Staphylococcus aureus has to cope with oxidative stress during infections. In this study, S. aureus was found to be resistant to 100 mM H2O2 during aerobic growth. While KatA was essential for this high aerobic H2O2 resistance, the peroxiredoxin AhpC contributed to detoxification of 0.4 mM H2O2 in the absence of KatA. In addition, the peroxiredoxins AhpC, Tpx and Bcp were found to be required for detoxification of cumene hydroperoxide (CHP). The high H2O2 tolerance of aerobic S. aureus cells was associated with priming by endogenous H2O2 levels, which was supported by an oxidative shift of the bacillithiol redox potential to −291 mV compared to −310 mV in microaerophilic cells. In contrast, S. aureus could be primed by sub-lethal doses of 100 µM H2O2 during microaerophilic growth to acquire an improved resistance towards the otherwise lethal triggering stimulus of 10 mM H2O2. This microaerophilic priming was dependent on increased KatA activity, whereas aerobic cells showed constitutive high KatA activity. Thus, KatA contributes to the high H2O2 resistance of aerobic cells and to microaerophilic H2O2 priming in order to survive the subsequent lethal triggering doses of H2O2, allowing the adaptation of S. aureus under infections to different oxygen environments. Staphylococcus aureus is a major human pathogen that can cause local skin and soft tissue infections, as well as life-threatening diseases, such as septicaemia, endocarditis, necrotizing pneumonia and osteomyelitis [S. aureus has to cope with reactive oxygen species (ROS), such as superoxide anion (O2•−) and hydrogen peroxide (H2O2) [2) to generate O2•−, which is dismutated to H2O2 either spontaneously or by superoxide dismutases (SODs), including the extracellular SOD3 [2O2 production [2•− to H2O2 upon infection [2O2 with chloride to the highly reactive hypochlorous acid (HOCl), which is the most potent oxidant and microbicidal agent released by activated neutrophils [S. aureus encounters endogenous ROS during aerobic respiration due to the stepwise one-electron transfer reactions to O2, leading to production of O2•− and H2O2 [2O2 reacts with free Fe2+ to generate the highly toxic hydroxyl radical (OH•), which can damage all cellular macromolecules, resulting in oxidation of proteins, lipids and carbohydrates [2O2 and HOCl can also function in redox signalling to activate or inactivate specific redox-sensitive regulators, which control defence mechanisms and confer resistance against the oxidants in bacterial pathogens [myelitis ,2,3. Dure (H2O2) , which ae (H2O2) ,6,7,8. Tlar SOD3 ,10,11. Btrophils ,10. In aand H2O2 . In the hydrates ,13,14. Hathogens .S. aureus uses various enzymatic and non-enzymatic ROS and HOCl detoxification systems, such as a unique catalase (KatA), several peroxiredoxins  and the low-molecular-weight (LMW) thiol bacillithiol (BSH) [EBSH) under oxidative stress in S. aureus [S. aureus is highly resistant to 100 mM H2O2, since the Brx-roGFP2 biosensor responded only weakly to high H2O2 levels, leading to small EBSH changes [2O2 detoxification enzyme, which conferred the constitutive H2O2-resistant phenotype to aerobically grown S. aureus cells [2O2 and organic hydroperoxides (OHPs) and contributed to nasal colonization [ol (BSH) ,16. BSH . aureus . This st changes . The catus cells ,19. KatAus cells ,20,21,22nization .P) and resolving Cys (CR), involving inter- or intramolecular disulfides, respectively [Escherichia coli. AhpC detoxification of H2O2 leads to formation of an oxidized AhpC dimer, which aggregates to an oligomer with chaperone functions [E. coli is induced by H2O2, OHPs and during aerobic growth and confers resistance against H2O2 and OHP stress [E. coli has been shown to catalyse detoxification of H2O2 and OHPs in vitro and is recycled by the Trx/TrxR system [S. aureus, Tpx responds strongly to H2O2 and other thiol-reactive compounds and was oxidized in the redox proteome under HOCl stress [S. aureus.In general, AhpC, Tpx and Bcp can be classified into typical (AhpC) or atypical  2-Cys peroxiredoxins based on their thiol-oxidation mechanism between the peroxidatic  medium. S. aureus COL strains were grown in RPMI medium to an optical density at 500 nm (OD500) of 0.5 and exposed to H2O2, cumene hydroperoxide (CHP) or hypochlorous acid (HOCl), followed by determination of colony-forming units (CFUs) in survival assays as previously described [t-test with the software Graph Prism. The biochemical compounds were purchased from Sigma Aldrich. The HOCl concentration was determined as previously described [Bacterial strains, plasmids and primers are described in escribed . Each exescribed .S. aureus COL ΔkatA mutant and katA complemented strains were previously constructed [S. aureus ΔahpC, Δtpx, Δbcp and ΔperR deletion mutants were constructed using the temperature-sensitive E. coli-S. aureus shuttle vector pMAD as previously described [BglII and SalI and ligated into pMAD. The constructs were electroporated into the restriction-negative S. aureus RN4220, followed by phage transduction using phage 81 into S. aureus COL [ahpCΔkatA double mutant, the plasmid pMAD-∆katA of S. aureus RN4220-pMAD-∆katA was transduced by the phage 81 into the S. aureus COL ΔahpC mutant. Selection of the ΔahpC, Δbcp, Δtpx, ΔperR and ΔahpCΔkatA deletion mutants was performed as previously described [The structed . The S. escribed . In brieescribed .S. aureus ahpC, bcp and tpx complemented strains was performed using the plasmid pRB473 as previously described [BamHI and KpnI/SacI, resulting in plasmids pRB473-ahpC-His, pRB473-bcp-His and pRB473-tpx-His, which were transduced in the ΔahpC, Δbcp and Δtpx deletion mutants. In addition, the plasmid pRB473-brx-roGFP2 [S. aureus COL ΔkatA, ΔahpC, ΔahpCΔkatA, Δtpx and Δbcp deletion mutants to construct the Brx-roGFP2 biosensor expressing catalase- and peroxiredoxin-deficient mutant strains.Construction of the His-tagged escribed . The genx-roGFP2  was intrS. aureus strains were grown aerobically under vigorous agitation in shake flasks in a shaking water bath at 150 rpm or microaerophilically in 50 mL Falcon tubes including 40 mL cultures with closed lids without shaking, as in previous publications [500 of 0.3, naïve S. aureus cells were primed by adding sub-lethal doses of 0.1 or 1 mM H2O2, respectively, to the bacterial culture for ~30 min. Subsequently, the lethal triggering doses of 10 or 40 mM H2O2, respectively, were added to the primed bacterial cultures, followed by counting of CFUs after 2 and 4 h of growth. For triggering only, naïve cells were treated with 10 or 40 mM H2O2 at an OD500 of 0.4, followed by counting of CFUs after 2 and 4 h of growth.For priming and triggering, the ications ,44. At aEBSH changes after H2O2 and CHP stress, we used the Brx-roGFP2 biosensor expressing WT, ΔkatA, ΔahpC, ΔahpCΔkatA, Δtpx and Δbcp mutant strains and performed injection assays with the oxidants. For measurements of Brx-roGFP2 oxidation during microaerophilic and aerobic H2O2 priming and triggering experiments, the S. aureus COL strain expressing Brx-roGFP2 was cultivated in LB medium to an OD540 of 0.3 and challenged with the priming dose of 0.1 mM H2O2 for 30 min, followed by the triggering dose of 10 mM H2O2, as described above. Samples were harvested from S. aureus cells in the naïve (C), primed (P), primed and triggered (PT) and triggered-only (T) states, alkylated with 10 mM N-ethylmaleimide (NEM), washed and resuspended in PBS with 10 mM NEM. The Brx-roGFP2 oxidation degree (OxD) and EBSH changes were determined in the S. aureus strains during oxidant injection or in samples harvested at C, P, PT and T as previously described [To monitor the escribed ,45. For escribed ,45.katA, dps and ahpCF in the S. aureus COL WT, ΔkatA, ΔahpC and ΔperR mutants using Northern blots, the S. aureus strains were grown in RPMI medium and harvested during the log phase at an OD500 of 0.4. To investigate katA and ahpC induction in the priming and triggering experiments, S. aureus WT cells were harvested in the naïve (C), primed (P), primed and triggered (PT) and triggered-only (T) states, as explained in the figure legends. RNA isolation was performed using the acid phenol extraction protocol as described previously [katA, ahpC and dps that were synthesized in vitro using T7 RNA polymerase and the corresponding primers katA-NB-for/rev and ahpC-NB-for/rev, as previously described [dps antisense RNA probe was constructed previously [To analyze transcription of eviously . Northerescribed . The dpseviously .S. aureus COL strains, protein extracts were prepared under native conditions and 50 µg of each sample was separated by non-denaturing 10% polyacrylamide gel electrophoresis. The gel was stained for catalase activity using 50 µg/mL horseradish peroxidase coupled with 5 mM H2O2 and 0.5 mg/mL diaminobenzidine, as described previously [To analyze catalase activities in eviously ,48.2O2 resistance of S. aureus COL, phenotype analyses of the ΔkatA, ΔahpC, ΔahpCΔkatA, Δtpx and Δbcp mutants and the complemented strains were performed during the aerobic growth under H2O2 stress (katA mutant was strongly impaired in growth after exposure to 10 mM H2O2 and did not survive doses of 40 mM H2O2 (S. aureus COL wild type (WT) was able to grow with low doses of 1 mM H2O2 and survived to 330 and 725% after 2 and 4 h, respectively  were not impaired in growth and survival under 0.1 mM H2O2 (2O2 compared to the triggering-only state (T)  B,D. Specc growth B,D. Howenditions C,E. Bothstimulus C,E.katA complemented strain did not recover the improved H2O2 resistance upon microaerophilic priming, resulting in growth inhibition and killing of PT and T bacteria  and triggering (10 mM) experiments as applied in the microaerophilic experiments  and triggering (40 mM) of S. aureus during the aerobic growth  compared to the OxD of 0.3 and the EBSH of −291 mV during aerobic growth (C1)  I,J. Thusbacteria I,J. Thespression I,J. Thes2O2 resistance and microaerophilic H2O2 priming was clearly revealed, the peroxiredoxins AhpC, Tpx and Bcp could also function in the resistance to organic hydroperoxides in S. aureus. Using growth and survival assays, the phenotypes of the catalase and peroxiredoxin-deficient mutants were analysed after CHP treatment (katA and Δbcp mutants was not affected by 0.15 mM CHP stress, the CHP-treated ΔahpC and Δtpx mutants showed slightly reduced growth rates (ahpC, Δtpx and Δbcp mutants showed significantly decreased survival rates after 4 h of CHP stress, supporting that the peroxiredoxins confer protection against CHP stress in S. aureus (ahpC, tpx and bcp complemented strains (katA mutant could not be reverted to WT levels in the katA complemented strain (While the role of KatA in aerobic Hreatment . While tth rates A–D, whicth rates E,F. In a. aureus G. These  strains H. Howeved strain G, probabd strain .EBSH in S. aureus, Brx-roGFP2 biosensor measurements were performed after 0.5 mM CHP stress (katA, ΔahpC, Δtpx and Δbcp mutants. However, while the WT and ΔkatA mutant could regenerate the reduced state of EBSH within 2 h, the ΔahpC mutant was unable to regenerate the basal level of EBSH during the recovery phase from CHP stress (bcp and Δtpx mutants showed significant delays in recovery of the reduced state of EBSH upon CHP stress as compared to the WT (EBSH during the recovery