This is consistent with theH-bond distances being generally shorter in the non-natural base pairs.The natural base pairs are energetically more stabilized in theirHoogsteen geometries than in their WC geometries. The Hoogsteen geometrymakes the A:T base pair slightly more stable, by −0.8 kcalmol–1, and it greatly stabilizes the G:C+ base pair, by −15.3 kcal mol–1. The G:C+ stabilization is mainly due to the fact that C has typicallyadded a proton when found in Hoogsteen geometries. By contrast, Hoogsteengeometries are substantially less favorable than WC geometries fornon-natural Hachimoji base pairs, by 17.3 (B:S) and 13.8 (P:Z) kcalmol–1.High-order quantum chemistry is appliedto hydrogen-bonded naturalDNA nucleobase pairs [adenine:thymine (A:T) and guanine:cytosine (G:C)]and non-natural Hachimoji nucleobase pairs triazin-4(1H)-one:6-amino-5-nitropyridin-2-one(P:Z)] to see how the intermolecular interaction energies and theirenergetic components  vary among the base pairs. Weexamined the Hoogsteen (HG) geometries in addition to the traditionalWatson–Crick (WC) geometries. Coupled-cluster theory throughperturbative triples [CCSD(T)] extrapolated to the complete basisset (CBS) limit and high-order symmetry-adapted perturbation theory(SAPT) at the SAPT2+(3)(CCD)δMP2/aug-cc-pVTZ level are usedto estimate highly accurate noncovalent interaction energies. Electrostaticinteractions are the most attractive component of the interactionenergies, but the sum of induction/polarization and London dispersionis nearly as large, for all base pairs and geometries considered.Interestingly, the non-natural Hachimoji base pairs interact morestrongly than the corresponding natural base pairs, by −21.8(B:S) and −0.3 (P:Z) kcal mol In biochemistry, the architecturesof biomacromolecules such as deoxyribonucleic acid (DNA), ribonucleicacid (RNA), and proteins are determined by NCI among the buildingblocks.3 Thisdouble helix structure assembles through H-bonding between the nucleobasesto form Watson–Crick (WC) base pairs: adenine (A) with thymine(T), and guanine (G) with cytosine (C). For RNA, T is replaced withuracil (U). A decade after Watson and Crick published their modelof the DNA double helix, Karst Hoogesteen discovered that A:T(U) andG:C+ can pair in a different geometrical orientation nowcalled Hoogsteen pairing (here C+ denotes a protonatedcytosine).4In 1953, James Watson and FrancisCrick reported a double helixstructure for double-stranded DNA.5 Further advances in synthetic biology have demonstratedthe creation of semisynthetic DNA- and RNA-like systems built fromeight nucleotide “letters,” the original four for DNA(or RNA), plus four non-natural so-called “Hachimoji”(HM) nucleobases, allowing a total of four orthogonal pairs in DNAor RNA.6 Analogous to natural double-strandedDNA and RNA, these non-natural nucleobases can also form double helices.Along with natural nucleobase H-bond pairs, Hachimoji DNA and RNAcan involve additional H-bond pairs resulting from new synthetic nucleobases:isoguanine (B), 2-aminoimidazotriazin-4(1H)-one (P),isocytosine (rS), 1-methylcytosine (S), and 6-amino-5-nitropyridin-2-one(Z).6 These are the Hachimoji nucleobasessynthesized by Hoshika and co-workers in early 2019.6 The non-natural DNA nucleobase S bonds with B, and P bondswith Z, while B bonds with rS in the non-natural RNA system. Thesenon-natural DNA building blocks P and B are analogues of the purine system, and S (and rS) and Z are analogues of the pyrimidine system. These synthetic systems meet the structuralrequirements needed to support Darwinian evolution, including a polyelectrolytebackbone, predictable thermodynamic stability, and stereoregular buildingblocks that fit a Schrödinger aperiodic crystal.6 Like natural DNA, Hachimoji DNA though non-naturalcan also support the evolution of organisms.4 Non-natural DNA has been suggested for numerous applications, includinginformation storage and drug design.9In 2014, Malyshev andco-workers opened up a novel technique inartificial (or synthetic) biology by introducing exogenous non-naturalnucleic acid base pairs into a living organisms DNA, demonstratingthe feasibility of propagating an augmented genetic alphabet.12 The H-bonds are stronger than the π-stackinginteractions, although both are essential for the stability of naturalDNA and RNA duplexes in aqueous solution.14 It has beenreported that H-bonding is primarily electrostatic in nature witha non-negligible dispersion contribution.16 Jurečka and co-workers have reported18 high-levelcomputations for H-bonding interaction energies of several DNA basepairs, including some with nonstandard substituents, using second-orderMøller–Plesset perturbation theory (MP2) with coupled-clustercorrections including noniterative triples contributions [CCSD(T)].19 Hesselmann and co-workers have examined20 the interaction energy between A:T and G:C basepairs and their physical components using density functional theory-basedsymmetry-adapted perturbation theory (DFT-SAPT),22 and Fonseca Guerra and Bickelhaupt conducted a similar study23 using an energy decomposition scheme of thesupermolecular DFT energy.It is well established that naturalDNA and RNA duplexes are primarilystabilized by the forces of WC H-bonding and nucleobase stacking (interstrandand intrastrand).However, to date, no high-level theoreticalstudy has directlycompared H-bonding in non-natural Hachimoji nucleobases to that innatural WC base pairs. Here, we present such an analysis using highorders of symmetry-adapted perturbation theory (SAPT) to obtain theelectrostatic, exchange-repulsion, induction/polarization, and Londondispersion contributions to the interaction in each base pair, complementedby CCSD(T) computations in the complete basis set (CBS) limit to obtainbenchmark-quality interaction energies. Our analysis includes Watson–Crick-typegeometries, as well as Hoogsteen-type geometries (newly proposed herefor the Hachimoji base pairs). We analyze interaction energies andtheir components in terms of the geometries of the base pairs andfind in general that interaction strength in these systems correlateswith the sum of H-bond and H-bond-like closest-contact distances.+) and HG-type HM base pairs (P:Z and B:S). We employB3LYP-D3(BJ)26 methods that have become routinely applied to NCIgeometry optimizations, with the popular correlation-consistent basissets of Dunning augmented with diffuse functions .27 All three basis sets provide essentially identical geometries. Thegeometry optimization was done using the Q-Chem code.28 Geometries were also optimized using (frozen core) MP2,and results were very similar to the DFT geometries. We used the B3LYP-D3(BJ)/aDZgeometries for further analysis. All results are obtained in the gasphase, which is sufficient to quantify the intrinsic attraction betweenthe nucleobases. In the condensed phase, additional interactions willbe present , but the details willvary depending on the particular environment. Moreover, such additionalinteractions are not expected to modify or “tune” thedirect nucleobase–nucleobase interactions.29In this present study, we have modeled a total ofeight base pairs:natural WC base pairs (A:T and G:C) and non-natural HM base pairs(P:Z and B:S), as well as Hoogsteen (HG) geometry base pairs (CCD)δMP2/aXZ, where X =D, T. This level of theory includes the following terms:At the DFT geometries, we obtained high-quality intermolecularinteraction energies. To obtain noncovalent interaction energies aswell as their physically meaningful components, we used the “goldstandard”30 Thislevel of SAPT is expected to provide accurate noncovalent interactionenergies and their components. Interaction energies should similarto those from the reliable CCSD(T)/CBS method.30 We also considered some lower levels of SAPT for comparisonpurposes.IE denotes aninteraction energy. The two numbers in superscriptsrefer to the order of the perturbation for the intermolecular interactionand the intramolecular electron correlation, respectively. Cross termslike exchange-induction and exchange-dispersion are accounted as inductionand dispersion, respectively. A detailed discussion of the above termscan be found in our previous paper.32 whereby the MP2/CBS limit  is corrected for higher-order electron correlation effectsby adding the difference between CCSD(T) and MP2 as computed in asmaller basis set, denoted by δMP2CCSD(T). In particular, these noncovalentinteraction energy values are computed at the MP2/CBS +δMP2CCSD(T)/X and MP2/CBS + δMP2CCSD(T)/X levels of theory, where X = aDZ oraTZ. These computations will be abbreviated here as CCSD(T)/CBSand CCSD(T)/CBS. This focal-point approach has beenwidely used to estimate CCSD(T) at the CBS limit for noncovalent interactionenergy values.49Our most accurate estimates of the interaction energiesare obtainedusing the focal-point approach to estimate the CCSD(T)/CBS limit,52 Core electronsare kept frozen in all interaction energy computations, which havebeen performed with the quantum chemistry program Psi4 v1.5.54CCSD(T) interaction energies are computed with and withouttheBoys–Bernardi counterpoise correction.Optimized structures of the four H-bonded nucleic acidbase pairs are shown in +, B:S, P:Z) nucleic acid base pairs.Table S1 of the Supporting Information additionallypresents CCSD(T)/CBS results without counterpoise correction, andalso using the smaller aDZ basis set for the CCSD(T) correction, todemonstrate basis set convergence. The difference in interaction energiesbetween the CCSD(T)/CBS and CCSD(T)/CBScomputations is very tiny and amounts to ≤0.02 kcal mol–1 for the WC- and HG-type base pairs, indicating thatthe MP2 portion of the interaction energy is very well converged.The differences between CCSD(T)/CBS and CCSD(T)/CBS(provided in the SI) are rather small and suggest that aTZ is probably sufficient to converge the coupled-clusterpart of the focal point procedure to within ∼0.1–0.2kcal mol–1. Additionally, the interaction energydifference between counterpoise correction and without counterpoisecorrection ranges from 0.1 to 0.2 kcal mol–1 forCCSD(T)/CBS and 0.0 to 0.1 kcal mol–1 for CCSD(T)/CBS for WC-type base pairs. This suggeststhat any remaining basis set superposition error in our best estimatesis rather small. We may also compare our best total interaction energyvalues at the CCSD(T)/CBS limit to the CCSD(T)/CBSresults reported by Jurečka and co-workers18 for the A:T and G:C base pairs. Despite using a methodologythat appears similar, by neglecting diffuse functions for the δMP2CCSD(T) correction,the interaction energies of Jurečka et al. are underbindingby 0.5–0.9 kcal mol–1 vs our