit , followed by single-end (1 × 100bp) sequencing on the Novaseq S2 flow cell. The sequencing depth is described in Additional file  g for 5 min at 4°C. Next, the cells were lysed in 20 mL cold lysis buffer  on ice for 30 min, followed by centrifuging at 760 g for 5 min to remove the supernatant. The pellet was washed in 1.25X NEB buffer2  and resuspended in 358 μL 1.25X NEB buffer2, followed by adding 11 μL of 10% SDS and incubating at 37 °C with shaking at 950 rpm for 30 min. Lastly, the SDS was quenched by adding 75μL of 10% Triton X-100 and incubating at 37 °C for 15 min with shaking. To digest chromatin using HindIII restriction enzyme, we added 12 μL of 100 U/μL HindIII  to each reaction, followed by shaking at 37 °C and 950 rpm overnight, followed by adding 5 μL of 100 U/μL HindIII  per reaction at 37 °C for 2 h on the following morning. To repair the digested overhangs, we added 6.1 μL 10X NEB buffer2, 25 μL molecular-grade water, 15.3 μL of 1 mM biotin-14-dATP , 1.56 μL of 10 mM dCTP, 1.56 μL of 10 mM dGTP, 1.56 μL of 10 mM dTTP, and 10.2 μL of 5U/μL DNA polymerase I, Large (Klenow) Fragment  and incubated the reaction at 37 °C for 1 h. In-nucleus ligation mix was prepared by adding 102μL 10X T4 DNA ligase buffer, 10.2 μL BSA , 350.9 μL molecular-grade water, and 25.5 μL 1U/ μL T4 DNA ligase , followed by incubation at 16 °C for 4 h in a thermomixer (shaking at 700 rpm for 5 s in every 2 min). Next, the nuclei were pelleted at 2500 g for 5 min and resuspended in 300μL cross-link reversal buffer , followed by adding 5 μL 10 mg/ml RNase A  at 37 °C for 30 min and 20 μL of 20 mg/ml Proteinase K  at 55 °C for 1 h and 68 °C overnight. The genomic DNA was extracted using cold ethanol and sodium acetate (pH 5.2) and resuspended in 130μL of 10 mM Tris–HCl. DNA was transferred to a microTUBE AFA fiber Pre-Slit Snap-Cap  and fragmented using a ME220 Covaris sonicator . The sonicated DNA was transferred to a 1.5-ml tube, and the total volume was brought to 200 μL by adding 70 μL of molecular-grade water. DNA size was double size selected using AMPure XP beads . First, 120 μL of beads were added to each reaction (ratio of AMPure beads to DNA: 0.6 to 1) and incubated for 5 min. The clear supernatant was transferred to a fresh tube through a magnetic separation stand. Next, 30 μl of fresh AMPure XP beads were added to the clear supernatant and incubated for 5 min at room temperature. Beads were separated on a magnetic separation stand and washed twice using 800 μL of 70% ethanol and dried at 37 °C, followed by eluting DNA in 300 μL 1X Tris buffer. DNA was used for library preparation with a normal size distribution between 300 and 500 bp and was visualized by TapeStation.Hi-C and Promoter Capture Hi-C (PCHi-C) library preparation was described in our previous publication with minor modifications . Ten milProcedures for Biotin/Streptavidin pull-down of the Hi-C ligation products, end repair, removal of biotin at the non-ligated DNA ends, adaptors ligation, Hi-C libraries amplification, and Hi-C size selection were described in our recent publication . The Hi-https://github.com/FelixKrueger/TrimGalore). FastQC program was used to check the quality of the data (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimmed reads were mapped to mouse reference genome (mm10) using Hisat2 (2.1.0) with default parameters [RNA-seq libraries were prepared for each time point. We included three biological and four technical replicates for each time point. Technical replicates were merged, and Trim Galore (0.6.2) pipeline was used to remove adapters and short reads from the data . JuiceBox was used to visualize Hi-C matrix heatmap and TAD structure [Hi-CFindTADs –correctForMultipleTesting fdr –thresholdComparisons 0.01 –delta 0.01) [Hi-C data were processed using HiCUP (v0.7.2) pipeline  with mm1tructure . Homer ptructure . The cortructure . Hi-C fitructure . TADs anta 0.01) .https://epigenomegateway.wustl.edu/) [peakEnrichemnt4Features function in CHICAGO) [PCHi-C mouse raw reads were processed by the HiCUP pipeline and were mapped to mm10 genome . Hi-CReptl.edu/) . To estiCHICAGO) .BEDTools (v2.26.0) “intersect” function was used to find PIR regions that overlap with H3K27ac peaks or H3K27me3 . To invefindPeaks function of HOMER package was used to call histone modification peaks by setting peak size 1000 bp and the remaining parameters were set to the default values [peakEnrichmen4Features [ p-value < 0.05 and fold change > 1.5 were used as threshold to identify induced C/EBPα binding sites. TSS of all genes were downloaded from Ensembl biomart (v100) [ChIP-seq data C/EBPα along with histone modification ChIP data were downloaded from GEO . Raw reat values . Peak cat values . To visut values . The enrFeatures . FurtherFeatures . p-valuet (v100) . Bedtoolt (v100) .t-test, Mann–Whitney U statistical test, and chi-squared test were performed using GraphPad Prism 8 and R. p-value < 0.05 was considered as significant unless specified and indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001.Unpaired Additional file 1: Fig. S1. Transcriptional profile changes during Pre-B transdifferentiation. a Principal component analysis (PCA) RNAseq of each replicate from different time points. b SNPsplit identifies the percentage of total