phase from CHP stress in S. aureus. In contrast, KatA does not contribute to CHP detoxification and resistance in S. aureus.To investigate the impact of the peroxiredoxins in the maintenance of the reduced state of P stress . The BrxP stress A,B. In ao the WT C,D. ThesS. aureus [katA, ΔahpC, Δtpx and Δbcp mutants under HOCl stress. However, none of these mutants showed significant defects in growth or survival after HOCl stress compared to the WT, indicating that the catalase and peroxiredoxins do not contribute to HOCl detoxification and resistance in S. aureus (Previous transcriptome analyses revealed an increased transcription of the PerR regulon under HOCl stress in . aureus . Thus, g. aureus .S. aureus during aerobic and microaerophilic growth. Our results revealed that S. aureus is not primable towards improved H2O2 resistance during aerobic growth due to its constitutive H2O2 resistance, which was shown to be dependent on the catalase KatA. Moreover, we found that microaerophilic S. aureus cells can be primed to acquire an enhanced resistance towards lethal H2O2 doses, which was also mediated by KatA (S. aureus to ensure the maintenance of the redox balance upon recovery from stress.Here, we investigated the roles of catalase and peroxiredoxins in the peroxide stress resistance and priming of  by KatA B,D. In aS. aureus have been previously demonstrated [2O2 detoxification and regeneration of reduced EBSH under oxidative stress. Without KatA, S. aureus cells are highly sensitive to oxidants and only able to remove low doses of 0.4 mM H2O2, which were shown to be detoxified by AhpC. In addition, the ΔahpCΔkatA double mutant showed an increased sensitivity towards H2O2 stress in survival assays as compared to the ΔkatA mutant. These data confirm that KatA and AhpCF have compensatory roles in H2O2 resistance to ensure the survival of S. aureus under oxidative stress [katA, bcp, dps and the ahpCF operon is controlled by the peroxide-sensing PerR repressor, which is inactivated by H2O2 due to Fe2+-catalysed histidine oxidation in S. aureus [ahpC mutant, katA and dps expression was elevated, as confirmed here using Northern blots and shown previously [ahpC mutant mediates the enhanced H2O2 resistance, confirming previous results in S. aureus and B. subtilis [The roles of KatA and AhpC in the peroxide resistance of nstrated . In thise stress . Express. aureus ,52,53. Deviously . Thus, tsubtilis ,54,55.S. aureus acquire an improved resistance to H2O2 during the stationary phase, which also depends on KatA. The oxidative stress resistance was also enhanced during the stationary phase in other bacteria, such as B. subtilis and E. coli [S. aureus and B. subtilis [S. aureus cells reveal that KatA is the major player that confers the strong constitutive H2O2 resistance during the log and stationary phases, while the peroxiredoxin AhpC plays an additional role of scavenging 0.4 mM H2O2 in the absence of KatA. Apart from its major role as an H2O2 scavenger, the catalase also provides heme and iron as cofactors for cellular metabolism, which could contribute to S. aureus survival under oxidative stress.In addition, we showed that aerobically grown  E. coli ,57. In asubtilis ,48. AltoS. aureus is primable with sub-lethal doses of 0.1 mM H2O2 to acquire an improved resistance towards otherwise lethal doses of 10 mM H2O2 during the microaerophilic growth. This microaerophilic H2O2 priming was found to be dependent on KatA, which was transcriptionally induced and showed a higher catalase activity upon challenge with the priming dose. These results are consistent with previous data showing increased KatA activity by exposure to 100 µM H2O2 during oxygen limitation [S. aureus. In contrast, due to their constitutive H2O2 resistance, aerobic S. aureus cells were not primable to acquire higher resistance towards lethal H2O2 doses. Aerobic priming was not possible with 0.1 mM or 1 mM H2O2 since katA transcription and catalase activity were already elevated in naïve cells and could not be further increased in primed cells. The higher basal level of EBSH of −291 mV in aerobic cells compared to the more reducing basal EBSH of −310 mV in microaerophilic cells strongly indicates an increased ROS level in aerobic cells due to aerobic respiration. These biosensor data support that S. aureus is primed by endogenous ROS generated by aerobic respiration to achieve their constitutive H2O2 resistance phenotype (2O2-resistant phenotype. We further showed that mitation . We showhenotype . Thus, tS. aureus during the aerobic growth was caused by the hypersensitive PerR repressor, which is poised by very low endogenous levels of H2O2 generated during aerobic respiration [2O2 concentration in aerobic E. coli cells was determined as ~50 nM [S. aureus is hypersensitive to endogenous H2O2 levels during aerobic growth, the PerR protein of B. subtilis is less sensitive and cannot sense such low H2O2 levels originating from aerobic respiration [B. subtilis can be primed during aerobic growth with 0.1 mM H2O2, leading to PerR inactivation and derepression of the PerR-controlled KatA, which confers an adaptive resistance against the otherwise lethal triggering dose of 10 mM H2O2 [This up-regulation of the PerR regulon in piration . The ends ~50 nM . While Ppiration . Thus, B mM H2O2 ,52,55,59E. coli and S. Typhimurium after exposure to 100 µM H2O2, leading to activation of the OxyR regulon, including the major catalase, which confers an adaptive and improved resistance towards lethal concentrations of 10 mM H2O2 [2O2 resistance depends on the sensitivity of the redox-sensing peroxide regulators. While many bacteria harbour less sensitive H2O2 sensors and are primable by sub-lethal H2O2 doses during aerobic growth, S. aureus PerR is hypersensitive to endogenous H2O2 levels during aerobic growth [2O2 resistance and microaerophilic priming to acquire improved H2O2 resistance only in oxygen-limited conditions (Similarly, aerobic priming is possible in  mM H2O2 ,57,60. Ic growth , resultinditions .2O2 and confers the high resistance to 100 mM H2O2 in aerobic S. aureus cells, the peroxiredoxin AhpC was shown to enable the detoxification of low levels of 0.4 mM H2O2, which could originate from the aerobic respiration. Thus, our results support that catalases are scavengers of high mM levels of H2O2, whereas peroxiredoxins can only detoxify physiological µM H2O2 levels. However, the levels of H2O2 experienced by S. aureus during the oxidative burst were determined in the range of ~2 µM inside the phagosomes of neutrophils and macrophages [2O2 concentration was estimated in the range of 1–100 nM, while the extracellular H2O2 was 100-fold higher [S. aureus might not experience such high doses of 10–100 mM H2O2 during interaction with immune cells. However, as a commensal bacterium, S. aureus colonizes the anterior nares and the nasopharynx together with competing microbes, such as Streptococcus pneumoniae, which generates millimolar levels of H2O2 by the lactate and pyruvate oxidases to kill competitive bacteria [S. aureus [katA mutant was impaired in survival towards H2O2 produced by S. pneumoniae during nasal colonization [2O2 priming of S. aureus might provide an advantage in its ecological niche to resist the high H2O2 levels generated by competing microbes.While KatA is responsible for detoxification of up to 100 mM Hrophages ,61,62. Id higher . Thus, Sbacteria ,67,68,69. aureus . Furthernization ,70. ThusS. aureus. However, KatA might contribute to lowering external H2O2 and, subsequently, HOCl levels in the neutrophil phagosome. In support of this notion, KatA was found to be induced upon macrophage infection and to be essential for survival of S. aureus inside macrophages [S. aureus strains with high catalase activity were more resistant to killing by neutrophils compared to strains with lower catalase activity [S. aureus against the respiratory burst of macrophages and neutrophils.In contrast, KatA and the peroxiredoxins are not directly involved in the defence against HOCl stress in rophages . In addiactivity . These dS. aureus cells. Using Brx-roGFP2 biosensor measurements, the peroxiredoxin-deficient ΔahpC, Δtpx and Δbcp mutants showed delayed regeneration of the reduced state of EBSH after recovery from CHP stress. Thus, AhpC, Tpx and Bcp function in CHP detoxification and contribute to the maintenance of the cellular redox balance. The role of AhpC in CHP resistance has been previously demonstrated in S. aureus [B. subtilis, E. coli and S. Typhimurium conferred resistance towards CHP stress [E. coli, the Δtpx and Δbcp mutants were more sensitive towards various OHPs, indicating that these peroxiredoxins are more specific to reduction of organic peroxide substrates [E. coli Tpx protein demonstrated the substrate specificity towards alkyl hydroperoxides over H2O2 [E. coli has a fivefold higher Vmax/Km value for linoleic acid hydroperoxide as a substrate compared to H2O2 [In addition, the peroxiredoxins AhpC, Tpx and Bcp were found to confer protection against CHP stress in . aureus . SimilarP stress ,71. In Ebstrates ,28. Kinever H2O2 . Similar to H2O2 .2O2 resistance in S. aureus and mediates priming to endogenous ROS levels generated during aerobic respiration to confer the constitutive H2O2 resistance towards the triggering stimulus in aerobic cells. In addition, KatA mediates microaerophilic priming by low H2O2 levels to prepare S. aureus cells for improved and adaptive resistance against otherwise lethal H2O2 doses. Furthermore, the peroxiredoxins AhpC, Tpx and Bcp were shown to contribute to CHP detoxification and resistance to ensure the survival and regeneration of the reduced EBSH in S. aureus (S. aureus.Taken together, we have shown that the catalase KatA is the major player in aerobic H. aureus . In futu