best estimateswith larger basis sets.High-quality total interaction energy values approachingthe CCSD(T)/CBSlimit, along with our highest level SAPT computations at the SAPT2+(3)(CCD)δMP2/aTZlevel of theory, are presented in –1 for WC-typenatural and non-natural base pairs. This indicates that our highestlevel of SAPT should be quite suitable for analyzing the various componentsof the interaction energy; we present this analysis below. In Tables S3–S4 and Figures S4–S11, we present a comparison between different levels of SAPT with aTZand aDZ basis sets. Changes of a few kcal mol–1 inthe interaction energies are found as we add more terms in SAPT. Themodest changes in interaction energy when the basis set increasesfrom aDZ to aTZ suggest that the SAPT results using the aTZ basisare fairly well converged with respect to the basis set. Finally, 20 for the A:T and G:C base pairs. Their results are underboundby 2.2 (A:T) and 3.2 (G:C) kcal mol–1 compared toour highest-level CCSD(T)/CBS results, for errors of about 10%–13%.SAPT2+(3)(CCD)δMP2/aTZ reduces the underbinding to 0.5 and 0.2kcal mol–1, respectively (or about 1%–3%),albeit at a significantly increased computational cost compared toDFT-SAPT.Results from our highest-level SAPTmethod, SAPT2+(3)(CCD)δMP2/aTZ,are quite close to our best CCSD(T)/CBS estimates. The range of differencesbetween SAPT2+(3)(CCD)δMP2/aTZ and CCSD(T)/CBSis from −0.11 to 0.54 kcal mol–1). Two of the bond lengths are very similar for B:Sand P:Z, but one is somewhat weaker (lengthened by 0.072 Å) inthe P:Z base pair (–1 (A:T), 5.436 Å and −33.0kcal mol–1 (G:C), 5.388 Å and −33.4kcal mol–1 (P:Z), and 5.291 Å and −39.2kcal mol–1 (B:S).The natural G:C base pair has nearly twice the interactionenergyas the natural A:T base pair 1. These ase pair 1. Compar20 is supported by –1 for the A:T, G:C, B:S, and P:Z base pairs,respectively (detailed energy component data are provided in the Supporting Information Tables S5 and S6). –1); for other base pairs, inductionis significantly larger than dispersion, by 7.8 kcal mol–1 or more. Overall, all four components are very important for determiningthe interaction energy. For the B:S base pair, the electrostatic contributionis the strongest among the base pairs. Furthermore, we note that theelectrostatic, exchange-repulsion, induction, and London dispersioncomponents for the B:S base pair are stronger by at least −8.0,7.7, – 4.6, and −1.3 kcal mol–1 thanin the A:T, G:C, and P:Z base pairs. SAPT interaction energy componentstend to be stronger for intermolecular interactions with shorter contacts,and as noted above, the sum of the H-bond contact distances is theshortest (5.291 Å) for B:S. P:Z and G:C have energy componentsthat are similar to each other, but smaller in magnitude than forB:S. This is consistent with the sum of H-bond contact distances beinglarger for these dimers than for B:S, but similar to each other (5.388Å for P:Z and 5.436 Å for G:C). Finally, the SAPT energycomponents are the smallest in magnitude for A:T, which has only tworeal H-bonds plus a weak C–H···O contact (summingto 6.374 Å).The point of view thatH-bonding is mainly stabilized by electrostatic contributions+(anti),by rotating the purine base in the WC-type base pair 180° aroundthe glycosidic bond torsion angle, so that it adopts a syn conformation rather than an anticonformation (−180° < χ< −90°), followed by the translation of the two basesby ∼2.0–2.5 Å, thus allowing the formation of aset of H-bonds .58 In a similar way, we create HG-typeHM base pairs to form B(syn):S(anti) and P(syn):Z(anti). Optimizedstructures of natural HG base pairs and a non-natural P:Z HG geometryare found to be planar, while the non-natural B:S HG geometry turnsnonplanar. We suspect that unfavorable H···H intermolecularinteractions focal-point approach and the SAPT2+(3)(CCD)δMP2 approach.The interaction energy results at the equilibrium geometriesofthe HG-type base pairs are tabulated in +) base pairs are more stabilized(have more negative interaction energies) than the WC-type base pairs.The interaction energy differences at our best level of theory, CCSD(T)/CBS,are −0.8 kcal mol–1 for A:T and −15.3kcal mol–1 for G:C+. For the HoogsteenG:C+ base pair, the N–H···O contactdistance is slightly shorter, and the N–H···Ndistance is significantly shorter than in the WC geometry. However,the final N–H··· O H-bond is replaced with aweak C–H···O contact (2.802 Å), which onemight expect to lead to a much weaker overall interaction energy inthe HG geometry. Instead, the interaction is much stronger in theHG geometry because of the protonation of C, leading to strong ion–dipoleinteractions. This explanation is confirmed by the SAPT analysis below.From 61 This result of a stronger interaction in the HG geometry is alsoconsistent with previous quantum chemical studies by Hobza et al.65The H-bond contact distances for A:T are similar in the HG geometryas in the WC geometry, consistent with its similar binding energy.The slightly stronger interaction of the A:T base pair in the HG geometrythan in the WC geometry is consistent with the experimental infrared-spectraanalysis of the A:T base pair in a nonpolar solvent.–1) vs the WC geometry  is consistent with elongated intermolecularH-bond contact distances. For the HG-type P:Z base pair, the weakerinteraction energy in the HG geometry  vs the WC-type geometry  is again consistent with longer intermolecular H-bond contacts.Although one N–H···O contact is medium length(1.815 Å), the other N–H···O contact islong (2.050 Å), and the remaining weak C–H···OH-bond is quite long (2.260 Å). Also note that both of the N–H···Ocontacts deviate significantly from the linear arrangement of thethree atoms that would be optimal for a strong H-bond. Nevertheless,as in the case of the WC geometries, we continue to see a correlationbetween the sum of the closest-contact H-bond distances and the interactionenergy, at least for A:T and P:Z: 6.389 Å and −18.2 kcalmol–1 for A:T and 6.125 Å and −19.6kcal mol–1 for P:Z. G:C+ deviates fromthis trend , and B:Shas only two significant H-bond-like contacts.In contrast to the natural base pairs, the non-natural Hachimoji(B:S and P:Z) base pairs are energetically less stable in their HG-typegeometries than in their WC-type geometries. As mentioned above, forthe HG-type B:S base pair, we observed a nonplanar titled configuration.The significantly weaker interaction in this HG geometry . This is due to the very strong ion–neutralinteraction in this base pair, as asserted above. We also note anunusually strong induction/polarization term for this base pair, asthe ion–induced dipole contribution will be very large comparedto dipole–induced dipole terms for the other base pairs. Thesestrongly attractive contributions pull the bases close together (leadingto two short H-bond contact distances noted above), thus also leadingto an increase in the exchange-repulsion energy.We analyzed the SAPT2+(3)(CCD)δMP2/aTZenergy components for the HG-type DNA base pairs 4. The la–1). For the WCgeometries, exchange-repulsion was larger in magnitude than electrostaticsfor every base pair. This could be due to generally shorter H-bonddistances in the WC geometries. Like in the WC base pairs, inductionand dispersion components contribute significantly to the total interactionenergies, with induction dominating over dispersion in all four basepairs, although the difference is <2.0 kcal mol–1, except for the G:C+ base pair where the difference is16.8 kcal mol–1.Looking more generally at –1 (P:Z) in the WC geometries, according to coupled-clustertheory [CCSD(T)] in the complete-basis-set limit. The stronger interactionin B:S vs A:T is due to the formation of a third H-bond in B:S.Noncovalentforces play a critical role in determining biomolecularstructure, including the structures of DNA and RNA. Here, we havepresented a detailed, high-level theoretical examination of H-bondingin both natural (A:T and G:C) and non-natural Hachimoji (B:S and P:Z)DNA base pairs, in their standard Watson–Crick (WC) type geometries,and also in Hoogsteen (HG) type geometries. Interestingly, the non-naturalHachomoji-type base pairs interact more strongly than the correspondingnatural base pairs, by −21.8 (B:S) and −0.3 kcal mol+ by −15.3 kcal mol–1. This stronger interaction of A:T in the HG-typegeometry more than the WC-type geometry is consistent with the experimentalinfrared-spectra analysis of A:T dimers in a nonpolar solvent.61 By contrast, HG-type geometries are substantially less stable than WC-type geometries for non-natural Hachimoji DNA basepairs, B:S by 17.3 and P:Z by 13.8 kcal mol–1. Wealso noted a general correlation between the total interaction energyand the sum of the closest-contact H-bond distances (including theweak C–H···O contact in A:T and in the HG geometryof P:Z).In addition, the natural base pairs are energetically more stabilizedin their HG-type geometries than in their WC-type geometries: A:Tby a minor −0.8 and G:C+ base pair than in any other basepair considered. This is consistent with very strong ion–dipoleinteractions in this complex which are absent in the other complexes,and it explains why the interaction energy is much stronger for thisbase pair even though it has only two true H-bonds.We have applied high-order quantum mechanical energycomponentanalysis, using symmetry-adapted perturbation theory (SAPT), to understandthe base pair interaction energies in terms of the physically meaningfulcomponents of electrostatics, exchange-repulsion, induction/polarization,and London dispersion. SAPT analysis confirmed that electrostaticinteractions are the most attractive in base pair interactions, consistentwith conventional wisdom about the nature of H-bonding interactions.However, the sum of induction and dispersion is nearly as large aselectrostatics for both natural and non-natural base pairs, and forWC- and HG-type geometries. The magnitude of the SAPT components alsotended to correlate with the sum of the closest-contact H-bond-typeinteractions. However, all SAPT terms are much larger in magnitudein the Hoogsteen G:CThe presentinteraction energies are expected to be the most accuratetheoretical values reported to date for these important prototypesystems, including for the newly proposed structures of Hoogsteen-typegeometries for the non-natural Hachimoji base pairs. As such, theinteraction energies may serve as valuable reference data. We alsohope that the detailed energetic analysis of the interaction energycomponents, and how they relate to structure, may aid future studiesof H-bonding between nucleobases and the design of additional non-naturalnucleobases.