RNA reads that align to Cebpa gene region that contain Mus musculus specific SNP (mouse specific), Rat specific SNP (Rat specific), regions that do not contain any SNP (Unassignable), conflicting SNP information (Conflicting). c Volcano plots show upregulated and downregulated differentially expressed genes (DEGs). d GO ontology 5 Fuzzy c-means clusters of DEGs e qPCR analysis of Pre-B cell and macrophage marker gene expression during Pre-B cell trans-differentiation. f Gene expression level (TPM) of Pre-B cell-specific genes , and macrophage-specific genes  during Pre-B trans-differentiation. Fig. S2. Hi-C data analysis. aHiCUP pipeline analysis HiC and PCHi-C data, including the following three categories: Cis < 10Kbp, Cis > 10Kbp, and trans-interactions across four-time points. b Heatmaps show Hi-C matrix at 100kb and 25 kb resolution. Fig. S3. Altered TAD and A/B compartment during Pre-B transdifferentiation. a Number of TADs and size distribution at each time point. b GO enrichment analysis of genes changed TAD boundaries from 0h to 48h. c Pearson correlation coefficient of PC1 values (50kb resolution) of the entire genome. d Gene expression level (TPM) in the A and B compartments at four different time points. A two-sided unpaired t-test was performed for the significance test. ePercentage of compartment change for DEGs .  f Log2 fold change of gene expression in dynamic compartments. stable (n= 19157), A to B (n= 450), and B to A (n= 474). g Gene ontology analysis of 2105 genes at 48h that switch from A to B and 1552 genes at 48h that switch from B to A. A two-sided unpaired t-test was performed for the significance test. Fig. S4. PCHi-C data analysis. a HiCUP pipeline analysis of PCHi-C data: Cis <10Kbp, Cis >10Kbp, and trans-interactions across four-time points. b Proportion of promoter-promoter (P-P) and promoter-other interactions (P-O) interactions as deduced from PCHi-C. c Expression values (log2(TPM+1)) of genes/promoters classified into 4 groups (at least one PIR overlaps with histone markers). H3K27ac: interaction between gene/promoter and PIRs only with H3K27ac marks. 0h (n=4829); 1h (n=5710);12h (n=5257); 48h (5735). H3K27Ac+H3K27me3: interaction between gene/promoter and PIRs with both H3K27ac and H3K27me3 marks. 0h (n=8075); 1h (n=7044); 12h (n=7926); 48h (n= 7727). H3K27me3: interaction between gene/promoter and PIRs only with H3K27me3. 0h (n=2757);1h (n=2286); 12h (n= 2545); 48h (n=2165). No marks: interaction between gene/promoter and PIRs without any histone modification. 0h (n=3667), 1h (n=4805); 12h (n=4403); 48h (n= 4291). The box plot represents 25 and 75 percentiles with the median. An unpaired two-sided t test is performed for the significance test. d Boxplot shows gene expression values (log2(TPM +1)) of promoters that do not contact any PIRs with H3K27ac categorized into four groups according to the number of PIRs marked by H3K27me3 at four different time points.  0: Promoters without any PIRs marked by H3K27me3. 0h (n=2551), 1h (n=2991); 12h (n=2711); 48h (n=2682).  1: Promoters with only one PIR marked by H3K27me3 0h (n=1247), 1h (n=1100); 12h (n=1188); 48h (n= 955). 1-5: Promoters with one to five PIRs marked by H3K27me3. 0h (n=749), 1h (n=672); 12h (n=697); 48h (n=567).  >5: Promoters with more than five PIRs marked by H3K27me3. 0h (n=80), 1h (n=63); 12h (n=103); 48h (n=95).  An unpaired two-sided t-test is performed for the significance test. The box plot represents 25 and 75 percentiles with the median. Fig. S5. Integrated analysis of promoter interactions and epigenetic features. a Maf1 and Ssbp1 region that shows promoter interaction changes accompanied by H3K27me3(green), H3K27ac (yellow), and H3K4me1 at interacting regions, along with gene expression TPM of Maf1 and Ssbp1 during Pre-B transdifferentiation. Multiple Maf1 promoters in two HindIII digested fragments b Log2 fold change of gene expression based on the number of H3K4me1 peaks on the increased PIRs (48h vs 0h). The number of histone mark peaks is classified into three groups.  <=1: Genes that gain chromatin interactions with less than one histone mark peaks (n=360). 1-5:  Genes that gain chromatin interactions with one to five histone mark peaks (n=313). >5: Genes that gain chromatin interactions with greater than five histone mark peaks (n=69). The unpaired two-sided t-test was performed to test significant differences between groups. The box plot represents 25 and 75 percentiles with the median. Fig. S6. Decreased promoter interactions at 48h. a GO term for genes that lost promoter interactions at 48h. b Examples of significantly decreased B cell-specific gene promoter interactions  across time points. Fig. S7. C/EBPα ChIPseq data analysis. a The number of identified C/EBPα ChIP peaks at 1h, 1h, 12 h and 48h. b Comparison of 1h, 12,h or 48h C/EBPα ChIP peaks signal to 0h. M value represents log2 fold change of read density between two samples, and A value represents the average signal intensity. The red dots represent induced C/EBPα binding sites at 1h, 12h, and 48h time points. The blue dots represent C/EBPα binding sites at 0h. The grey dots represent unchanged C/EBPα binding sites at four time points. Fig. S8. Uncropped blots.Additional file 2: Table S1.Additional file 3: Table S2.Additional file 4: Table S3.