GLUT10G128E variant (GLUT10G128E mice), which are highly sensitive to high-fat diet (HFD)-induced metabolic dysregulation. The genetic variant has high relevance to human populations, as genetic polymorphisms in glucose transporter 10 (GLUT10) are associated with a T2DM intermediate phenotype in nondiabetic population.The development of type 2 diabetes mellitus (T2DM) is highly influenced by complex interactions between genetic and environmental (dietary and lifestyle) factors. While vitamin C  has been suggested as a complementary nutritional treatment for T2DM, evidence for the significance and beneficial effects of AA in T2DM is thus far inconclusive. We suspect that clinical studies on the topic might need to account for combination of genetic and dietary factors that could influence AA effects on metabolism. In this study, we tested this general idea using a mouse model with genetic predisposition to diet-induced metabolic dysfunction. In particular, we utilized mice carrying a human orthologous GLUT10G128E mice fed with a normal diet or HFD. Overall, the beneficial effects of AA on metabolism were greater in HFD-fed GLUT10G128E mice than in HFD-fed WT mice. At early postnatal stages, AA improved the development of compromised epididymal white adipose tissue (eWAT) in GLUT10G128E mice. In adult animals, AA supplementation attenuated the predisposition of GLUT10G128E mice to HFD-triggered eWAT inflammation, adipokine dysregulation, ectopic fatty acid accumulation, metabolic dysregulation, and body weight gain, as compared with WT mice.We investigated the impacts of AA supplementation on metabolism in wild-type (WT) mice and GLUT10G128E mice than HFD-fed WT mice. As such, AA plays an important role in supporting eWAT development and attenuating HFD-induced metabolic dysregulation in GLUT10G128E mice. Our results suggest that proper WAT development is essential for metabolic regulation later in life. Furthermore, when considering the usage of AA as a complementary nutrition for prevention and treatment of T2DM, individual differences in genetics and dietary patterns should be taken into account.Taken together, our findings suggest that AA has greater beneficial effects on metabolism in HFD-fed The online version contains supplementary material available at 10.1186/s12263-022-00713-y. Type 2 diabetes mellitus (T2DM) is a major medical problem worldwide, and its development is highly affected by complex interactions between genetic and environmental (dietary and lifestyle) factors . While gAntioxidants, such as vitamin C , have been proposed to prevent the T2DM at least partially by attenuating white adipose tissue (WAT) inflammation . WAT actGLUT10G128E mice are highly sensitive to HFD-induced T2DM [SLC2A10) lead to a rare autosomal recessive connective tissue disorder called arterial tortuosity syndrome  [SLC2A10 locus has been associated with T2DM in sib-pair studies [SLC2A10 locus is associated with T2DM intermediate phenotypes in nondiabetic human subjects [SLC2A10 (GLUT10G128E mice); the study revealed that GLUT10G128E mice have impaired WAT development and are highly sensitive to HFD-induced obesity and metabolic dysregulation compared with WT mice [GLUT10G128E mice might require supplementation of AA to sustain normal physiological function and metabolism when fed with a HFD.We have previously shown that 208,050) . We and 208,050) . Interes studies , 17; how studies –23. Alon WT mice , we suspGLUT10G128E mice fed with a normal diet (CD) or HFD. We found that AA supplementation differentially affects metabolism depending on the genetic variant and diet. Our study therefore provides strong support for the idea that clinical studies on the effects of AA in T2DM prevention should account for interactions between diet and specific genetic variants.To test whether AA effects on metabolism might be influenced by combinations of genetic and environmental factors, we determined whether AA supplementation might differentially affect metabolism in wild-type (WT) mice and GLUT10G128E mice on a CD or HFD, we began supplementing the drinking water (3.3 g/L AA) of breeding pairs, nursing females, and mice after weaning. This protocol of AA supplementation was previously demonstrated to maintain optimal physiological AA levels (75 μm in serum) in AA synthesis-deficient mice [GLUT10G128E mice at 3 and 20 weeks of age. We found that AA supplementation of pregnant and nursing female mice led to significantly increased serum AA levels in both WT and GLUT10G128E pups at 3 weeks of age  levels in the mice after HFD feeding. The FBG levels were significantly higher in HFD-fed GLUT10G128E mice than in HFD-fed WT mice after 15 weeks of HFD-feeding (20 weeks of age)  and insulin resistance test (ITT), respectively , and inGLUT10G128E mice. First, we determined the effects of AA supplementation on overall body fat and lean compositions in WT and GLUT10G128E mice on a HFD. The AA group had a trend toward reduced body fat composition in HFD-fed WT mice, but the difference did not reach statistical significance  is associated with local and systemic inflammation and predisposes individuals to metabolic dysregulation [GLUT10G128E mice. To evaluate the effects of AA supplementation on HFD-induced inflammation in eWAT, we first examined the crown-like structures (CLSs) that surround dead adipocytes and are indicative of inflammation in WAT [GLUT10G128E mice compared with HFD-fed WT mice  of gulation , 28, we n in WAT . No CLSsice Fig. A. AA supice Fig. A. In conGLUT10G128E mice by analyzing the serum levels of adipokines that control systemic energy homeostasis, including adiponectin, leptin, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α); these adipokines were previously shown to be highly dysregulated in eWAT of HFD-fed GLUT10G128E mice [GLUT10G128E mice  and total cholesterol (TCHO), leading to lipid deposition in other organs, including liver and interscapular brown adipose tissue (iBAT) –32. We hice Fig.  A and B.ice Fig.  C–H. In ice Fig.  A–H. ThuGLUT10G128E mice. As GLUT10G128E mice have compromised eWAT development, which plays a critical role in predisposing the mice to HFD-induced metabolic dysregulation [GLUT10G128E mice. The eWAT deposits arise during late-embryonic and neonatal development [GLUT10G128E pups  and increasing the percentage of large adipocytes (> 250 area μm2), according to quantification of adipocyte size in the eWAT sections  [GLUT10G128E mice at 3 weeks of age. Pref-1 is highly expressed in preadipocytes and absent after adipocyte differentiation [GLUT10G128E eWATs had higher levels of Pref-1 protein than did WT eWATs, and AA supplementation reduced the Pref-1 protein levels in GLUT10G128E eWATs  have greatly contributed to the increased prevalence of obesity and accelerated the spread of T2DM epidemic worldwide. At least partly based on its antioxidant properties, AA has been considered as a complementary nutritional treatment for T2DM. However, evidence for the significance and beneficial effect of AA in T2DM has thus far been inconclusive , 9. We sice Fig. . TogetheWe found that AA supplementation significantly increases serum AA levels in pups at 3 weeks of age (before weaning). However, we did not observe significantly increased serum AA levels in mice at 20 weeks of age, even though the AA supplementation was continued Fig. . Like thGLUT10G128E mice. The mechanisms by which AA reduces obesity and improves metabolic dysregulation in different obese models may be complex. AA supplementation in pregnant and nursing females significantly increases serum AA levels and improves the development of eWATs in the GLUT10G128E pups  [GLUT10G128E pups, the anti-oxidative and anti-inflammatory activities may participate with AA-mediated reductions in lipid accumulation to generate the final outcome of reduced obesity and improved metabolic dysregulation in HFD-fed GLUT10G128E mice.Several mechanisms have been proposed to link AA with reduced obesity, and metabolic dysregulation has been proposed in different rodent models of obesity. AA supplementation has been shown to combat obesity and metabolic syndrome in cafeteria diet-induced obese rats , obese mob mice) , and ovaob mice) , withoutob mice) –49. Furtob mice) , 50. SinCebpa and Pparg, increasing their expression [GLUT10G128E eWATs via DNA demethylation. Whether the other functions of AA might also influence adipose function and metabolism in the context of HFD feeding remains to be investigated.AA is also an enzyme cofactor that is required for many important biological functions . In thispression . These rGLUT10G128E mice carrying an orthologous human GLUT10G128E variant with compromised GLUT10 function. These mice have increased serum HbA1c levels under CD diet and are genetically predisposed to HFD-induced metabolic dysregulation. Similarly, genetic polymorphisms in the GLUT10-encoding human gene (SLC2A10) locus are associated with increased serum HbA1c levels in a nondiabetic population [GLUT10G128E MEFs but also in GLUT10-knockdown 3T3-L1 cells [SLC2A10 gene that are predicted to affect GLUT10 function, our results suggest that other variants in SLC2A10 associated with compromised GLUT10 expression or function might also negatively affect eWAT development, and AA could potentially attenuate HFD-induced metabolic dysregulation in carriers of these variants.In this work, we utilize Overall, our findings in this study provide a proof of concept that AA has differential effects on metabolism in the presence or absence of genetic and environmental T2DM-risk factors. We expect these results will stimulate more sophisticated population studies to accurately assess whether AA is an appropriate treatment or preventative agent for T2DM in susceptible populations.GLUT10G128E mice were generated on a C3HeB/FeJ background [GLUT10G128E mice in this study were on the C57BL/6 J background and maintained by heterozygous mattings. Mice were housed in a specific pathogen-free controlled environment with a 14-h light/10-h dark cycle at 21–23 °C. AA-supplemented groups received AA (3.3 g/L) and 0.01 mM EDTA in the drinking water, which was changed once per week. The stability of AA in drinking water was examined by determining the AA levels in the water by HPLC as described previously [All animal protocols were approved by the Institutional Animal Care and Utilization Committee at Academia Sinica (Protocol no. 14–12-795). ckground  and wereckground . WT and eviously . The levFor fasting blood glucose measurements, blood samples were collected from the tail vein after overnight fasting. The GTT and ITT were performed as previously described . BrieflyMice were housed individually for measurement of food intake and water intake, using Tecniplast® Metabolic Cage . The metabolic rate was measured using the CLAMS-home cage (CLAMS-HC) system  in the Taiwan Mouse Clinic at Academia Sinica. The first readings were taken after a 48-h acclimation period. Heat production, RER, oxygen consumption rate (VO2), and carbon dioxide production (VCO2) rates were determined. VO2, VCO2, and heat were measured every 17 min during a 76-h period at the indicated temperature and were normalized to body weight.For blood chemistry and adipokine assays, blood was collected from cardiac puncture at the conclusion of experiments. The TCHO levels were analyzed from serum samples using Fuji biochemical slides and a Fuji Dri-Chem 4000i analyzer  in the Taiwan Mouse Clinic at Academia Sinica. Plasma levels of adiponectin, leptin, IL-6, and insulin were measured using mouse ELISA kits . The plasma free fatty acids were measured using an ELISA kit , and blood HbA1c was measured using the mouse Hemoglobin A1c (HbA1c) Assay Kit .Serum AA levels were determined using an Ascorbic Acid Assay Kit .Tissue sections were stained with hematoxylin and eosin (H&E).Total protein lysates from tissues were used for analysis. Proteins were transferred to PVDF membranes . Membranes were then incubated with the primary antibodies against Pref-1 (DLK1) , PPARγ 1 , or β-actin  and the appropriate secondary antibodies. The signal was detected by enhanced chemiluminescence .Mouse body composition was analyzed with Bruker’s Minispec LF50 Body Composition Analyzer in the Taiwan Mouse Clinic at Academia Sinica.P-values less than 0.05 were considered statistically significant.Statistical analyses were performed in GraphPad Prism 7 . Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. A compact letter display was used to indicate significant differences in pairwise comparisons. Additional file 1: Figure S1. Mice from different genotypes or treatment groups exhibit no significant differences in food intake, water intake, physical activity, or energy expenditure. Figure S2. AA supplementation has no obvious effect on histology of sWAT of HFD-fed GLUT10G128E and WT mice.