AppNL-G-F mice, a mouse model of AD, have three familial AD mutations in the amyloid-β precursor gene and exhibit age-dependent AD-like symptoms and pathology. Gut-brain interactions have attracted considerable attention and inflammatory bowel disease (IBD) has been associated with a higher risk of dementia, especially AD, in humans. However, the underlying mechanisms and the effects of intestinal inflammation on the brain in AD remain largely unknown. Therefore, we aimed to investigate the effects of intestinal inflammation on AD pathogenesis.Alzheimer’s disease (AD) is one of the neurodegenerative diseases and characterized by the appearance and accumulation of amyloid-β (Aβ) aggregates and phosphorylated tau with aging. The aggregation of Aβ, which is the main component of senile plaques, is closely associated with disease progression. AppNL-G-F mice at three months of age were fed with water containing 2% dextran sulfate sodium (DSS) to induce colitis. Immune cells in the brain were analyzed using single-cell RNA sequencing (scRNA-seq) analysis, and the aggregation of Aβ protein in the brain was analyzed via immunohistochemistry.Wild-type and AppNL-G-F mice with acute intestinal inflammation. Detailed scRNA-seq analysis of immune cells in the brain showed that neutrophils in the brain increased after acute enteritis. Eliminating neutrophils by antibodies suppressed the accumulation of Aβ, which increased because of intestinal inflammation.An increase in aggregated Aβ was observed in the brains of These results suggest that neutrophils infiltrate the AD brain parenchyma when acute colitis occurs, and this infiltration is significantly related to disease progression. Therefore, we propose that neutrophil-targeted therapies could reduce Aβ accumulation observed in early AD and prevent the increased risk of AD due to colitis.The online version contains supplementary material available at 10.1186/s41232-023-00257-7. Alzheimer’s disease (AD), the most common form of dementia, is a progressive age-related neurodegenerative disorder. The early stages of AD are characterized by amyloid-β (Aβ) production due to the abnormal cleavage of amyloid precursor protein (APP) by β- and γ-secretases, which oligomerize and aggregate into extracellular Aβ plaques called senile plaques. The disease is characterized by senile plaques, neurofibrillary tangles, and neuroinflammation, including microgliosis and astrogliosis . These p+ T cells [+ CD4+ T cells [Neurodegenerative diseases are closely related to the immune system , and the T cells  and IFNγ T cells  influenc T cells , 7. Neut T cells . Thus, AAppNL-G-F mice, which may result from the decrease in phagocytosis by microglia [Interactions between the gut microbiota and brain have been demonstrated, and the molecular mechanisms are beginning to be elucidated. These interactions are called the gut-brain axes and closely related to many diseases . Inflammicroglia . HoweverAppNL-G-F mice, and immune cells in their brains were analyzed by scRNA-seq analysis. We found that acute colitis increased neutrophil infiltration into the brain, resulting in the accumulation of Aβ. Our results suggest that neutrophil-targeted therapies could reduce Aβ accumulation observed in early AD and prevent the increased risk of AD due to colitis.In this study, we investigated the effects of intestinal inflammation on the pathogenesis of AD. Colitis was induced in wild-type and AppNL-G-F mice (RBRC06344) were obtained from Dr. Takaomi C Saido and Dr. Takashi Saito at the RIKEN BioResource Center, Japan. These knock-in mice carry the humanized Aβ sequences with the addition of Swedish (NL), Arctic (G), and Iberian (F) mutations. This results in increased Aβ production as well as Aβ aggregation and increases the Aβ42/Aβ40 ratio [AppNL-G-F mice were maintained under appropriate conditions, including a 12-h light:12-h dark cycle and free access to food and water at Laboratory of Embryonic and Genetic Engineering, Medical Institute of Bioregulation, Kyushu University. Animal experiments were performed in strict accordance with the recommendations in the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. All experiments were approved by the Animal Research Committee and Ethics Committee of Kyushu University. Unless otherwise indicated, 3-month-old male and female C57BL/6J and AppNL-G-F mice were used. It is important to consider gender differences when studying AD, as epidemiological studies have shown that females have a higher prevalence and incidence of AD and that females have a greater Aβ plaque load in mouse models [40 ratio , 16. Wile models , 18. Howe models . TherefoFor flow cytometric analysis, fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein-cyanine 5.5 (PerCP-Cy5.5), allophycocyanin (APC), PE-Cy7, APC-Cy7, Brilliant Violet (BV421), and BV711-conjugated antibodies were purchased from BD Biosciences, , BioLegend  or eBioscience . The following antibodies were used: anti-mouse CD45 (30-F11), anti-mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse Gr1 (RB6-8C5), anti-mouse Ly6C (HK1.4), anti-mouse Ly6G (1A8). Fixable Viability Dye eFluor 780 (FVD780) (eBioscience) was used to remove dead cells.For immunohistochemistry, the following antibodies were used: anti-human Aβ , anti-mouse IBA1 , anti-mouse GFAP , anti-mouse MRP8 , Alexa Fluor 488-, 546-, or 633-conjugated goat anti-rabbit, mouse, or rat IgG , and Alexa Fluor 488-conjugated donkey anti-goat IgG . Anti-mouse Ly6G  (1 mg/kg), anti-rat kappa immunoglobulin light chain  (1 mg/kg), and rat IgG2a isotype control  (2 mg/kg) were used as neutralizing antibodies. DNase I (Roche) (8 mg/kg), matrix metallopeptidase 9 (MMP-9) inhibitor II  (10 mg/kg), and N-acetyl-l-cysteine (NAC) (Wako) (100 mg/kg) were used by intraperitoneal injection.AppNL-G-F mice were housed in cages according to experimental group. The control groups received only water, the acute colitis groups received DSS  in their drinking water for one week, and the chronic colitis group received three cycles of alternating 2% DSS then only water, each for 5 days. To determine if colitis was induced, body weight was measured at the timing of the 2% DSS and drinking water exchanges and for one week prior to the day of mouse dissection. Weight loss was calculated as a ratio of the weight on each specific day over the weight on day 0. The photographs of the colons were taken, and the lengths were measured using ImageJ software .WT and AppNL-G-F mice were intraperitoneally administered 0.1 mL of the allergen (0.5 mg/mL in PBS) mixed with 0.1 mL of Alum adjuvant. This was administered once per week for a total of three times. For 7 consecutive days after the above sensitization, 250 mg/mL of allergen was administered orally using a sonde at a dose of 0.2 mL per animal.Ovalbumin (OVA) (Sigma) were used as allergens. OVA was used as the food allergen only for the single-cell RNA sequencing experiments. WT and Mice were anesthetized by inhalation with isoflurane and perfusion was performed by injecting PBS into the left ventricle followed by perfusion fixation via administration of 4% paraformaldehyde (PFA). After decapitation, the heads were immersed in 4% PFA and fixed at 4 °C for 24 h while the skin was cut and the skull was exposed. For frozen sections, the brains were removed and transferred in turn to 10%, 20%, and 30% sucrose solutions until completely submerged. The brains were then embedded in O.C.T. compound (Sakura Finetek) and rapidly frozen on dry ice and stored at – 20 °C. Frozen sections (10 μm) were prepared using a cryostat. For paraffin-embedded sections, coronal slices from the brain in cassettes were prepared by the Laboratory for Research Support, Medical Institute of Bioregulation, Kyushu University.Brain cryosections and paraffin-embedded sections were treated on slides for 1 h at room temperature (25 °C) using Blocking One Histo . The sections were incubated with primary antibody in 5% BSA/PBS overnight at 4 °C, washed twice with PBS containing 0.1% Triton X-100 (PBS-T) and once with PBS, and incubated with the secondary antibody for 1 h at room temperature. The brain sections were sealed with cover glasses using a mounting agent. The images were taken with BZ-X700 (Keyence) and image analysis was performed using Keyence software.The cecum to distal colon was dissected and removed from the mice that had been perfused with PBS and stored in PBS. After fat and muscle were removed and photographs were taken side by side for each treatment group, the cecum was removed and washed internally with PBS. The colon was opened and rolled before immersion in 4% PFA. The Laboratory for Research Support of the Institute of Bioregulatory Medicine processed the tissue samples up to paraffin permeabilization. The samples were then paraffin-embedded and cut into 5 μm sections using a microtome, placed on glass slides, and then stretched using a paraffin stretching machine. The glass slides were placed in a staining basket and deparaffinized with xylene and ethanol. After staining with Meyer’s hematoxylin solution for 3 min and eosin solution for 1 min, they were dehydrated with ethanol and xylene and sealed with Paramount D .g for 5 min the supernatant was collected and stored at − 80 °C. Enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific) were then used according to the manufacturer’s instructions to measure IL-6 levels.Mice were anesthetized by inhalation with isoflurane, blood in the heart was collected with a 26-G needle and 1 mL syringe and left at room temperature for 30 min. After centrifugation at 7000×+CD11b+Ly-6Gintra+) are shown in Supplementary Figure SMice were anesthetized by inhalation with isoflurane, and blood from the hearts of mice was placed in heparin-coated tubes and treated with RBC lysis buffer. The following monoclonal antibodies were used for flow cytometric analysis at a dilution of 1/400: anti-CD45, anti-CD11b, anti-Ly6G, anti-Ly6C, anti-Ly6G/Ly6C (Gr-1) antibodies. For intracellular staining, after staining with antibodies against cell surface proteins, cells were washed three times with MACS buffer, fixed with eBioscience™ IC Fixation Buffer (Thermo Fisher Scientific), permeabilized with eBioscience™ Permeabilization Buffer, and then stained with an antibody against an intracellular marker. Cells not stained with FVD780 were categorized as live cells. Data were acquired with LSRFortessa  and analyzed with FlowJo. The gating strategies of neutrophils , cells were isolated by centrifugation with slow acceleration/deceleration 800×g for 20 min and resuspended with filtered 0.5% BSA/PBS. Staining was performed with anti-CD45.2 and anti-CD11b antibodies. Totalseq-Hashtag antibody (BioLegend) was incubated with each sample, and CD45.2+ cells were sorted by FACSMelody (BD Biosciences).Three-month-old WT and nd HEPES . After sLibraries were prepared according to the protocol of the Chromuim Next GEM Single Cell 5' Reagent Kits v2  using a Chromium controller (10× Genomics). Briefly, GEMs were prepared by loading barcoded Single Cell VDJ 5' Gel Beads, master mix containing cells and RT enzymes, and Oil onto Chromium Next GEM Chip K. Barcoded cDNA was then generated from the GEM. This cDNA was amplified by PCR reaction to generate V(D) J, 5' Gene Expression and Feature Barcode libraries. Sequencing was then performed using Novaseq 6000  at the Laboratory for Research Support of the Institute of Bioregulatory Medicine, Kyushu University.FASTQ files were generated from the NGS data by Cell Range (v6.1.2) (10× Genomics). Feature barcode matrices were created from the FASTQ data by Cell Ranger Multi , and then converted to Seurat objects.Analysis was performed using Seurat (v4.0.0) in r (v.4.1.3)  . Seurat objects for each dataset were created with the CreateSeuratObject function . Cells with more than 5% of mitochondrial genes and cells with nFeature_RNA less than 200 were filtered out. Expression data were normalized with the normalizeData function  and scaled with the ScaleData function. Highly variable genes in each dataset were identified using the FindVariableFeatures function. Principal component analysis (PCA) was performed on the identified highly variable genes using the RunPCA function. Enrichment of each PC was calculated using the JackStraw and ElbowPlot functions, and enriched PCs were selected for clustering and dimensionality reduction analysis. Clustering was performed using the FindClusters function (resolution = 0.5) and dimensionality reduction was performed using the RunUMAP function. Uniform manifold approximation and projection (UMAP) was used for dimensionality reduction and visualization. Statistically significant marker genes for each cluster were identified using the FindAllMarkers function . Each identified cluster was then manually annotated with marker genes that have been previously reported as marker genes for defining cell subsets , 22. TraAppNL-G-F mice were treated with 2% DSS for 7 days and anti-Ly6G antibody and anti-rat kappa light chain antibody or isotype control were administered starting on day 3 of DSS treatment. Anti-Ly6G antibody (1 mg/kg) and anti-rat kappa immunoglobulin light chain (anti-rat κIgL antibody) (1 mg/kg), or rat IgG2a isotype control (2 mg/kg) were injected intraperitoneally. These antibodies were administered daily (anti-Ly6G and IgG2a isotype control) or every other day (anti-rat κIgL) over a 4-day period. For inhibitor treatment, DNase I (8 mg/kg), MMP-9 inhibitor II (10 mg/kg), and NAC (100 mg/kg) were administered intraperitoneally daily for 4 days starting on day 3 of DSS treatment.This protocol was based on that from a previously reported study . Three-mt test or Mann-Whitney U test to analyze differences between two groups. P < 0.05 was considered to represent a significant difference . Statistical analysis was performed using Prism 9.0 (GraphPad Software)Data are expressed as mean ± s.e.m. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple-comparisons tests to analyze differences among three or more groups, and by unpaired Student’s AppNL-G-F and WT mice  in 3-month-old ice Fig. A. Both Went Fig. A. As colent Fig. C, D. HisAppNL-G-F mice was performed. Compared to the control group, the number of Aβ plaques in the cortex was slightly higher in the acute colitis model, lower in the food allergy model, and comparable to that in the chronic colitis model  revealed the presence and heterogeneity of various immune cells  was found to be increased in both the acute and chronic colitis models  did not change, but the percentage of Ly6Gintra+Ly6Gint/lo neutrophils (immature neutrophils) increased in AppNL-G-F mice with acute colitis  or isotype control (isotype: iso) were administered starting on day 3 of DSS treatment  and cytotoxic enzymes contained in granules including several matrix metalloproteinases (MMPs), which can cause tissue injury . As MMP-P-9 Fig. E, F. Mmpls Fig. S. These rAppNL-G-F mice  or AppNL-G-F mice (C) induced acute DSS colitis, chronic DSS colitis, and food allergies. D The protein levels of IL-6 in the serum of AppNL-G-F mice with colitis. Data points are individual mice from one representative of two independent experiments . P values were determined by one-way ANOVA. Data are shown as the mean ± s.e.m. Supplementary Figure S2. Reduction of Aβ plaques in the brains of AppNL-G-F mice with acute colitis by neutrophil depletion. AppNL-G-F mice drinking with fresh water or 2% DSS water were injected with anti-Ly6G antibody plus anti-rat kappa light chain antibody or isotype control antibody. A The photos of colon of AppNL-G-F mice on day 7. B The percentage of Aβ plaques area in cortex and hippocampus by immunohistochemical staining. Data points are individual mice in one experiment. Supplementary Fig. S3. Cluster-specific gene expression in neutrophils. Dot plots for selected differential expressed genes (DEG) and cell-type specific markers for the indicated clusters. Supplementary Figure S4. Gating strategy for neutrophils in the blood. Gating strategy to analyze young and adult neutrophils in the blood by flow cytometry analysis as shown in Fig.


Pek RH, Yuan X, Rietzsche N, Zhang J, Jackson L, Nishibori E, Ribeiro A, Simmons W, Jagadeesh J, Sugimoto H, Alam MZ, Garrett L, Haldar M, Ralle M, Phillips JD, Bodine DM, Hamza I. 2019. Hemozoin produced by mammals confers heme tolerance. Published 1 October 2019PubPeer - Hemozoin produced by mammals confers heme tolerance) that we duplicated the loading controls of the spleen Western Blots for the liver in Fig. 1C. After checking the first author’s lab notebooks and the raw figure files, we found the original images for the liver samples, which show the correct ponceau S stained nitrocellulose membranes. We can confirm that the duplication was inadvertent, and we have now corrected Fig. 1C by using the correct ponceau S staining image. During carefully proofreading the manuscript, we identified two textual errors in Figure legends 4 and 6. These changes neither impact the results nor the conclusions in the manuscript.It was brought to our attention through PubPeer  bone marrow Ter-119+populations II+III and (D) splenic Ter-119+subpopulations.”1. The original figure legend stated “…Gating of (C) splenic Ter-119+ populations II+III and (D) bone marrow Ter-119+ subpopulations.”The correct figure legend for Figure 6—figure supplement 1 should be “…(A) Representative images of intracellular reactive oxygen species (ROS) in WT and KO BMDMs at the indicated timepoints post-treatments. (B) Representative images of WT and KO BMDMs post-EP.”2. The A) Representative images of WT and KO BMDMs post-EP. (B) Representative images of intracellular reactive oxygen species (ROS) in WT and KO BMDMs at the indicated timepoints post-treatments.”The original figure legend was “…(corrected figure 1C is shown below:The originally published Figure 1 is shown for reference:The Here are the source images for reference.


Drosophila.Ubiquitylation (ubi) of the intracellular domain of the Notch ligand Delta (Dl) by the E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1) on lysines (Ks) is thought to be essential for the its signalling activity. Nevertheless, we have previously shown that DlK2R-HA, a Dl variant where all Ks in its intracellular domain (ICD) are replaced by the structurally similar arginine (R), still possess weak activity if over-expressed. This suggests that ubi is not absolutely required for Dl signalling. However, it is not known whether the residual activity of DlK2R-HA is an effect of over-expression and, if not, whether DlK2R can provide sufficient activity for the whole development of DlattP-DlK2R-HA, a knock-in allele into the Dl locus. Our analysis of this allele reveals that the sole presence of one copy of DlattP-DlK2R-HA can provide sufficient activity for completion of development. It further indicates that while ubi is required for the full activity of Dl in Mib1-dependent processes, it is not essential for Neur-controlled neural development. We identify three modes of Dl signalling that are either dependent or independent of ubi. Importantly, all modes depend on the presence of the endocytic adapter Epsin. During activation of Dl, direct binding of Epsin appears not to be an essential requirement. In addition, our analysis further reveals that the Ks are required to tune down the cis-inhibitory interaction of Dl with Notch.To clarify these issues, we generated and analysed Our results indicate that Dl can activate the Notch pathway without ubi of its ICD. It signals via three modes. Ubi is specifically required for the Mib1-dependent processes and the adjustment of cis-inhibition. In contrast to Mib1, Neur can efficiently activate Dl without ubi. Neur probably acts as an endocytic co-adapter in addition to its role as E3 ligase. Endocytosis, regulated in a ubi-dependent or ubi-independent manner is required for signalling and also suppression of cis-inhibition. The findings clarify the role of ubi of the ligands during Notch signalling.The online version contains supplementary material available at 10.1186/s12915-023-01759-z. Drosophila, and is thought to require endocytosis of the ligands interacting in trans with the Notch receptor 70]. The ribed in  Antibodies used:Alexa-Fluorochrome-conjugated secondary antibodies were purchased from Invitrogen/Molecular Probes. Images were acquired with a Zeiss AxioImager Z1 Microscope equipped with a Zeiss Apotome.2.Adult wings, legs and embryos were mounted in Hoyers Medium and Images were acquired using a Zeiss Axiophot equipped with a Zeiss MRC digital camera.Adult flies were imaged with a Leica MZFLIII equipped with a Zeiss MRC digital camera.Image analysis was performed using Fiji(ImageJ).Dmon1 mutant clones, the association of the anti HA-signal with the anti Nextra signal was analysed by thresholding the Nextra signal using Fiji(ImageJ) thresholding tool. To obtain regions of interest (ROI) encompassing the accumulations of Notch in endosomes, particle analysis was performed. The ROI sets were measured, and the median fluorescence of anti HA was determined for each ROI (endosome). Three discs of each genotype, were analysed (Dl-HA and DlK2R-HA). The total number of vesicles analysed for each genotype was n = 1043 and n = 983, respectively. The obtained data were statistically analysed using Prism Graphpad 7. The comparison of Gbe + Su(H)-lacZ, anti HA and GFP fluorescence intensities in DlattPDl-HA (+ ubiGFP) and DlattP-Dl-K2R-HA mosaic discs was done by drawing a ROI and subsequently analysing the fluorescence using Fiji (ImageJ) plot profile tool.For the analysis of endosomes in Cuticle of homozygous embryos were prepared according to Van der Meer (1977) .DlattP-landing site as described in [The constructs Dl-HA and Dl-K2R-HA were seamlessly cloned into the original vector pGE-attB-Dl::EGFP via replacement of the ICD::GFP part by Gibson Assembly . An addiribed in .Primer used: Dl_ICD_attP _for: GCA AGC AGT GCG ATG AGG AGT CCT ACG ATT C GGT GAC CTT CGA TGC CCA CC.Dl_ICD_attP _rev: TTC CCA GGA GCC CTT CCG GAT TTT TGG AG TTA AGC GTA GTC TGG AAC GTC.NdeI_HA_XbaI_3'UTR of Dl_for: GCACTCCGCATATGGCCTACCCATACGACGTTCCAGACTACGCTTAATCTAGAAACTCCAAAAATCCGG.3'UTR of Dl_NheI-rev: GTTTACACATACGCGTTGGCTACGGCTAGCATTAATATTTAC.lqf-GFP rescue construct [The onstruct  was obtaAdditional file 1: Fig. S1. Expression of DlattP-constructs in various tissues compared to Dl::GFP.  Expression in various imaginal discs of the late third instar stage.  Expression in the eye imaginal disc.  Expression in the haltere, wing and leg disc. Expression in the larval brain of the late third instar stage. The arrowhead highlights expression in the optic lobes.  Expression in stage 11 embryos.  Expression in the adult gut. Expression is largely restricted to escargot (esg-GFP) positive ISCs . Fig. S2. (A-B’’) Comparison of expression of DlattP-Dl-HA and DlattP-DlK2R-HA on the cell surface. Clonal analysis was used to twin clone that express either of the DlattP-variants in homozygosity, as described in (A).  An example of a disc bearing the clones. The arrowhead points to a Dl-HA homozygous clone. The abundance of Dl is much reduced in the cells of Dl-HA homozygous in comparison to cells of DlK2R homozygous or heterozygous clones. (B’’) Pixel density measurements of the channels in the apical membrane region highlighted by the thin line in . It revealed that Dl-HA is significantly less abundant than DlK2R-HA in the membrane. (C-C’’’) Co-depletion of Rab4 and Rab11 by expression of RNAi constructs for 46h using a combination of ciGal4 and tubGal80ts. It efficiently suppresses the expression of a mCherry-Rab4 construct (C’), as well as Rab11 (C’’).  Co-depletion of Rab4 and Rab11 had no effect on the expression of DlK2R-HA, suggesting that endosomal recycling is not involved in the increase of it abundance in the membrane. Fig. S3. Three additional examples of wing discs with twin clones homozygous for DlattP-DlK2R-HA or DlattP-Dl-HA crossing the region of stripe2 at different points. Fig. S4. (A-C’’’) Analysis of SOP formation in DlattP-DlK2R-HA/Df (B-B’’’) and DlattP-Dl-HA/Df (C-C’’’) wing discs. (A-A’’’) The expression of the used marker in the wildtype is shown for comparison. (B-C’’’) No significant differences in the expression of the markers are detected, indicating that the SOP selection occurs normally in DlattP-DlK2R-HA/Df flies.  Depletion of neur function by neur-RNAi with ciGal4 in DlattP-DlK2R-HA/Df flies causes the development of a neurogenic phenotype (arrow). (F) Homozygous DlattP-Dl-NEQN2A-HA flies die during embryogenesis and display the neurogenic cuticle phenotype. The arrow point to the small patch of remaining cuticle. (G-G’’) Notal region of a wing disc bearing DlattP-Dl-NEQN2A-HA homozygous clones. The clones are labelled by the loss of GFP.  magnification of the region highlighted by the arrows in (G). It reveals the neurogenic phenotype and the accumulation of DlattP-Dl-NEQN2A-HA in the plasma membrane of the SOPs (arrow). This accumulation is much less obvious in the SOP developing in the heterozygous DlattP-Dl-NEQN2A-HA/+ region .