Seasonal influenza and the threat of global pandemics present a continuing threat to public health. However, conventional inactivated influenza vaccines (IAVs) provide little cross-protective immunity and suboptimal efficacy, even against well-matched strains. Furthermore, the protection against matched strains has been shown to be of a short duration in both mouse models and humans. M2SR (M2-deficient single-replication influenza virus) is a single-replication vaccine that has been shown to provide effective cross-protection against heterosubtypic influenza viruses in both mouse and ferret models. In the present study, we investigated the duration and mechanism of heterosubtypic protection induced by M2SR in a mouse model. We previously showed that M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) significantly protected C57BL/6 mice against lethal challenge with both influenza A/Puerto Rico/8/34  and influenza A/Aichi/2/1968 , whereas the inactivated influenza vaccine provided no heterosubtypic protection. The homosubtypic protection induced by M2SR was robust and lasted for greater than 1 year, whereas that provided by the inactivated vaccine lasted for less than 6 months. The heterosubtypic protection induced by M2SR was of a somewhat shorter duration than the homosubtypic protection, with protection being evident 9 months after vaccination. However, heterosubtypic protection was not observed at 14 months post vaccination. M2SR has been shown to induce strong systemic and mucosal antibody and T cell responses. We investigated the relative importance of these immune mechanisms in heterosubtypic protection, using mice that were deficient in B cells or mice that were depleted of T cells immediately before challenge. Somewhat surprisingly, the heterosubtypic protection was completely dependent on B cells in this model, whereas the depletion of T cells had no significant effect on survival after a lethal heterosubtypic challenge. While antibody-dependent cellular cytotoxicity (ADCC) has been demonstrated to be important in the response to some influenza vaccines, a lack of Fc receptors did not affect the survival of M2SR-vaccinated mice following a lethal challenge. We examined the influenza proteins targeted by the heterosubtypic antibody response. Shortly after the H1N1 M2SR vaccination, high titers of cross-reactive antibodies to heterosubtypic H3N2 nucleoprotein (NP) and lower titers to the stalk region of the hemagglutinin (HA2) and neuraminidase (NA) proteins were observed. The high antibody titers to heterosubtypic NP persisted one year after vaccination, whereas the antibody titers to the heterosubtypic HA2 and NA proteins were very low, or below the limit of detection, at this time. These results show that the intranasal M2SR vaccine elicits durable protective immune responses against homotypic and heterosubtypic influenza infection not seen with intramuscular inactivated vaccines. Both the homo- and heterosubtypic protection induced by the single-replication vaccine are dependent on B cells in this model. While the homosubtypic protection is mediated by antibodies to the head region of HA, our data suggest that the heterosubtypic protection for M2SR is due to cross-reactive antibodies elicited against the NP, HA2, and NA antigens that are not targeted by current seasonal influenza vaccines. Despite the availability of vaccines, influenza is still a major cause of morbidity and mortality worldwide. The current seasonal influenza vaccines primarily depend on a close match between the vaccine immunogen and circulating viruses in order to be effective, meaning they are relatively ineffective against newly emerging influenza viruses or viruses that have drifted away from the vaccine strain . Inactivp = 0.0505) and reduced illness [We previously described an intranasal vaccine platform, M2SR (M2-deficient single-replication influenza virus), which delivers influenza RNA to the mucosa, with the subsequent production of influenza antigens that stimulate broad host immune responses ,15,16. M illness . In a su illness .In this study, we evaluated the mechanism of M2SR’s durable protective immune responses using a mouse model. The longevity of the homosubtypic protection was evaluated in comparison with an inactivated whole influenza vaccine. We next evaluated the duration and mechanism of the heterosubtypic protection provided by H1N1 M2SR against the H3N2 challenge. 2. The derivation of M2CK cells—MDCK cells that stably express the influenza M2 protein—was described previously [In total, 293T human embryonic kidney cells (ATCC CRL-3216), Madin–Darby canine kidney , and M2CK  cells weInfluenza A/A/Puerto Rico/8/34 (PR8) and influenza A/Aichi/2/1968 (Aichi) were amplified in MDCK cells in media containing 1 µg/mL trypsin/TPCK. M2SR, a recombinant PR8 virus lacking M2 protein expression, was generated using plasmid-based reverse genetics, as previously described . The M2SThe M2SR virus was generated using a plasmid rescue system described previously ,21,22. Ttm1RavFcer1g) and matched control wildtype mice were obtained from Taconic Bioscience . The genotype of the FcRγ−/− mice was verified via PCR on DNA from tail snips, using the vendor’s protocol. The MuMT- (B6.129S2-tm1CgnIghm/J) mice were obtained from Jackson Laboratories, Sacramento, CA, USA. The genotype of the muMT- mice was confirmed via flow cytometry on CD19-stained splenocytes or serum antibody titers. All study protocols were approved by the FluGen or BRISC Institutional Animal Care and Use Committees and all experiments were performed in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. Six- to eight-week-old C57BL/6J mice at 17–20 g in weight  were used in all experiments unless noted otherwise. The FcRγ−/− (B6.129P2-6 plaque forming units (PFU) of M2SR virus in 30 μL of sterile PBS. The mock-infected control mice received 30 μL of PBS alone. For comparison, in some experiments the mice were vaccinated with 1 μg of purified inactivated whole influenza A/PR/8/34 (H1N1) antigen  intramuscularly. At specified intervals after immunization, the sera were collected for an antibody titer or the mice were challenged intranasally with 40 mouse-lethal doses of 50 (MLD50) PR8 or 40 MLD50 Aichi. In some experiments, the T cells were depleted from groups of either wildtype C7BL/6 mice or B-cell-deficient muMT mice, using 0.5mg per mouse of each antibody, GK1.5 anti-CD4, 2.43 anti-CD8 and 30H12, anti-Thy1, or 1.5 mg control rat IgG  3 times per week for 2 weeks prior to the challenge. This protocol depleted >99% of the T cells. Their survival was monitored for 14–21 days after the challenge. The mice were anesthetized with either isoflurane or 2,2,2 tribromoethanol and were infected intranasally with 10The serum immunoglobulin titers were measured using an ELISA. Here, 96-well high-protein-binding plates were coated with 200 ng/well purified inactivated whole influenza A/PR/8/34 (H1N1) antigen , purified influenza A/Aichi/2/1968 (H3N2) neuraminidase/NA , purified influenza A/Aichi/2/1968 (H3N2) nucleoprotein/NP , or purified influenza A/Hong Kong/4801/2014 (H3N2) hemagglutinin subunit 2/HA2 amino acids 325–529 . The antibody was detected using horseradish-peroxidase-conjugated goat anti-mouse IgG followed by development with SureBlue TMB Microwell Peroxidase Substrate . The endpoint antibody titers are given as the reciprocal of the dilution with an OD reading of 0.3 at 450 nM and three standard deviations above the mean of the background. A standard hemagglutination inhibition (HI) assay as described previously  was perfA statistical analysis was performed using Student’s t test or a Mann–Whitney test (depending on whether the data followed a Gaussian distribution) for antibody titers and a Mantel–Cox log rank test  and Prism software .M2SR has been shown to elicit strong humoral and cellular immune responses to influenza . While ip < 0.001, Mantel–Cox log rank test), there was no significant difference in survival between B-cell-deficient M2SR-vaccinated mice and unvaccinated controls  has been implicated in the action of antibodies targeting the conserved stem region of the influenza hemagglutinin (HA) molecule . As we hAs durable protection is the key to any vaccine strategy, we examined the longevity of the homosubtypic and heterosubtypic protection elicited by M2SR and the homosubtypic protection induced by intramuscular vaccination with inactivated virus. The homosubtypic protection induced by M2SR was maintained for over a year, in contrast to that induced by the inactivated virus, which lasted less than 27 weeks , consistWe and others have previously shown that the inactivated virus does not induce any heterosubtypic protection ,28; therTo investigate which viral proteins were targeted by the heterosubtypic antibody response to influenza in the H1N1-M2SR-vaccinated mice, we determined the ELISA titers to more conserved proteins or protein regions from the H3N2 influenza viruses. The latter included the neuraminidase (NA) and nucleoprotein (NP) proteins and the conserved HA2 region of the HA (hemagglutinin) molecule. The H1N1-M2SR-induced cross-reactive antibody titers against H3N2 NA, HA2, and NP were detectable at one month after vaccination . As expeThe M2SR single-replication influenza vaccine has been shown to induce both homosubtypic and heterosubtypic protection against influenza, in contrast to the inactivated influenza vaccine, which offers little cross-protection against heterologous viruses ,28. FurtM2SR induces high HAI titers and sterilizing immunity to homosubtypic influenza viruses ,16. ThesIn contrast to the homosubtypic immunity, M2SR demonstrates heterosubtypic protection in the absence of HAI antibodies ,16, suggThus, the heterosubtypic protection appeared to be dependent on B cell responses. The reason that T cells are unable to mediate heterosubtypic protection in this model is unclear, but as lethality commences from day 5 after challenge, it is possible that the T cell response in the respiratory tract develops too slowly to prevent the widespread infection of cells and subsequent lung damage due to the cell lysis by either the virus or the T cells targeting infected cells. Consistent with this hypothesis, our previous data showed that while low numbers of virus-specific T cells were present at day 4 after challenge, they were considerably higher at day 7 . As antibody-dependent cellular cytotoxicity (ADCC) has been implicated in the action of antibodies targeting the conserved stem region of the influenza hemagglutinin (HA) molecule, and as we have shown that H1N1 M2SR induces antibodies that bind to this region of the H3N2 HA molecule, we investigated the protection mediated by M2SR in Fc receptor γ chain-deficient mice ,26. HoweAlthough both the homosubtypic and heterosubtypic protection induced by M2SR appeared to be mediated by B-cell-dependent antibody responses, the homosubtypic protection was effective for at least 12 months, whereas the heterosubtypic protection declined after 9 months. To gain further insight into the heterosubtypic antibody response, we investigated the longevity of the responses to different viral proteins. Cross-reactive antibodies to the HA2 stem region of the hemagglutinin ,38,39,40In conclusion, our study shows that while the homo- and heterosubtypic immunity induced by M2SR and the homosubtypic protection induced by IAV are mediated by B cells, the lengths of the responses differ considerably, and they may be directed against different viral proteins. Therefore, this provides a useful model for investigating why some antibody responses last longer than others and for studying the mechanisms by which the different types of B cell responses decline. Additional studies will be required to further investigate the mechanism of heterosubtypic immunity and the basis for the differences in the longevity of homosubtypic antibody responses to M2SR and inactivated influenza vaccines and homo- and heterosubtypic antibody responses to M2SR. Understanding the basis for the decline in B cell responses over time and the methods for prolonging such immunity are key to developing long-lasting highly effective influenza vaccines, including those that confer heterosubtypic immunity, and may lead to the identification of new immune correlates of protection.