NF-κB is an inflammatory protein that contributes to the low rate of survival observed in pancreatic cancer patients. The activation of NF-κB causes high expression of proteins that drive and sustain pancreatic cancer growth. Thus, it is important to understand how NF-κB is regulated to help with the diagnosis and treatment of pancreatic cancer. In this study, we discovered that the typical NF-κB subunit p65 is modified by O-GlcNAc at serines (S)550 and S551. To characterize the role of O-GlcNAcylated p65 at S550 and S551, we overexpressed p65 serine-to-alanine (S-A) mutants, such as S550A, S551A, and S550A/S551A, in pancreatic cancer cells. Using this model, we show that the p65 mutants reduce NF-κB transcriptional activity, nuclear translocation, p65 phosphorylation, and target gene expression. We also observed that the p65 mutants blocked pancreatic cancer cell growth and migration. This suggests the contribution of p65 O-GlcNAcylation at S550 and S551 to pancreatic cancer phenotypes. Taken together, our study uncovers a novel aspect of NF-κB regulation, which could aid future therapeutic development by targeting O-GlcNAc transferase (OGT) in pancreatic cancer.Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a mere 5-year survival of ~10%. This highlights the urgent need for innovative treatment options for PDAC patients. The nuclear factor κB (NF-κB) is a crucial transcription factor that is constitutively activated in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds great promise for PDAC diagnosis and effective therapeutics. Here, we report a novel finding that the p65 subunit of NF-κB is O-GlcNAcylated at serine 550 and 551 upon NF-κB activation. Importantly, the overexpression of either serine-to-alanine (S-A) single mutant (S550A or S551A) or double mutant (S550A/S551A) of p65 in PDAC cells impaired NF-κB nuclear translocation, p65 phosphorylation, and transcriptional activity, independent of IκBα degradation. Moreover, the p65 mutants downregulate a category of NF-κB-target genes, which play a role in perpetuating major cancer hallmarks. We further show that overexpression of the p65 mutants inhibited cellular proliferation, migration, and anchorage-independent growth of PDAC cells compared to WT-p65. Collectively, we discovered novel serine sites of p65 O-GlcNAcylation that drive NF-κB activation and PDAC phenotypes, thus opening new avenues by inhibiting the NF-κB O-GlcNAcylation enzyme, O-GlcNAc transferase (OGT), for PDAC treatment in the future. Pancreatic Ductal Adenocarcinoma (PDAC), which accounts for over 90% of pancreatic cancer cases, is experiencing a rapid increase in incidence. Its overall five-year survival rate stands at a dismal 12% . DespiteNF-κB is a master regulator of inflammation known to be constitutively active in PDAC . This ubNotably, in PDAC, aberrant signaling of the canonical NF-κB pathway contributes to various cancer hallmarks, such as metastasis, chemoresistance, cell proliferation, and cell survival, among others . SpecifiO-GlcNAcylation involves the transfer of an N-acetylglucosamine (GlcNAc) molecule from the donor molecule, UDP-GlcNAc, to an acceptor protein’s serine or threonine residue by O-linked N-acetyl-glucosaminyl transferase (OGT). This modification can be reversed by the O-linked N-acetyl-glycosaminidase (OGA) . ElevateIn this study, we have uncovered O-GlcNAcylation of p65 at S550 and S551, both located within the TAD domain of p65. We further elucidate the mechanistic function of these termini serine residues by showing that overexpression of S550A and/or S551A p65 mutants leads to significantly reduced NF-κB transcriptional activity, NF-κB target gene expression, and nuclear localization in PDAC cell lines. Moreover, p65 mutants significantly decrease PDAC cellular proliferation, migration, and anchorage-independent growth. Collectively, our findings unveil novel O-GlcNAcylated sites on p65 and their consequential effects on NF-κB signaling and PDAC phenotypes. This discovery of functional O-GlcNAcylated sites on the NF-κB protein holds promise for advancing the development of effective PDAC therapies targeting the OGT/NF-κB axis.vol/vol). A linear gradient of acetonitrile from 4 to 40% in water in the presence of 0.1% formic acid over a period of 45 min was used at a flow rate of 300 nL/min. The spectra were acquired by data-dependent methods, consisting of a full scan (m/z 400–2000) and then tandems on the five most abundant precursor ions. The previously selected precursor ions were scanned once during 30 s and then excluded for 30 s. The obtained data were analyzed by Mascot software  against a customized p65 protein database with a setting of 10 ppm for precursor ions and 0.8 Da for product ions. Carbamidomethylation of cysteine was set as a fixed modification, while oxidation of methionine, O-GlcNAcylation of serine, and threonine were set as variable modifications. The tandem mass spectra of candidate-modified peptides were further interpreted manually.A coomassie-stained SDS-PAGE gel band containing FLAG-tagged WT-p65 (FLAG-WT-p65) protein was subjected to in-gel tryptic digestion. FLAG-WT-p65 gel pieces were subjected to destaining and reduction of cysteine residues using 50% acetonitrile in 100 mM ammonium bicarbonate and 100% acetonitrile, followed by treatment, followed by 20 mM DTT at room temperature for 60 min. Alkylation with 55 mM iodoacetamide for 30 min was performed in the dark. The solution was removed, and the gel pieces were washed with 100 mM ammonium bicarbonate and dehydrated in acetonitrile. Gel pieces were then dried in a SpeedVac centrifuge and proteolytically digested by rehydration overnight at 37 °C in 50 mM ammonium bicarbonate containing sequencing grade modified trypsin . Extracted peptides were treated with 50% acetonitrile in 5% formic acid, dried, and reconstituted in 0.1% formic acid for mass spectrometry analysis. Analysis of proteolytic digests was performed by using an LTQ Orbitrap XL linear ion-trap mass spectrometer , coupled with an Ultimate 3000 HPLC system . The digests were injected onto a reverse-phase C18 column  equilibrated with 0.1% formic acid/4% acetonitrile  and cultured in Cytiva HyClone™ Dulbecco’s High Glucose Modified Eagles Medium (DMEM) supplemented with 5 mL of 10,000 units/mL penicillin, 10,000 units/mL streptomycin, and 5% fetal bovine serum (FBS). The FLAG-S550A, S551A, and S550A/S551A mutants of p65 were generated with the QuikChange II XL Site-Directed Mutagenesis Kit from Agilent Technologies. Primers were designed using the Agilent Technologies QuikChange Primer Design online software. Constructs were transfected into PDAC cell lines as described previously  using LiCells were cultured to about 90–95% confluence before lysis. Whole cell samples were collected and lysed using Radio Immunoprecipitation Assay buffer , 50 mM Tris-HCl pH 8.0, and protease inhibitors). Whole cell lysates were separated by SDS/PAGE gels, and proteins were probed by Western blotting. Antibodies used to detect the target proteins include anti-p65 , anti-FLAG , anti-O-GlcNAc , anti-IκBα , anti-pS536 , and anti-beta actin . Quantifica-tions were performed using ImageJ on three inde-pendent western blot images.5 PANC1 or MIA PaCa2 cells were seeded onto coverslips in a 24-well plate. The next day, the cells were treated with or without IL-1β for 1 h. After treatment, the cells were fixed with 4% formaldehyde for 30 min, and the reaction was quenched by 100 mM glycine for 5 min. The cells were gently washed with 1× PBS and then blocked and permeabilized with blocking buffer (1XPBST and 1% BSA) and permeabilizing buffer (Blocking buffer with 0.2% Tritonx-100), respectively. Cells were further probed with anti-FLAG antibodies for FLAG-tagged WT-p65 or S550A, S551A, and S550A/S551A and Alexa Fluor 488 (green) goat antimouse IgG. Before sealing the coverslips, mounting medium with DAPI was used to stain the nucleus. The slides were examined under a Leica DMI6000B series fluorescent microscope at 40× magnification.1 × 10NF-κB luciferase assays were conducted by transiently transfecting the κB-luciferase construct ELAM-luc into parental control or FLAG-WT-p65 stable cell lines. Cells were treated with or without OSMI-1  for 24 h. Luciferase activity was quantified 48 h after transfection using the Luciferase Assay System with Reporter Lysis Buffer kit . The ELAM-luc plasmid contains three NF-κB binding sites derived from the E-selectin gene located upstream of a luciferase reporter gene. A β-galactosidase construct was used to normalize for transfection efficiency. Luciferase activity was measured using a Synergy H1 Multi-Mode Reader .v/v), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 20 µM aprotinin, 1 mM phenylmethanesulfonyl fluoride, and 1 mM pepstatin A). FLAG-tagged p65 proteins were then immunoprecipitated with anti-FLAG-M2 beads , using immunoprecipitation methods previously described [Cells stably expressing FLAG-tagged p65 proteins were cultured to ~80% confluency and then treated with Thiamet G  for 24 h. Following treatment, cells were lysed in co-immunoprecipitation buffer  was used to prepare 3% Matrigel and media cell suspension. 250 cells were seeded and cultured for 5 days at 37 °C and 5% CO2. On day 5, formed spheroids were imaged and captured using a Canon EOS Rebel T3i Digital SLR camera and treated with Alamar Blue  at 10% culture volume. Fluorescence intensity was read using the Synergy H1 Multi-Mode Reader  at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.For cell proliferation assays, PDAC-stable cells overexpressing FLAG-WT-p65 and mutant p65 constructs were seeded in triplicate at 2 × 105 cells were seeded in the top of the insert (upper chamber), while serum-rich media (10% serum) was supplied in the well below as a chemoattractant. After 24 h, migrated cells were fixed with 4% formaldehyde and stained with crystal violet. Stained cells were visualized with a light microscope and quantified. Images were captured using a Canon EOS Rebel T3i Digital SLR camera. Quantification was performed using imageJ.Migration assays were conducted using Boyden chambers. Briefly, a Boyden chamber consists of 8 μm pore size cell culture inserts in a 24-well plate. Each insert was coated with gelatin on the side facing the lower chamber. In serum-free media, 2 × 10qPCR experiments were carried out as previously described (6). Briefly, established PDAC stable cells were cultured to ~90% confluence, and total RNA was isolated using Trizol reagent . Total isolated RNA was used to prepare cDNA using the SuperScript III First-Strand Synthesis PCR System . qPCR was carried out using FastStart Universal SYBR Green Master ROX . Primers were designed by the NCBI Primer BLAST tool. Primer sequence is as shown: CD274-Forward: 5′-GGTGCCGACTACAAGCGAAT-3′; CD274-Reverse: 5′-TGACTGGATCCACAACCAAAATT-3′; FGF8-Forward: 5′-AGTACCGACCCGCACGCTCTT-3′; FGF8-Reverse: 5′-GACCAGCAAGTGCAACAGCCACG-3′.A list of genes downregulated by S550A, S551A, and S550A/S551A was analyzed by the IPA software. The setting and filter were as follows: reference set: ingenuity knowledge base ; relationship to include: direct and indirect; includes endogenous chemicals; filter summary: consider only molecules where species were human, rat, or mouse.NF-κB is subjected to several PTMs that fine-tune its function. This phenomenon highlights one of the complexities of the NF-κB signaling pathway in cancer . Thus, tThe constitutive activation of NF-κB transcriptional activity enhances PDAC progression . PreviouThe regulation of p65 by PTMs also results in differential gene expression . CurrentThe global elevation of cellular O-GlcNAcylation has been reported to increase the nuclear accumulation of p65 in murine embryonic fibroblasts (MEFs) . ConsideNumerous studies have highlighted the intricate interplay between O-GlcNAcylation and phosphorylation in proteins . NotablyOur IPA findings revealed a noteworthy observation: numerous genes downregulated by S550A and/or S551A are intricately linked to cancer-related functions C. In ligO-GlcNAcylation is regulated by only one pair of enzymes, namely OGT and OGA . InteresAs proposed in NF-κB is a well-established link between inflammation and cancer, including PDAC. The aberrant signaling of NF-κB in PDAC is driven by different factors, such as PTMs, which adds a significant layer of complexity to the outcomes of NF-κB signaling . NotablySince its discovery in 1983, O-GlcNAcylation has been reported to modify about 4000 proteins, including inflammatory proteins such as NF-κB, IKKβ, STAT3 , TAK-1, NFAT (Nuclear factor of activated T cells), Sp1, etc. ,31. ThusInterestingly, the connection between high blood glucose, O-GlcNAcylation, and proinflammatory cytokine production has also been documented. For instance, Ferrer et al. suggested that O-GlcNAcylation controls major metabolic and signaling pathways known to promote cancer hallmarks . Cancer In recent years, the NF-κB signaling pathway has emerged as a prime target to inhibit tumor progression in PDAC. One attractive approach to inhibiting NF-κB is by targeting its regulators. Given the plethora of reports on the significance of p65 site-specific O-GlcNAcylation, it is important to elaborate on the mechanistic knowledge of this PTM in PDAC. Previous studies have demonstrated that O-GlcNAcylation of p65 at T352 and T322 increases anchorage-independent growth in PDAC, with no further evaluation of its molecular impact on NF-κB regulation . Other sIn our study, for the first time, we show that S550A, S551A, and S550A/S551A downregulate the majority (~70%) of the NF-κB-dependent genes in PDAC cells. Most of these genes are involved in key oncogenic molecular and cellular functions and are regulated by NF-κB activators . For exaSimilarly, our findings have revealed that S550A and S551A mutations may affect the dynamics of one of the major p65’s major phosphorylation sites in the TAD, S536. This observation was made in both PANC1 and MIA PaCa2 cells, as illustrated in Furthermore, it is worth noting that the S550 and S551 residues are evolutionally conserved across multiple species, indicating the significance of the termini serine residues to p65 function E. From aIn summary, our study reveals that p65 is O-GlcNAcylated at S550 and S551 and is critical to the effective function of the NF-κB signaling pathway in PDAC cells. For the first time, we have provided extensive mechanistic insights into the role of site-specific O-GlcNAcylation on p65 in PDAC. As a result, we anticipate that our findings will serve as a basis for using OGT as a potential and effective therapeutic target to mitigate the oncogenic impact of O-GlcNAcylated p65 in PDAC patients.


Specifically, it modulates mitochondrial activity, and H2S oxidation in mitochondria produces various reactive sulfur species, thus modifying proteins through sulfhydration. This review focused on highlighting the neuron modulation role of H2S in regulating neurodegenerative diseases through anti-oxidative, anti-inflammatory, anti-apoptotic and S-sulfhydration, and emphasized the importance of H2S as a therapeutic molecule for neurological diseases.Neurotoxicity is induced by different toxic substances, including environmental chemicals, drugs, and pathogenic toxins, resulting in oxidative damage and neurodegeneration in mammals. The nervous system is extremely vulnerable to oxidative stress because of its high oxygen demand. Mitochondria are the main source of ATP production in the brain neuron, and oxidative stress-caused mitochondrial dysfunction is implicated in neurodegenerative diseases. H Neurodegenerative diseases in both humans and animals are usually complicated. They can be induced by pharmaceuticals and various environmental chemicals, including antiviral drug tilorone, typhaneoside, metronidazole, homocysteine, paraquat and some heavy metals . In the 2S) has been accepted as a novel gas transmitter, followed by nitric oxide (NO) and carbon monoxide (CO). The earliest discovery of H2S in 1989 reported the endogenous production of H2S and its bioactive properties in mitochondria fraction of rats’ brain .,38.2S frs system  44]. Mo. Mo2S frchondria  9]. Exc. Exc2S fhe brain ,46,47. T2S production are tissue-specific enzymes involving certain biochemical pathways in humans . In. In2S isproteins ,56. Moretabolism . Thus, t2S synthesis are mainly catalyzed by the enzymes CBS, CSE and 3-MST. DAO is a newly discovered enzyme that can produce H2S in mitochondria. H2S oxidation occurs in mitochondria, and H2S is oxidized to persulfide (SQR-SSH) by SQR via accepting a thiol (-SH), after which the persulfide is oxidized to SO32− by ETHE1. The SO32− is oxidized to SO42− and S2O32− by SO and TST, respectively. In addition, H2S is methylated by thiol S-methyltransferase (TMST) to form methanethiol and dimethyl sulfide. The dimethyl sulfide is oxidized to SO42− by rhodanese. Transsulfuration pathway (TSP), which involves the transfer of sulfur from homocysteine to cysteine, can integrate with sulfide-associated metabolic pathways, including methionine cycle, folate cycle, glutathione synthesis and nucleotides metabolism to maintain a physiological balance of H2S. H2S, hydrogen sulfide; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; CAT, cysteine aminotransferase; DAO, D-amino acid oxidase; 3MST, 3-mercaptopyruvate sulfurtransferase; SQR, sulfide quinone oxidoreductase; ETHE1, ethylmalonic encephalopathy 1; TST, thiosulfate sulfurtransferase; SO32−, sulfite; SO42−, sulfate; S2O32−, thiosulfate; TSP, transsulfuration pathway. The pink arrows indicate the transsulfuration pathway (TSP) and related metabolic pathways. This model was created using BioRender .H2S donors NaHS and disodium sulfide (Na2S) can quickly release H2S and display cytoprotective effect by regulating mitochondrial biogenesis and function [2S dependent on acidic pH and higher temperatures [2S generation, which could inhibit endothelial growth factor-A-related diseases [2S donors, H2S can also be generated from natural plants. For example, diallyl disulfide (DADS) and diallyl trisulfide (DATS) derived from Allium plant garlic function as H2S donors, impairing the mitochondrial function [2S donors to clinical experiments, SG1002, a novel H2S prodrug, was clinically used in patients with heart failure. It was found that H2S is essential for vascular homeostasis by preserving mitochondrial functions and increasing myocardial vascular density [2S donor, could improve preeclampsia state through lowering blood pressure and attenuating renal damage in mice model C57Bl/6 J of preeclampsia [2S can protect mitochondrial activity against a high level of antiangiogenic-factor-sFlt-1-induced mitochondrial respiration inhibition and superoxide production in endothelial cells [2S producer sodium thiosulfate exhibits anti-inflammatory and antioxidative effects and has already been investigated in phase I trials for potential benefits in patients with an acute coronary syndrome undergoing coronary angiography [Hfunction ,59. GYY4eratures . It can eratures . Furtherdiseases . Besidesfunction  or decrefunction . Concern density ,66. MZe7clampsia ,68. Meanal cells . H2S proiography .2S donors whose application can regulate glucose-oxidase-induced oxidative stress and improve mitochondrial function in endothelial cells [2S donors can potentially be used in the treatment of neurological diseases in the near future.Recently, it has been reported that both AP39 and AP123 are mitochondrial-targeted Hal cells . It has al cells . Thereby2S in human brain tissue are nearly 50–160 μM, which suggests that H2S may exert a possible biological function in neurons [2S in the brain mainly include potentiating long-term potential (LTP) through activation of the NMDA receptors [2+ influx waves in astrocytes [2S in regulating neurodegenerative diseases through anti-oxidative, anti-inflammatory, or anti-apoptotic effects , resulting in oxidative stress and neurodegeneration [2S may have a vital role in AD. Indeed, it was found that the H2S levels in both plasma and the brain of AD patients were much lower than those in healthy individuals [2S level was negatively correlated with the severity of AD [2S is synthesized from homocysteine via CBS catalyzation in the transsulfuration pathway, and SAM is the activator of CBS [2S might be associated with decreased activity of CBS [2S signaling axis in the AD brain. Thus, dietary adjustment has been proposed to improve symptoms of AD patients, as a diet rich in taurine, cysteine, folate, B12, and betaine could promote H2S synthesis in the brain [AD has been characterized by memory deterioration and cognition impairment, mainly caused by neuropathologic hallmark events, which include enlargement of β-amyloid plaque size and hyperphosphorylation of tau protein in the brain . AD-relaneration ,85. Therividuals ,87. Furtr of CBS . In AD pr of CBS . Therefoy of CBS ,88, whiche brain .2S protected PC12 cells against Hcy or formaldehyde (FA)-induced neurotoxicity by preserving mitochondrial membrane potential (MMP) and attenuating intracellular ROS accumulation [2S donor, has been found to exert a neuroprotective effect in a dose-dependent manner in APP/PS1 neurons by increasing energy production and cell viability, protecting mitochondrial DNA, and decreasing ROS accumulation at 100 nM concentrations, which reduces Aβ deposition in the brain, inhibits brain atrophy, and ameliorates memory deficiency in the Morris water maze experiment [2S-mediated sulfhydration of ATP synthase helps to maintain mitochondrial bioenergetics [2S in modulating AD development. Thus, it is possible that the exogenous application of appropriate concentration of H2S preserves mitochondrial dysfunction in AD by improving mitochondrial redox homeostasis and maintaining energetic production.Mitochondrial dysfunction is characterized as a pathogenesis of AD. Previous studies found that Hmulation ,91. AP39ergetics , implyin2S treatment alleviated the excitotoxicity-triggered oxidative stress by decreasing levels of malondialdehyde in the cerebral cortex of the 3xTg-AD mice model [2S has been explored, revealing that H2S exerts antioxidative effects by activating nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and glutathione S-transferase (GST) in APP/PS1 transgenic mice [2S donator, preserves mitochondrial function and prevents Hcy-induced apoptosis by inhibiting cytochrome C releasing, ROS accumulation, and caspase-3 activation in PC12 cells of the AD mouse model [Meanwhile, it was found that Hce model ,95. Morenic mice . In addinic mice . It has se model .2S exerts its anti-inflammatory effect via several signal pathways, including preserving mitochondrial function in a p38 and JNK-MAPK dependent manner in microglia [2S exerts protective effects through different neuroprotective pathways. For example, it was found that the application of NaHS and Tabiano’s spa water (rich in H2S) can slow down the progression of learning and memory impairment in three experimental models of AD, including brain injection of β-amyloid1-40 (Aβ) or streptozotocin-induced rat models, and in an AD mouse model harboring human transgenes APPSwe, PS1M146V and tauP301L (3xTg-AD mice), by lowering phosphorylation level of tau protein, preventing oxidative and nitrosative stresses in the cerebral cortex, upregulating Bcl-2 and downregulating BAX in the hippocampus [2S donor (NaHS) into APP/PS1 transgenic mouse improves spatial memory acquisition through shifting from the plaque-forming β pathway to the non-plaque forming α pathway of APP cleavage, a non-amyloidogenic processing of APP [2S decreased Aβ deposition in mitochondria by inhibiting γ-secretase activity [2S donor could clearly reduce the size of β-amyloid plaques, and reduce activities of c-jun N-terminal kinases (JNK), extracellular signal-regulated kinases and p38 involved in tau phosphorylation, inflammatory response and apoptosis in the cortex and hippocampus, thus finally protecting AD mice against cognitive impairment [2S by targeting multiple pathophysiological events in AD.Hicroglia , inhibiticroglia , suppresicroglia  in mousepocampus . Moreoveg of APP . It was activity . Overallpairment . These d2S has been shown to exert a neuroprotective effect on account of its antioxidative, anti-inflammatory, and anti-apoptotic properties, and that administration of H2S donors is beneficial for AD, the exact molecular mechanisms underlying the neuroprotective effect are still largely unknown. Glycogen synthase kinase 3β (GSK3β) promotes hyperphosphorylation of tau protein, which finally leads to AD .GSK3β binds to Tau protein and hyper-phosphorylates Tau, which subsequently leads to AD pathology. The application of H2S has an essential anti-oxidative effect in dealing with PD patients suffering from oxidative stress [2S exerts its neuroprotective effects by acting as an antioxidant in the brain of PD patients.PD has been characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and dopamine deficiency in the striatum . Oxidatie stress ,108,109.2S can attenuate rotenone-induced cell apoptosis by preserving mitochondrial function and regulating the JNK-MAPK pathway [2S level was decreased in the SN. On the other hand, the exogenous application of H2S promoted the increase in endogenous H2S and inhibited the movement disorder by reversing the depletion of tyrosine–hydroxylase-positive neurons in the SN. This protective effect may be mediated by promoting leptin signaling and increasing malondialdehyde levels in the striatum [+)-induced mitochondrial transmembrane potential