B and FluPolA) are PB1-PB2-PA heterotrimers that perform transcription and replication of the viral genome in the nucleus of infected cells. Initiation of viral mRNA synthesis requires a direct association of FluPol with the host RNA polymerase II (RNAP II), in particular the repetitive C-terminal domain (CTD) of the major RNAP II subunit, to enable “cap-snatching” whereby 5’-capped oligomers derived from nascent RNAP II transcripts are pirated to prime viral transcription. Here, we present the first high-resolution co-crystal structure of FluPolB bound to a CTD mimicking peptide at a binding site crossing from PA to PB2. By performing structure-based mutagenesis of FluPolB and FluPolA followed by a systematic investigation of FluPol-CTD binding, FluPol activity and viral phenotype, we demonstrate that IBVs and IAVs have evolved distinct binding interfaces to recruit the RNAP II CTD, despite the CTD sequence being highly conserved across host species. We find that the PB2 627 subdomain, a major determinant of FluPol-host cell interactions and IAV host-range, is involved in CTD-binding for IBVs but not for IAVs, and we show that FluPolB and FluPolA bind to the host RNAP II independently of the CTD. Altogether, our results suggest that the CTD-binding modes of IAV and IBV may represent avian- and human-optimized binding modes, respectively, and that their divergent evolution was shaped by the broader interaction network between the FluPol and the host transcriptional machinery.During annual influenza epidemics, influenza B viruses (IBVs) co-circulate with influenza A viruses (IAVs), can become predominant and cause severe morbidity and mortality. Phylogenetic analyses suggest that IAVs (primarily avian viruses) and IBVs (primarily human viruses) have diverged over long time scales. Identifying their common and distinctive features is an effective approach to increase knowledge about the molecular details of influenza infection. The virus-encoded RNA-dependent RNA polymerases  co-circulate with influenza A viruses (IAVs) and can cause severe outcomes. The influenza polymerase is a key drug target and it is therefore important to understand the common and distinctive molecular features of IBV and IAV polymerases. To achieve efficient transcription and replication in the nucleus of infected cells, influenza polymerases closely cooperate with the cellular RNA polymerase II (RNAP II) and interact with the repetitive C-terminal domain (CTD) of its major subunit. Here we gained new insights into the way IBV and IAV polymerases interact with the CTD of RNAP II. High-resolution structural data was used to perform structure-based mutagenesis of IBV and IAV polymerases followed by a systematic investigation of their interaction with RNAP II, transcription/replication activity and viral phenotype. Strikingly, we found that IBVs and IAVs have evolved distinct interfaces to interact with the host transcriptional machinery, in particular with the CTD of RNAP II. We provide evidence that these differences may have evolved as a consequence of the differences in IBV and IAV host range. Our findings are of significant importance with regard to the development of broad-spectrum antivirals that target the virus-host interface. Orthomyxoviridae family and are classified into four genera: influenza A, B, C and D viruses. Influenza A viruses (IAVs) and influenza B viruses (IBVs) are of public health importance, as they co-circulate in humans with a seasonal epidemic pattern and cause a significant morbidity and mortality, especially in the aged or immunocompromised population -GTP. For the endonuclease assay 25 nM FluPolA and 50 nM FluPolB were incubated with 1.2-fold molar excess of v5’ RNA  and the capped RNA in reaction buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 2 mM tris(2-carboxyethyl)phosphine (TCEP). Transcription reactions were performed with 50 nM FluPolA or FluPolB in the reaction buffer, supplemented with 150 nM v3’ template RNA  and 250 μM NTP mix (ThermoFisher). 50 μM CTD peptides were added at concentrations corresponding to at least a 10-fold excess over the KD of the lowest measured affinity for a two-repeat peptide. Two- and four-repeat phosphoserine 5 (pS5) CTD peptides were purchased from Covalab and six-repeat pS5 CTD peptide was synthesised at the Chemical Biology Core Facility at EMBL Heidelberg. Reactions were incubated at 30 °C for 30 min and quenched with RNA loading dye , supplemented with 50 mM EDTA and boiled at 95 °C. The reaction products were separated on 20% denaturing acrylamide gel (containing 8 M urea) in Tris-Borate-EDTA (TBE) buffer, exposed on a Storage Phosphor screen and recorded with a Typhoon reader. DECADE marker was used as ladder.RNA for the activity assays was produced escribed . 23 nt RHomo sapiens (NP_000928.1), Sus scrofa (XP_020923484.1), Equus caballus (XP_014584045.2), Canis lupus (XP_038521325.1), and Mus musculus (NP_001277997.1) host species were obtained from the RefSeq database ..4 [B, depS5aP6aS7a-Y1bS2bP3bT4bpS5bP6bS7b-Y1cS2cP3cT4cpS5cP6c) are visible in site 2B in both structures, including two well-defined phosphoserines (in bold). The N-terminal part of the CTD peptide (Y1a-S2b) forms a compact structure comprising two successive proline turns stabilised by four intra-peptide hydrogen bonds, with P3a stacked on Y1b and P6a stacked on PA/Y597 (B-specific PA R608 is in a four-residue insertion (606-GDRV-609) compared to FluPolA, with hydrogen bond interactions from PA D607 and N611 positioning the side-chain of PA Y597 under the CTD peptide. This configuration of residues seems specifically designed to accommodate the compactly folded CTD peptide. Interestingly, recently identified FluPolB-like polymerases from fish and amphibians [B, are all the functional residues Y597, D607, R608, N611 conserved [andThree repeats  of the CTD peptide  polymerase  and C/Jorespectively. The PB2 and PA sequence are very conserved, with > 94% identity among the A/WSN/1933(H1N1), A/NT/60/1968(H3N2) and A/Zhejiang/DTID-ZJU01/2013(H7N9) viruses  [B compared to FluPolA (A were mutated (PA K289A and R638A [bottom). Although the wt and S5A CTD showed similar steady-state levels of expression, the binding of the S5A CTD to FluPolB and FluPolA was significantly decreased compared to WT CTD. Consistently, pharmacological inhibition of CDK7, which represents the major kinase for CTD S5 phosphorylation [A/B binding to the CTD but not to the FluPolA interaction partner NUP62 [To confirm the structural findings of this study and investigate the distinctive features of CTD binding sites in FluPol and G2) . The ful and G2) . Upon co FluPolA . The intnd R638A ), the inrylation , specifier NUP62   which buttresses the CTD at the junction between the PA-Cter and PB2 627 domains in FluPolB is absent in FluPolA. The PA R608A mutation significantly decreased FluPolB CTD binding in our cell-based complementation assay . Consistently, the corresponding recombinant mutant IBV could not be rescued upon reverse genetics (B showed reduced polymerase activity (right panel). We then mutated to alanines the residues W552 and R555 (W553 and R556 according to IBV numbering), which are located on the PB2 627 domain, make contact with the CTD pS5 in the FluPolB co-crystal and are conserved between IBVs and IAVs  could make it easier for the FluPolB PB2 627 domain to adopt a CTD-bound conformation. To rule out any CTD binding activity on the FluPolA PB2 627 domain, we deleted the whole domain as described before [B but not to FluPolA , and the flexible PB2 C-terminus [B and not to FluPolA. Therefore, we tested the impact of pS5 CTD mimicking peptides of varying lengths  on FluPolB and FluPolAin vitro transcriptional activity (Bin vitro endonuclease activity (lane 4) and elongation activity (lane 8) were increased in the presence of the six-repeat pS5 CTD mimicking peptide compared to the mock control (lanes 1 and 5), and a similar trend was observed with FluPolA. These data complement previous reports that CTD pS5 binding facilitates FluPolA and FluPolC transcriptional activity [A PB2 627 domain has no CTD binding activity  indicates that FluPolA has evolved a divergent mechanism by which the CTD stabilizes the FluPol transcriptase. It also questions whether bridging of the PB2 627 and PA-Cter domains per se is needed for transcriptional activation of FluPols.We asked whether this major difference between FluPolterminus . Our obsactivity . The Fluactivity , lane 4 activity , lane 8 activity  and streactivity . Howeveractivity  indicateA/B can interact with the two major RNAP II subunits  independently of the CTD, using the split-gaussia luciferase complementation assay. The G2-RPB1 and RPB2-G2 fusion proteins were co-expressed with G1-tagged FluPol (PA-G1) by transient transfection as described above. Both combinations resulted in robust and comparable interaction signals . Moreover, as the G2-RPB1ΔCTD protein accumulates at substantially higher levels than the endogenous RPB1 protein  and site 2B mutations and  host species susceptible to IAV or IBV infection . It is t signals . Interes nucleus  and brid protein , it is l protein  had only protein . The sam protein  and siteB and an avian (isolated from human) FluPolA bound to pS5 CTD mimicking peptides. We uncover the conformation and directionality of the CTD peptide bound to FluPolB at a site that crosses over from the PA-Cter to the PB2 627 domain (site 2B), and has no counterpart on FluPolA or FluPolC. Two CTD binding sites have been characterised on FluPolA (sites 1A and 2A)  [B co-crystal structure, site 1B is similar to site 1A, whereas site 2B is distinct from site 2A and 2C.Here we report co-crystal structures of a human FluPoland 2A) ( and this and 2A) . On the B and FluPolA followed by a systematic investigation of FluPol-CTD binding, FluPol transcription/replication activity and viral phenotype, we confirm that CTD binding involves the same key residues at site 1AB for FluPolB and FluPolA, but distinct and specific residues at site 2A for FluPolA and site 2B for FluPolB, respectively. In particular, we demonstrate that the PA 606–609 loop, which buttresses the CTD at the junction between PA and PB2 in the FluPolB co-crystal structure and is not conserved in FluPolA or FluPolC, is an essential component of site 2B.By performing structure-based mutagenesis of FluPolA CTD binding mode presumably reflects an avian-optimised mode and co-evolved with protein interfaces between avian host factors and the PB2 627 domain, known to restrict avian IAV replication in humans .Our data and others’ ,33 demonSchizosaccharomyces pombe, Candida albicans and Mus musculus were shown to bind directly S5 CTD repeats with very distinct binding interfaces and distinct conformations of the bound CTD [Another example of a functional interaction with RNAP II being achieved through distinct CTD binding is provided by the cellular mRNA capping enzyme (CE). The CEs from ound CTD . These dound CTD  which coThere is considerable evidence, however, that the FluPol-CTD interaction is only part of a more complex interaction pattern between the viral and cellular transcription machineries, raising the possibility that interactions between the FluPol and less conserved components of the cellular transcriptional machinery could have indirectly shaped the evolution of distinct CTD binding modes. We observed that a truncated RPB1 subunit, which lacks the CTD, retains partial binding to FluPol . Mass-spin vitro transcriptional activity of FluPolB is facilitated by the addition of CTD pS5 mimicking peptides, as reported previously for FluPolA and FluPolC [C [B with PA-PB2 bridging occurring at site 2B. Our data show that it does not apply to FluPolA, unless another yet unidentified domain of PB2, distinct from the PB2 627 domain, is involved.We show that the  FluPolC . The mecuPolC [C ,33 in whA and FluPolB is of significant importance with regard to the development of broad-spectrum antivirals and need to be taken into account when targeting the FluPol-CTD binding interface for antiviral intervention.As underlined by the different sensitivity of IAV and IBVs to cap-binding inhibitors related to differences in the cap-binding mode of their PB2 subunits , a detaiS1 FigA. CTD peptide bound in site 2B of FluPolB (B/Memphis/13/2003) polymerase. Fo-Fc omit map shown at 2.5 σ with clear density for two phosphoserines (pS5a and pS5b). B. CTD peptide bound in site 2A of FluPolA (A/Zhejiang/DTID-ZJU01/2013(H7N9)) polymerase. Fo-Fc omit map shown at 2.7 σ with clear density for one phosphoserine.(TIF)Click here for additional data file.S2 FigA. Superposition of the PB2-627 domains of CTD-bound  and unbound (wheat) FluPolB (B/Memphis/13/2003) polymerase, showing induced-fit conformational changes of three key hydrophobic residues . B. Ribbon diagram looking down the 2-fold axis of the symmetrical homodimeric A/H7N9 polymerase core with CTD peptide bound in site 2A. One monomer is coloured with PA (green), PB1 (cyan), PB2 (red) with CTD peptide (slate spheres) and 5’ vRNA hook (violet). The second monomer has paler versions of these colours. C. Details of the binding of the CTD peptide (slate blue) in site 2A of FluPolA (A/Zhejiang/DTID-ZJU01/2013(H7N9) core) polymerase. D. Comparison of site 1A configuration for CTD bound form of FluPolA  [A (A/NT/60/1968 (H3N2), [A (A/Zhejiang/DTID-ZJU01/2013(H7N9) core, light blue, this work). The FluPolA (H7N9) polymerase core is the symmetrical dimer with each polymerase in the open, ‘dislocated’ state [(H17N10) , PDB: 5M (H3N2), , PDB: 6Rd’ state . Due to (TIF)Click here for additional data file.S3 Fighttps://www.uniprot.org/), aligned with SnapGene 6.0 and visualized by Espript 3.0 [Protein sequences from A/WSN1933(H1N1), A/NT/60/1968(H3N2), A/Zhejiang/DTID-ZJU01/2013(H7N9) and A/little yellow-shouldered bat/Guatemala/060/2010(H17N10), were obtained from UniProt (ript 3.0 . Identic(TIF)Click here for additional data file.S4 FigSame as in (TIF)Click here for additional data file.S5 Fighttps://www.uniprot.org/), aligned with SnapGene 6.0 and visualized by Espript 3.0 [Protein sequences were obtained from UniProt (ript 3.0 . Identic(TIF)Click here for additional data file.S6 FigA. HEK-293T cells were transfected with G2-CTD. At 24 hpt cells were treated for 1 h with 20 μM BS-181-HCl . Cell lysates were analysed by western blot with antibodies specific for pS5 or pS2 CTD and tubulin. B.In vivo CTD binding of FluPolA (A/WSN/1933) and FluPolB (B/Memphis/13/2003). G2-tagged CTD was expressed by transient transfection in HEK-293T cells together with the viral polymerase subunit PB2, PB1 and PA-G1. At 24 hpt cells were treated for 1 h with 20 μM BS-181-HCl or 0.2% DMSO before cell lysis and measurement of G. princeps luciferase activity as described in the Materials and Methods section. As a control, the previously described FluPolA−NUP62 interaction was investigated by co-transfection of G2-NUP62, PB2, PB1 and PA-G1. RLUs are expressed as percentages relative to DMSO treated cells. The data shown are mean ± SD of five independent experiments performed in technical triplicates. ***p ≤ 0.002, ***p ≤ 0.001 .(TIF)Click here for additional data file.S7 FigA-C.In vivo CTD binding of the indicated site 1AB (A), site 2A (B) and site 2B (C) mutants of FluPolA  and FluPolB . The G2-tagged CTD was expressed by transient transfection in HEK-293T cells together with PB2-G1, PB1 and PA. RLUs are expressed as percentages relative to wt FluPolA/B. The data shown are the mean ± SD of at least three independent experiments performed in technical triplicates. **p ≤ 0.002, ***p ≤ 0.001 . (n.d.) not determined.(TIF)Click here for additional data file.S8 FigCharacterisation of recombinant IBV (B/Brisbane/60/2008) PA R608A mutant virus. Recombinant viruses with the indicated mutations were generated by reverse genetics as described in the Materials and Methods section. Reverse genetic supernatants were titrated on MDCK cells and stained at 72 hpi by crystal violet. The pictures show one representative plaque assay with the indicated ten-fold dilution.(TIF)Click here for additional data file.S9 FigSus scrofa (wild boar), Equus caballus (horse), Homo sapiens (human), Canis lupus (wolf), Mus musculus (house mouse), Gallus gallus (chicken) and Anas platyrhynchos (wild duck) were obtained as described in the Materials and Methods section, aligned with SnapGene 6.0 and visualised by Espript 3.0 [The RPB1 CTD sequences of ript 3.0 . The CTD(TIF)Click here for additional data file.


The Smc5/6 complex plays multiple roles in DNA replication and repair. Its genome-protecting functions rely on its interaction with DNA; however, how this complex engages DNA is poorly understood. We report on a cryogenic electron microscopy structure of DNA-bound budding yeast Smc5/6 complex, revealing that its subunits form a clamp to encircle a double-helical DNA. We define the multi-subunit interactions forming the DNA clamp and the DNA binding sites distributed among subunits. We identify subunit transformations upon DNA capture and functional effects conferred by its multiple DNA contact sites. Our findings, in conjunction with studies on other structural maintenance of chromosomes (SMC) complexes, suggest a common SMC DNA-clamp mechanism with individual complex specific features that enable diverse genome organization and protection functions by SMC family complexes. Saccharomyces cerevisiae Smc5/6 complex containing five of its core subunits, including Smc5, Smc6, and the Nse1-3-4 subcomplex. Intricate interactions among these subunits support the formation of a clamp that encircles the DNA double helix. The positively charged inner surface of the clamp contacts DNA in a nonsequence-specific manner involving numerous DNA binding residues from four subunits. The DNA duplex is held up by Smc5 and 6 head regions and positioned between their coiled-coil arm regions, reflecting an engaged-head and open-arm configuration. The Nse3 subunit secures the DNA from above, while the hook-shaped Nse4 kleisin forms a scaffold connecting DNA and all other subunits. The Smc5/6 DNA clamp shares similarities with DNA-clamps formed by other SMCs but also exhibits differences that reflect its unique functions. Mapping cross-linking mass spectrometry data derived from DNA-free Smc5/6 to the DNA-bound Smc5/6 structure identifies multi-subunit conformational changes that enable DNA capture. Finally, mutational data from cells reveal distinct DNA binding contributions from each subunit to Smc5/6 chromatin association and cell fitness. In summary, our integrative study illuminates how a unique SMC complex engages DNA in supporting genome regulation.Structural maintenance of chromosomes (SMC) complexes are essential for chromatin organization and functions throughout the cell cycle. The cohesin and condensin SMCs fold and tether DNA, while Smc5/6 directly promotes DNA replication and repair. The functions of SMCs rely on their abilities to engage DNA, but how Smc5/6 binds and translocates on DNA remains largely unknown. Here, we present a 3.8 Å cryogenic electron microscopy (cryo-EM) structure of DNA-bound  N- and C-terminal domains to link the head of one SMC to the head-proximal arm region (neck) of another SMC, forming a trimeric SMC-kleisin structure. In cohesin and condensin, two large U-shaped HEAT  repeat HAWK (HEAT proteins associated with kleisins) subunits attach to the middle region of the kleisin. By contrast, the Smc5/6 kleisin (Nse4) binds to smaller WH (winged helix)-containing KITE (kleisin interacting tandem WH elements) subunits (Nse1 and Nse3)  complexes are essential genome regulators in both prokaryotes and eukaryotes. In eukaryotes, the cohesin and condensin SMC complexes organize DNA, while the Smc5/6 complex (referred to as Smc5/6) directly regulates DNA replication and repair . At the nd Nse3) .SMC-mediated functions depend on interactions with DNA. Recent cryogenic electron microscopy (cryo-EM) structures of DNA-bound cohesin and condensin revealed that their HAWK subunits and the SMC head-neck regions form a clamp to enclose a single DNA double helix –7. DNA c7710Saccharomyces cerevisiae Smc5/6 complex reveals that the head-neck Smc5-6 regions and the Nse1-3-4 subcomplex together form a clamp entrapping the DNA helix. The structure further reveals protein subunit folds and association, as well as how the subunits collaborate to entrap DNA. Comparison of CLMS analyses of DNA-free Smc5/6 with the structure of the DNA-bound Smc5/6 unveils large scale, multi-subunit conformational changes that enable Smc5/6 to encircle DNA. Finally, our mutational data suggest distinct contributions from each of the DNA binding regions to Smc5/6 chromatin association and cellular fitness. Comparison of our findings with those of other SMCs reveals that diverse SMC complexes use a similar DNA clamping strategy despite structural differences, and that Smc5/6 possesses unique features distinct from cohesin, condensin, and prokaryotic SMCs. Our work lays the foundation for an in-depth understanding of how Smc5/6 fulfills unique roles in genome protection.Here we address the molecular mechanisms by which this unique SMC complex binds DNA using an integrative approach, coupling a cryo-EM-based structural characterization with CLMS analyses and functional investigation. Our atomic structure of a DNA-bound SI Appendix, Fig. S1) using an expression system published and kindly provided by the Stephan Gruber laboratory  A and theboratory . The comSI Appendix, Figs. S2 and S3A; and Table S1) allowed us to generate an atomic structure of DNA-bound Smc5/6 , whereas other parts of the complex were not visualized due to flexibility . While the DNA double helix could be placed into its density, it could only be modeled as an oligoA-oligoT duplex due to inability to definitively identify individual bases. In this structure, a single DNA double helix is encircled inside a clamp formed by the Smc5 and 6 head and neck regions in conjunction with the Nse1-3-4 subcomplex. We describe below how the DNA clamp is formed and how DNA is stabilized in the interior of the clamp. We also highlight the common and distinct features of Smc5/6 compared with DNA-bound structures of cohesin  reconstruction from cryo-EM particles , but different from the juxtaposed alignment of two head domains seen in DNA-free condensin  . A similar tilt is seen for cohesin and condensin, but not the prokaryotic SMC complex MukBEF  A. This iig. S3E) , 18. Theig. S3E) B and C.  