loss, oxidative stress and cell apoptotic in the PD cell model [2S production from the CBS-H2S axis enhances 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or MPP+-induced neurotoxicity in the PD model [2S donor) or overexpression of CBS in the striatum attenuates hallmark pathological event of nitrated α-synuclein in the PD model and rescues motor disorder induced by MPTP [2S application may preserve mitochondrial activity and cell viability in the brain of PD by attenuating nitrate stress induced by post-translational modification of α-synuclein.In addition, rotenone is used to establish models of PD disease in animals. It has been reported that H pathway , which sstriatum ,111. NaHll model . AccordiPD model ,113. Mor by MPTP ,114. Sin2S production by strictly controlling CBS synthesis in astrocytes exhibits anti-inflammatory and neuroprotective effects [2S inhibited microglial activation in SN and prevented the accumulation of inflammatory factors in the striatum in PD model induced by rotenone [2S generated from gut bacteria promotes the formation of α-synuclein fibrils by releasing cytochrome C from mitochondria and stimulating ROS accumulation [2S may contribute to PD disease. According to these findings, H2S exerts an essential physiological role in the brain, mainly depending on strictly controlling physiological H2S concentrations. Except for the application of H2S-releasing donors, it has been reported that inhaled H2S protects MPTP-induced movement disorder in PD mouse model against neurodegeneration via antioxidative, anti-inflammatory, and antiapoptotic signaling in the brain [2S in animal models and protection of dopaminergic neurons associated with PD [Proper endogenous H effects . It was rotenone . Howevermulation , thus suhe brain , which w with PD .2S has been defined as the sulfhydration of specific target proteins. It has been recently reported that H2S increases expression level of silent information regulator-1 , and enhances the activity of SIRT1 through H2S-mediated sulfhydration, which could increase autophagy flux and attenuate cell injury in PD mouse model induced by MPP+ [2S-induced sulfhydration of SIRT1 promotes the deacetylation of PGC-1α, finally resulting in the amelioration of mitochondrial activity and improvement of PD -induced diabetic rat models, and significantly ameliorated the diabetic-associated cognitive disorders by inhibiting ER stress, including preventing expressions of glucose-regulated protein 78 (GRP78), cleaved caspase-12, and C/EBP homologous protein (CHOP) [SIRT1 inhibits the ER stress pathway [2S could improve diabetes-associated cognitive deficits by inhibiting hippocampal ER stress through upregulating SIRT1. However, the mechanisms through which SIRT1 regulates ER still remain unclear. Because the activity of SIRT1 is associated with mitochondrial biogenesis and mitophagy, the ER and mitochondria are tightly contacted at specific subdomains via tethering mediated by mitochondria-associated ER membrane (MAM) proteins, which are critical for determining cell fate [2S may modulate diabetes-related learning and memory decline by regulating ER-mitochondria signaling. Moreover, it was reported that exogenous H2S had anti-apoptotic and anti-inflammatory effects in ameliorating the diabetes-associated cognitive deficit by downregulating mitochondria-mediated pro-apoptotic genes cleaved Caspase-3, cleaved Caspase-9, Bax and cytochrome C, and expressions of IL-23/IL-17 [2S donors with the regulation of mitophagy could be used as a new strategy to improve diabetes-associated cognitive impairment.Numerous studies have pointed out that patients with diabetes are more likely to develop cognitive impairment than healthy individuals , resultidiabetes ,141, sugn (CHOP) . Meanwhin (CHOP) . In the n (CHOP) . Conside pathway , it is pell fate . It can 23/IL-17 . Therefo2S exerts its antioxidant functions by ameliorating oxidative stress and preserving mitochondrial function in neurodegenerative diseases [2S are generally cytoprotective, whereas higher doses are detrimental. For example, amyotrophic lateral sclerosis (ALS) is defined as the selective degeneration of upper and lower motor neurons caused by mutations of Cu/Zn superoxide dismutase 1 (SOD1) genes and dysregulation of mitochondrial complexes II and IV [2S concentrations were increased in astrocytes and microglia and contributed to motor neuron death by inducing intracellular Ca2+ accumulation in motor neurons, and activating Nrf-2-mediated oxidative stress response and peroxiredoxins [2S on neurodegenerative diseases mainly depends on the type of H2S donors and modulators, and the timing of therapy [As is well known, Hdiseases ,108. HowI and IV ,148. Sturedoxins ,150. Intredoxins . Based o therapy ,153,154.2S in protecting neurons by regulating antioxidative, anti-inflammatory and anti-apoptotic responses  or downregulating (marked in red) the antioxidant, antiapoptotic, and anti-inflammation genes, or influencing activities of specific proteins through sulfhydration. Strictly controlling the endogenous H2S production through the administration of mitochondrial-targeted H2S donors and modulation of enzymatic or non-enzymatic mediated H2S metabolism would be beneficial for antioxidative stress and preserving mitochondria activity and biogenesis, thus subsequently improving neurological diseases. This model was created using BioRender .Oxidative damage and mitochondria dysfunction are regarded as the primary cause of neurodegenerative diseases, including AD, PD, HD, DACD, and ALS. H


Drosophila protamines. During spermiogenesis, somatic-type chromatin is taken apart and replaced with sperm chromatin in a multistep process leading to an extraordinary condensation of the genome. During fertilization, the ova face a similarly challenging task of SNBP eviction and reassembly of nucleosome-based chromatin. Despite its importance for the animal life cycle, sperm chromatin metabolism, including the biochemical machinery mediating the mutual replacement of histones and SNBPs, remains poorly studied. In Drosophila, Mst77F is one of the first SNBPs loaded into the spermatid nuclei. It persists in mature spermatozoa and is essential for sperm compaction and male fertility. Here, by using in vitro biochemical assays, we identify chaperones that can mediate the eviction and loading of Mst77F on DNA, thus facilitating the interconversions of chromatin forms in the male gamete. Unlike NAP1 and TAP/p32 chaperones that disassemble Mst77F–DNA complexes, ARTEMIS and APOLLO, orthologs of mammalian importin-4 (IPO4), mediate the deposition of Mst77F on DNA or oligonucleosome templates, accompanied by the dissociation of histone–DNA complexes. In vivo, a mutation of testis-specific Apollo brings about a defect of Mst77F loading, abnormal sperm morphology, and male infertility. We identify IPO4 ortholog APOLLO as a critical component of sperm chromatin assembly apparatus in Drosophila. We discover that in addition to recognized roles in protein traffic, a nuclear transport receptor (IPO4) can function directly in chromatin remodeling as a dual, histone- and SNBP-specific, chaperone.DNA in sperm is packed with small, charged proteins termed SNBPs (sperm nuclear basic proteins), including mammalian and  Drosophila is a powerful alternative model to study the histone-to-protamine transition. Drosophila melanogaster sperm chromatin comprises several highly basic, protamine-like SNBPs that all share an MST-HMG-Box motif , Prtl99C and male-specific transcript 77F (Mst77F), generally cooperate to achieve proper sperm chromatin organization  that replace nucleosomes during spermiogenesis, the differentiation of haploid spermatids. While mammalian protamines are the best characterized SNBPs, ox motif , 2, 3. Gnization , 5, 6. IKaryopherins (importins) are a superfamily of conserved receptor proteins that mediate the post-synthetic transport of large (>50 kDa) nuclear proteins from the cytoplasm across the nuclear pore complex into the nucleus , 8. AccoD. melanogaster genome encompasses tandem duplicate genes Apollo (Apl) and Artemis (Arts), encoding nearly identical  proteins APOLLO/APL and ARTEMIS/ARTS, orthologs of a mammalian importin β, importin-4/IPO4 . The Mst77F–DNA complex was reconstituted by dialysis with a supercoiled plasmid and used as a substrate for remodeling by embryonic S-190 extract in vitro (B). The association of Mst77F-V5 with the plasmid DNA was assayed by sucrose gradient sedimentation and V5 western analyses of the gradient fractions (B and C). Similar to protamine–DNA complexes . Since molecular chaperones that remove Mst77F from DNA likely remain associated with it upon eviction (B and C) and analyzed them by SDS-PAGE . As a control, pulldown experiments were also performed with equivalent fractions from Mst77F-free, S-190-only sucrose gradients . The identities of purified polypeptides were established by mass-spec sequencing (A).To isolate chaperone molecules for Mst77F, we followed the approach previously utilized to identify chaperones for nd ProtB . We exprnd ProtB A. The Msin vitro B. The asractions , B and Complexes , the Msteviction , we puriquencing A.Figure Drosophila ortholog of IPO4. To confirm their physical interactions with Mst77F, we expressed recombinant ARTS, NAP1, NLP, and TAP/p32 (B) and examined them by co-immunoprecipitation (co-IP) with free Mst77F-V5 (C). We discovered that each individual polypeptide directly interacted with Mst77F in solution.In addition to V5-tagged Mst77F, the immunoprecipitated proteins included NAP1, TAP/p32, and NLP, the histone chaperones that were previously implicated in the remodeling of ProtA/B-DNA substrates . Moreove TAP/p32 B and exast77F-V5 C. We disin vitro (A). In these experiments, we used size-exclusion chromatography to separate released Mst77F from the Mst77F–DNA complex (A–C), instead of sucrose gradients (C), which are more suitable for large-scale bulk reactions. As expected, the Mst77F-DNA substrate alone fractionated in early (heavy) fractions . However, when treated with super-stoichiometric recombinant NAP1 or TAP/p32, the Mst77F–DNA complex was dissociated, and Mst77F fractionated in later (light) fractions (B). In contrast, ARTS and NLP alone failed to efficiently remodel the substrate.To further characterize the molecular functions of the identified Mst77F chaperones, we examined the activities of recombinant proteins in the eviction of Mst77F from its complex with DNA in vitro A. In the complex , A–C, inradients C, which ractions B, top. Hractions B. In conC) and the discovery of native ARTS and NLP in association with Mst77F that was released from the Mst77F–DNA complex upon remodeling by S-190 (D). However, the contradiction may be resolved if, in the S-190 remodeling reactions, Mst77F is exchanged between NAP1 or TAP/p32 (the true eviction factors) and ARTS or NLP. For instance, when