MukBEF 3, 17.Xenopus counterparts, Nse1 and 3 interact through their WHA domains  that may serve a regulatory role . Second, a short segment of Nse4 (Nse1-BD) binds to Nse1 through an area formed by residues spanning all its three domains  featuring two helices that interact with Nse3 . Such sequence-nonspecific binding can permit genome surveillance with no sequence restrictions. The electrostatic surface representation of the complex reveals five distinct DNA binding patches . Corresponding residues for a subset of these in the metazoan Nse3 proteins have been noted to affect in vitro DNA binding . These are all located within the N-lobe of the head region in a similar fashion as the DNA binding sites in other SMCs, suggesting that SMC head-DNA binding is conserved across SMC family members  mutated for DNA binding residues in Patches 1 and 2  mutated for those in Patch 5  mutated for residues in Patch 3  mutated for residues in Patch 4 . The two DNA-free Smc5/6 datasets gave similar and internally consistent conclusions. They reveal that the majority of violated CLs in DNA-bound Smc5/6 structure occur between subunits, while satisfied CLs are located mostly within the same subunit, with the exception of Nse4. These results suggest that while subunits shift position relative to each other upon DNA binding, individual subunit structures do not undergo major changes, with the exception of Nse4. Specifically, we identified four groups of changes upon DNA binding, as well as a pivot point that may serve as an anchor during transformation .We applied unified criteria to reanalyze CLMS datasets of DNA-free Smc5/6 from our group  and thosSI Appendix, Table S2). This result confirms arm-opening upon DNA binding as mentioned above and provides a validation for our analysis.First, all 14 cross-links connecting Smc5 and Smc6 arms in DNA-free complex violate cross-link distances when mapped to the DNA-bound Smc5/6 structure, with cross-linked residues now separated by up to 86 Å . We conclude that DNA engagement entails moving Nse3 from head/neck regions toward the direction of the hinge.Second, we found that Nse3 shifts position relative to Smc5 and 6 B. Eight SI Appendix, Fig. S6). A total of 13 cross-links connecting Nse1 or Nse3 with Nse4-WHD and its linker regions are violated CLs, with a maximal Cα-Cα pair distance of 113 Å in the current structure . Among these are three DNA-binding lysine residues in the Nse3-WHA domain , suggesting that they move away from Nse4-WHD in order to engage with DNA . We further identified seven satisfied CLs connecting Nse4 around its Nse3-BD domain to either Nse3 or Smc6 neck region, indicating interactions retained during structural transformation .Third, Nse1 moves along with Nse3 upon DNA binding as shown by their similar cross-linking changes relative to Nse4 . This is in striking contrast to CLs within Smc5, Smc6, Nse1 and Nse3, which are all satisfied CLs when mapped to our structure  or HAWK subunits in cohesin  and condensin   . In thisig. S7C) . This isig. S7C) . When coig. S7C) . Anotherig. S7C) . The sigig. S7C) .SI Appendix, Fig. S7). Detailed differences noted above regarding Smc5/6 may confer specificity of Smc5/6-mediated functions. Testing this hypothesis in the future will help further elucidate SMC-based DNA manipulation.Overall, comparing the DNA-bound Smc5/6 structure with those of cohesin, condensin, and MukBEF, a fundamental shared feature of the SMCs appears to be DNA clamping in the E-K compartment  to pull down the complex tagged with 3C-Twin-Strep. After washing the column with 50 mL lysis buffer, Smc5/6 was eluted with lysis buffer supplemented with 2.5 mM desthiobiotin and loaded on Heparin column (1 mL). After washing the column with 10 mL lysis buffer, Smc5/6 was eluted with 50 mM Tris⋅HCl, pH 7.5, 800 mM NaCl, 5% glycerol. The elution was concentrated and applied to a superpose 6 increase column equilibrated with 25 mM Hepes, pH 7.5, 250 mM NaCl, 1 mM DTT to obtain peak fractions containing the complex .The complex composed of Smc5 (E1015Q), Smc6 (E1048Q), and Nse1-4 was purified as described . Briefly2 and 1.5 μM dsDNA (5′-TGGTTTTTATATGTTTTGTTATGTATTGTTTATTTTCCCTTTAATTTTAGGATATGAAAACAAGAATTTATC-3′ and its complementary strand) at 4 °C for 2 h. This sample was applied onto glow-discharged UltrAuFoil 300 mesh R1.2/1.3 grid. Grids were blotted for 2.0 s at 4 °C, 100% humidity, and flash frozen in liquid ethane using a FEI Vitrobot Mark IV. All images were collected on a FEI Titan Krios electron microscope operated at an acceleration voltage of 300 kV with a Gatan K3 camera with a 1.069 Å pixel size. Movies were recorded in counting mode at an electron dose rate of 30 e−/pixel/s with a total exposure time of 2 s and frames rate 50 ms per /frame, for an accumulated electron dose of 53.55 e−/Å2. Motion correction was performed with MotionCor2 (https://pymol.org/2/) or UCSF Chimera  was incubated with 1 mM ATP, 2 mM MgCltionCor2 , and contionCor2 . All othtionCor2  and CryotionCor2 . After btionCor2  picking tionCor2 . The modtionCor2 . All fig Chimera . DetailsCLMS data used for analysis was taken from Yu et al. (2021)  and fromRAD5. To generate DNA binding site mutations for each gene studied, DNA fragment containing the mutations was synthesized and then fused with a tag containing Flag and a selection marker using PCR-based method. The resultant PCR product was used to transform diploid yeast cells. The transformants were screened for the correct gene replacement of one of the two alleles by PCR-based diagnosis method. The positive clones were further analyzed by sequencing the entire targeted gene locus to identify those only containing the desirable mutations and no other mutation. Such diploids were sporulated and examined by tetrad analyses that involved at least 12 tetrads. In all cases, two independent biologically isolated diploids were examined, and they gave the same results. In the case of Nse4 and Smc5 DNA binding mutants, haploid cells were spotted on plates in the presence or absence of MMS to assess genotoxic sensitivity. In all cases, yeast cells were grown at 30 °C. The controls with tagged wild-type genes were examined in the same manner as the cells containing the mutated genes.All yeast strains are in W303 background containing wild-type Diploid cells containing one copy of the DNA binding mutant tagged with a Flag module and one copy of untagged wild-type gene for Nse3, Smc5 and Smc6 were examined for protein levels using TCA (trichloroacetic acid) method as described . In brie3VO4, 1× Sigma protease inhibitors, 1% Triton X-100) for 5 min on ice. Lysates were centrifuged at 16,000 × g for 15 min on a sucrose cushion. Chromatin pellets were washed and resuspended with extraction buffer. Protein loading buffer was added to all fractions and boiled for 5 min followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Flag-tagged proteins were detected by anti-Flag antibody (Sigma). Histone H3 was used as the marker for chromatin-associated proteins and was detected by an anti-H3 antibody . Pgk1 was used as a marker for nonchromatin-associated proteins and was detected by an anti-Pgk1 antibody . For Nse4, haploid cells containing either the Flag tagged wild-type of mutant proteins were examined by TCA method for protein levels and by chromatin fractionation.Chromatin fractionation was performed as described in diploid cells for Nse3, Smc5 and Smc6 . BrieflySupplementary File


Genetic mapping for drought-induced Pro and H2O2 production under field conditions is very limited in crop plants since their phenotyping with large populations is labor-intensive. A genome-wide association study (GWAS) of a diversity panel comprised of 184 bread wheat cultivars grown in natural field (control) and rain-out shelter (drought) environments was performed to identify candidate loci and genes regulating Pro and H2O2 accumulation induced by drought.Proline (Pro) and hydrogen peroxide  of Pro and H2O2 were identified on 5B and 1B chromosomes, respectively. Total 143 significant MTAs were identified including 36 and 71 were linked to drought and 2 and 34 were linked to STI for Pro and H2O2, respectively. Next, linkage disequilibrium analysis revealed minor alleles of significant single-markers and haplotypes were associated with higher Pro and H2O2 accumulation under drought. Several putative candidate genes for Pro and H2O2 content encode proteins with kinase, transporter or protein-binding activities.The GWAS identified top significant marker-trait associations (MTAs) on 1A and 2A chromosomes, respectively for Pro and H2O2 biosynthesis underlying drought adaptation lay a fundamental basis for functional studies and future marker-assisted breeding programs.The identified genetic factors associated with Pro and HThe online version contains supplementary material available at 10.1186/s12870-022-03943-9. Pro is an amino acid that is synthesized in plants cells both under control and stress conditions  for Pro content under drought stress, the STI, and Pro accumulation under control conditions. The significant MTAs were observed across different chromosomes  located on chromosome 1A (8.23 Mbp), established a haplotype block (Pro_1A_Hap1). This chromosome harbored 9 significant markers. The hotspot region of significant markers for Pro content under drought stress was located on chromosome 4A and comprised of 12 markers across 41.95 to 46.12 Mbp. We observed that 19 SNPs out of 36 overlapped with candidate genes that possessed 52% of significant SNPs.Marker-trait association (MTA) analysis revealed a combined total of 125 markers that passed the significance threshhold  was set as the significance threshold instead. SNPs that satisfied the threshold P-value were considered true positives and used for candidate gene search. The data was visualized as Manhattan plots using the R package ‘CMplot’.Pro and Hications , 62 wereications . Associaications . After tications . In accoications , 67, a PD′ values ranging between > 0.98 to 0.7 [2O2 are listed in Supplementary Table SBased on the significant markers identified in the GWAS, LD analysis was performed using Haploview 4.2 to define candidate loci/haplotype blocks . A LD he8 to 0.7 . Generalhttps://wheat-urgi.versailles.inra.fr) [Arabidopsis was conducted with Triticeae-Gene Tribe through one to one selection [http://wheat.cau.edu.cn/TGT/).Genes  coinciding with the identified loci were selected as candidate genes. A comprehensive list is shown in Supplementary Table Sinra.fr) . Candidaelection  (http://Additional file 1.Additional file 2.Additional file 3.