the eviction factors are limiting (substoichiometric to Mst77F), this exchange would liberate NAP1 and TAP/p32 for additional rounds of Mst77F eviction, thus stimulating the reaction. Indeed, sub-stoichiometric NAP1 remodels the Mst77F-DNA substrate inefficiently . However, an excess of NLP in addition to NAP1 strongly stimulated the activity of NAP1 .This finding poses an apparent contradiction with the efficient binding of recombinant ARTS and NLP to Mst77F C and theby S-190 D. HoweveC, middle). It also partially reversed the near complete remodeling of the Mst77F–DNA substrate by super-stoichiometric NAP1 and TAP/p32 (cf.2B). This result is consistent with the ability of ARTS to load Mst77F on the DNA, rather than release it from the Mst77F–DNA complex. The proposed molecular roles of NAP1, TAP/p32, ARTS, and NLP in the metabolism of the Mst77F–DNA complex are schematized in Figure 2D.In contrast, an excess of ARTS failed to stimulate the NAP1-mediated release of Mst77F, but, to the contrary, strongly inhibited the reaction C, middle/p32 cf. and 2B. In vivo, ARTS is primarily expressed in the ovary (A) and performed loading experiments in vitro. Briefly, Mst77F–APL complexes were incubated with plasmid DNA, and Mst77F association with the DNA was analyzed by size-exclusion chromatography and western blot of fractions (A). As anticipated, both APL and ARTS facilitated the assembly of the Mst77F–DNA complex in vitro, unlike NAP1, TAP/p32, or NLP (B). For instance, in the absence of an acceptor DNA template, APL cofractionated with Mst77F-V5 (B). However, upon addition of the plasmid DNA, Mst77F-V5 was loaded on the template, whereas APL remained in the low molecular mass column fractions (B).he ovary . Therefohe ovary . As oppohe ovary A and perractions A. As ant, or NLP B. For inst77F-V5 B. Howeveractions B.Figure in vivo. Importantly, the physiological substrate of APL is a nucleoprotein complex, rather than naked DNA. Therefore, we examined the loading of Mst77F on the in vitro reconstituted oligonucleosome substrate and observed an efficient association of Mst77F with DNA/chromatin in the presence of APL (C). Furthermore, Mst77F deposition in vivo must be accompanied by the disassembly of chromatin and removal of core histones (and other chromatin components). It is possible that APL can mediate both the Mst77F deposition and core histone eviction. Indeed, when gel filtration fractions were analyzed by SDS-PAGE, we discovered that core histones were removed from oligonucleosomes by APL and cofractionated with it (D). The nucleosome disassembly did not depend on Mst77F and could be mediated by APL alone .This observation suggests that APL may mediate the deposition of Mst77F on DNA e of APL C. Furthe with it D. The nuin vitro, APL mediates the exchange of core histones for Mst77F (E). This mechanism indicates that APL also functions as a core histone chaperone. It further suggests that APL must exhibit a higher affinity toward core histones than toward Mst77F. To further confirm this idea, we examined the binding of APL to Mst77F and/or purified core histones by glycerol gradient sedimentation. Whereas both free Mst77F and core histones sedimented at the top of the gradient (C), substoichiometric APL interacted with a fraction of Mst77F and shifted its sedimentation toward the middle of the gradient (D). As predicted, core histones, when mixed with Mst77F, efficiently competed with it for binding to the limiting APL (E). This result strongly supports the proposed model for the function of APL in the concomitant disassembly of nucleosomes and assembly of Mst77F–DNA complexes (E).Thus, r Mst77F E. This mgradient C, substogradient D. As preting APL E. This romplexes E.Drosophila IPO4 orthologs ARTS and APL may play in sperm chromatin metabolism and loading of Mst77F in sperm chromatin in vivo, we used CRISPR/Cas9 to prepare fly mutant alleles that inactivated both Apl and Arts. A guide RNA complementary to both genes (A) was expressed in the female germline together with the Cas9 enzyme (Apl and Arts were recovered (A). Df(3L)IPO4[1] encompasses a deficiency completely deleting Apl (and flanking sequences) as well as a small deletion (indel) in Arts, resulting in a frameshift in its open reading frame. Df(3L)IPO4[2] encompasses a deficiency uncovering the central portion of the Apl–Arts locus; there is also an indel producing a frameshift in the putative Apl–Arts “fusion gene”. Additionally, IPO4[3] contains frame-shifting indels in both genes (A). Since the frameshifts eliminate the protein-binding KAP95 domains of both proteins, we expected these mutations to result in the loss of function for both genes.To analyze the roles that th genes A was exp9 enzyme . Three aecovered A. Df(3L)th genes A. Since A) that recognizes both recombinant APL and ARTS (A). When lysates of whole adult males (strongly expressing APL in the testis) and adult females (strongly expressing ARTS in ovaries) were examined by western blot, the antibody specifically stained an ∼120-kDa band in wildtype animals but failed to detect APL or ARTS in Df(3L)IPO4[1] and Df(3L)IPO4[2] (B). Consistent with the putative function of APL in loading of Mst77F into sperm chromatin, IPO4-C antibody strongly stained elongating spermatid nuclei of wildtype males (B). In contrast, no nuclear staining was evident in mutant spermatids (B). Also, as expected, robust cytoplasmic staining of ARTS was observed in wildtype but not Df(3L)IPO4[1] or Df(3L)IPO4[2] ovaries (C).We also raised a polyclonal antibody IPO4-C, A that reand ARTS A. When l)IPO4[2] B. Consispe males B. In conermatids B. Also,  ovaries C.C). In addition, no mature, needle-shaped sperm was observed in their seminal vesicles (D). Early spermatogenesis appeared normal in homozygous Df(3L)IPO4[2] males, but shortly after the histone-to-SNBP transition, spermatid nuclei all appeared abnormally shaped and condensed. The spermiogenic phenotype appeared much more severe in Df(3L)IPO4[1] mutants, with testes containing rare, scattered spermatid nuclei. Interestingly, in both mutants, while ProtB was nevertheless loaded in spermatid nuclei, Mst77F remained undetected by immunofluorescence (D). It has been previously reported that Apl null mutations do not affect the loading of Mst77F in spermatid chromatin . However, by intentionally increasing the intensity of red florescence detection on confocal images (10-fold over that used for control samples), mRFP1-Mst77F could be detected in mutant spermatids (D), indicative of a very low-level deposition of Mst77F. This result may explain the apparent discrepancy between the two studies.Homozygous mutant males were completely sterile, producing no progeny in crosses with control females C. In addvesicles D. Early rescence D. It hashromatin . In cont reduced E. Howeveermatids D, indicaDf(3L)IPO4[1] mutation impairs the female fertility (E), despite neither allele expressing the full-length ARTS (B and C). The result seemingly contradicts the previous report that Arts is required for female fertility . Thus, the truncated protein is likely sufficient to mediate the function(s) of ARTS in the egg. We also noted that the female sterile Df(3L)IPO4[1] fails to manifest the “round egg” phenotype observed for the progeny of homozygous Arts[null1] mothers (data not shown). Combined, these observations indicate that the female sterility of Df(3L)IPO4[1] does not arise from the mutation of Arts. Despite a high homology between spd-2 and CR18217 nucleotide sequences flanking Apl near the distal breakpoint in Df(3L)IPO4[1], CR18217 transcript cannot be translated into a functional protein due to the presence of multiple stop codons. For instance, there is a small 31-bp deletion in the CR18217 sequence, compared to that of spd-2 (F). This deletion is retained in the Df(3L)IPO4[1] genome and predicted to bring about a premature termination of any hypothetical fusion protein and thus inactivation of spd-2 (F). Notably, spd-2-null mutant has been shown to exhibit male and female sterility (spd-2 may explain the sterility of Df(3L)IPO4[1] females (E) as well as the stronger spermiogenesis failure phenotype in the Df(3L)IPO4[1] testis (D).Unexpectedly, whereas all three mutant alleles are male sterile, only ertility E, despitgth ARTS , B and Certility . However protein A. Thus,  mothers   and replacement-type (H3.3) histones . Thus, iApl or IPO4 mutants (not shown). This finding is consistent with Mst77F representing only a fraction of the SNBP composition of sperm chromatin, so that only a percentage of total histones need to be evicted to accommodate for the newly deposited Mst77F. The deposition of Mst77F in the late canoe stage of spermiogenesis largely coincides with that of the transition protein Tpl94D , predictably so, considering the near structural identity of the two proteins. However, it is highly unlikely that this biochemical activity of ARTS has physiological consequences for chromatin structure in the Drosophila egg, where it is abundantly present. First, in steady-state Mst77F binding experiments with embryonic extracts (D), Mst77F-dissociating chaperones NAP1 and TAP/p32 appear to be in >5-fold molar excess compared to the Mst77F-loading ARTS. The reaction equilibrium (D and 3E) is further shifted toward the removal of Mst77F by a much greater concentration of core histones  than that of Mst77F (contributed by a single sperm cell in the fertilized egg). Thus, rather than the disassembly of nucleosomes and deposition of Mst77F, ARTS may be required in the embryo to play other roles, such as the regulation or nuclear transport of essential components of actin networks, as proposed  in SW-28 rotor (Beckman) at 4 °C. As controls, S-190 extract alone or DNA-Mst77F mixed with 20 mg nuclease-free BSA (New England Biolabs) were similarly analyzed by sucrose gradient sedimentation. The gradients were cut in 3-ml fractions, and the fractions  were analyzed by anti-V5 western blotting. Mouse monoclonal V5 antibody (Sigma) and infrared dye-labeled secondary antibody (LI-COR Bioscience) were used at 1:5000 and 1:10,000, respectively.DNA-Mst77F substrate see  equivaleFor purification of putative Mst77F chaperones, gradient fractions from the top of the gradients that contained V5-immunoreactive material were pooled and immunoprecipitated with 20 μl anti-V5 agarose (Sigma) as described . SimilarPhysical interactions between Mst77F and chaperones were analyzed by co-IP and glycerol gradient cosedimentation of recombinant proteins. The association of Mst77F with and its release from DNA was examined by size-exclusion chromatography and western with anti-V5 antibody as described . See SupDrosophila testes and ovaries were stained with IPO4-C, anti-Mst77F, anti-Mst35B, or anti-histone antibodies exactly as described (escribed , 30. SeeAll data are included in the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.