Transcription factors bind to specific DNA sequences, modulate the transcription of target genes, and regulate various biological processes, including peripheral nerve regeneration. Our previous analysis showed that SS18L1, a gene encoding the transcription factor SS18-like protein 1, was differentially expressed in the distal sciatic nerve stumps after rat sciatic nerve transection injury, but its effect on peripheral nerve injury has not been reported. In the current study, we isolated and cultured primary Schwann cells, and examined the role of SS18L1 for the biological functions of the cells. Depletion of SS18L1 by siRNA in Schwann cells enhanced cell proliferation and inhibited cell migration, as determined by EdU assay and transwell migration assay, respectively. In addition, silencing of SS18L1 inhibited Schwann cell differentiation induced by HRG and cAMP. Bioinformatics analyses revealed an interaction network of SS18L1, including DF2, SMARCD1, SMARCA4, and SMARCE1, which may be implicated in the regulatory functions of SS18L1 on the proliferation, migration and differentiation of Schwann cells. In conclusion, our results revealed a temporal expression profile of SS18L1 in peripheral nerve injury and its potential roles during the process of nerve recovery. A transcription factor is a sequence-specific DNA binding protein that regulates the transcription rates of target genes , 2. To dPeripheral nerve injury is an important clinical problem that can be induced by a variety of causes, including accidental trauma, physical injury, pathological damage, and degenerative diseases , 11. Varin situ hybridization, immunohistochemistry and Western blotting analysis all showed that the expression of SS18L1 is enriched in the brain  protein, which is a chromatin remodeling protein. The SS18L1 protein consists of three functional domains, an N-terminal auto-regulatory domain that suppresses transactivation in the basal state, a central methionine-rich domain involved in protein-protein interaction, and a C-terminal glutamine-rich domain responsible for transactivation . Northerhe brain . SS18L1 he brain . It playhe brain . SS18L1 he brain . In addihe brain . Furtherhe brain , 17. We he brain , but itsAdult and neonatal Sprague-Dawley (SD) rats were purchased from the Laboratory Animal Center of Nantong University. All experimental procedures were ethically approved by the Institutional Animal Care and Use Committee of Laboratory Animals center of Nantong University (Inspection No: S20210105-013). A total of 30 6- to 8-week adult male SD rats, weighing 180-200 g were randomly separated into 5 groups  at 6 rats per group, and used for sciatic nerve crush as previously described (® III RT SuperMix for qPCR (+gDNA wiper) . Briefly, 1 μg of RNA sample and 4 μl of 4 × gDNA wiper Mix were incubated at 42°C for 3 min, then, 4 μl of 5 × HiScript III qRT SuperMix was added and the reaction mixture was incubated at 37°C for 15 min and 85°C for 5 s. The cDNA was subjected to real-time PCR analysis of gene expression using SYBR Green Premix Ex Taq (TaKaRa) on a StepOne Real-time PCR machine . The thermocycling program was as follows: initial denaturation at 95°C for 5 min; 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 45 s and extension at 72°C for 30 s; final extension at 72°C for 5 min. The relative gene expression level was calculated using the 2ΔCt−Δ method with GAPDH as the reference gene. Primers of rat SS18L1, P0, MBP, or GAPDH were designed by National Center for Biotechnology Information Primer-BLAST specific primer designing tool and synthesized by Sangon Biotech . The sequences of primers were as follows: SS18L1 (NCBI accession Nos. NM_138918) (forward) 5′-TGCAGAGCCCATGAGTCAAC-3′ and (reverse) 5′-GTCGTAGCTCTGCTCTGTGT-3′; P0 (NCBI accession Nos. NM_001314068) (forward) 5′-CGTGATCGGTGGCATCCTC-3′ and (reverse) 5′-GGCATACAGCACTGGCGTCT-3′; MBP (NCBI accession Nos. NM_001025289) (forward) 5′-CCGACGAGCTTCAGACCATC-3′ and (reverse) 5′-AGTACTTGGATCCGTGTCGC3′; and GAPDH (Accession Nos. NM_017008) (forward) 5′-AACGACCCCTTCATTGAC-3′ and (reverse) 5′-TCCACGACATACTCAGCAC-3′. The experiment was repeated three times.Total RNA was isolated from cultured Schwann cells using RNA-Quick Purification Kit  and then reversely transcribed into cDNA using HiScript® . Immunostaining images were obtained under a fluorescence microscopy . The fluorescence intensity were conducted by calculations of the crushed areas using Image J software (https://imagej.nih.gov/ij/index.html). The experiment was repeated three times.Collected sciatic nerves were fixed with 4% paraformaldehyde, dehydrated with 30% sucrose, embedded in optimal cutting temperature (OCT) compound, and cut into 12 μm tissue sections using a cryostat. Nerve sections were blocked with Immunol Staining Blocking Buffer  for 30 min, and incubated with primary antibodies mouse anti-S100β antibody  and rabbit anti-SS18L1 antibody , rabbit anti-P0 antibody  or rabbit anti-MBP antibody  overnight at 4°C followed by incubation with secondary antibodies goat anti-mouse IgG-Alex-488  and goat anti-rabbit IgG-Cy3  at room temperature for 2 h. Nuclear staining was performed using DAPI Fluoromount-G2 incubator at 37°C. The dishes were pre-coated with poly-L-lysine (Sigma). Then the medium was replaced with an equal volume of DMEM/10% FBS supplemented with 10 μM Ara-C (Sigma) for additional 2 days to eliminate fibroblasts. After removing debris by gently rinsing three times with PBS, the cells were maintained in DMEM/10% FBS supplemented with 10 ng/ml human heregulin-β1  and 2 μM forskolin (Sigma) for 3 days and then purified with anti-Thy1.1 antibody (Sigma) and rabbit complement (Sigma) to remove residual fibroblasts. The purity of Schwann cells was assessed by S100β immunostaining and the cells were used for experimental analysis when the purity reached 95%.A total of 60 1-day-old neonatal SD rats were used to isolate Schwann cells as previously described . BrieflyCultured primary Schwann cells were transfected with 3 siRNAs against SS18L1  or a non-targeting negative control (NC-siRNA: GGCTCTAGAAAAGCCTATGC)  for 48 h using Lipofectamine RNAiMAX reagent  according to the manufacturer's instruction. The gene silencing efficiency was determined by real-time RT-PCR analysis. The experiment was repeated three times.4 primary Schwann cells were suspended in 100 μl cultured medium and seeded onto 96-well plates pre-coated with poly-L-lysine at a density of 2 ×105 cells/ml. The cells were incubated for additional 12 h after adding 50 μM EdU (RiboBio), and then fixed in 4% paraformaldehyde  in PBS for 30 min. The proliferation of Schwann cells was determined using the Cell-LightTM EdU DNA Cell Proliferation Kit (Ribobio) according to the manufacturer's protocol. The numbers of EdU-positive cells and total cells were determined by Apollo 567 fluorescent dyes and Hoechst 33342 staining. The proliferation rate of Schwann cells was calculated by dividing the numbers of EdU-positive cells by the numbers of total cells. Images were taken using a DMR fluorescence microscope . The experiment was repeated three times. The three non-overlapping fields in each well were used for statistics.A total of 2 ×104 primary Schwann cells were suspended in 100 μl DMEM medium and seeded onto the upper chamber of a 6.5 mm transwell chamber with 8 μm pores  at a density of 4 ×105 cells/ml. Schwann cells were incubated for additional 24 h after filling the bottom chamber with 500 μl culture medium. The bottom of the transwell chamber was pre-coated with 10 μg/ml fibronectin . After cleaning cells left on the upper surface, the transwell chambers were stained with 0.1% crystal violet (Beyotime) for 20 min at room temperature. Images were taken using a DMR inverted microscope (Leica Microsystems) and the relative migration ability of Schwann cells was calculated by measuring crystal violet-stained areas in randomly selected fields. The areas of migratory Schwann cells were calculated using Image J software. The experiment was repeated three times.A total of 4 ×10Schwann cell differentiation was induced by treatment of HRG and cAMP as previously described . Brieflyhttps://string-db.org/). Gene expression heatmap was generated using MeV software v4.9.0 (http://www.tm4.org/). Potential regulatory network of SS18L1 was predicted using the Ingenuity Pathways Knowledge Base (IPKB) of the Ingenuity pathway analysis (IPA) software .SS18L1-centered genetic network was constructed using the ClueGo (v2.5.8) and CluePedia (v1.5.8) plug-ins of the Cytoscape software (v3.8.2). CluePedia was used to identify the potential target genes of SS18L1. Gene ontology  and Kyoto Encyclopedia of Genes and Genomes  by GluoGO were used to enrich GO categories and KEGG pathways of SS18L1 and potential target genes, with a P <0.05 were selected and listed. An interaction network of SS18L1, potential target genes, and involved GO categories and KEGG pathways were constructed using the Cytoscape software. Protein-protein interaction network was constructed using STRING database  were indicated by asterisks.Statistical analysis was conducted with GraphPad Prism 6.0 . One-way ANOVA followed by Previous analyses revealed the down-regulation of SS18L1 mRNA in rat distal sciatic nerve stumps after sciatic nerve transection . Here, rImmunohistochemistry analysis indicated a predominant expression of SS18L1 in Schwann cells. To confirm this observation, we isolated and cultured primary Schwann cells and then detected the expression of SS18L1 by immunofluorescence staining. The purity of the cells was first verified by co-staining with the Schwann cell marker S100β and DAPI, and the result showed that more than 95% of the cells were Schwann cells. Further immunostaining of cultured Schwann cells with SS18L1 showed that the majority of SS18L1 staining overlapped with S100β staining, confirming that SS18L1 protein was expressed in Schwann cells .The proliferation of Schwann cells after peripheral nerve injury are critical for subsequent peripheral nerve regeneration. To investigate the biological roles of SS18L1 in Schwann cell proliferation and peripheral nerve regeneration, we first transfected the cultured Schwann cells with three different siRNAs against SS18L1 or a siRNA control. RT-PCR analysis showed all the SS18L1 siRNAs could effectively inhibit SS18L1 mRNA expression , of whicTo assess whether SS18L1 has any effect on cell migration, we transfected Schwann cells with SS18L1 siRNA or siRNA control and performed transwell migration assays . MigrateThe expressions of the myelin-related genes P0  and MBP are two of the well-defined markers of matured Schwann cells. Immunohistochemistry analysis showed nerve crush significantly decreased the expression of both P0  and MBP To evaluate underlying biological functions regulated by SS18L1 in Schwann cell proliferation, migration and differentiation, we used the ClueGo tool of Cytoscape software to explore potential targets of SS18L1. The analysis showed that a total of 17 genes, including DPF1 (Double PHD Fingers 1), ARID1B (AT-Rich Interaction Domain 1B), PHF10 (PHD Finger Protein 10), SMARCC2 , SMARCA2 , ACTL6B (Actin Like 6B), DPF3 (Double PHD Fingers 3), SMARCC1 , SMARCD1 , ARID1A (AT-Rich Interaction Domain 1A), ACTL6A (Actin Like 6A), DPF2 (Double PHD Fingers 2), SMARCB1 , SMARCA4 , SMARCD3 , SMARCE1  and SMARCD2  could be potential targets of SS18L1. The analysis indicated that these genes are associated with several biological processes including nBAF complex, SWI/SNF superfamily-type complex, histone H4 acetylation, brahma complex and positive regulation of glucose mediated signaling pathway . In addiSchwann cells are unique glial cells in the peripheral nervous system and play important roles during peripheral nerve repair and regeneration , 22. PerTo recognize essential factors for the plasticity of Schwann cells during peripheral nerve repair and injury, our laboratory has constructed a rat sciatic nerve injury model and determined gene expression patterns in the sciatic nerve stumps after rat sciatic nerve injury , 28, 29.The mutation of the SS18L1 gene is associated with amyotrophic lateral sclerosis (ALS) , 34. Thein vitro. The exact function of SS18L1 in the differentiation remains unknown. It is possible that SS18L1 facilitates debris removal and creates a favorable microenvironment for peripheral nerve regeneration.Schwann cells have two time-dependent phenotypes during peripheral nerve injury . In the Besides the functional assessment of SS18L1, we also predicted potential genetic and protein networks of SS18L1 that may regulate Schwann cell proliferation, migration and differentiation. It has been shown that SS18L1 can physically interact with DF2, SMARCD1, SMARCA4, and SMARCE1 . These STaken together, through morphological, cellular and molecular experiments, we explored the expression pattern of the transcription factor SS18L1 and its effects on Schwann cell proliferation, migration and differentiation after peripheral nerve injury in rats. Furthermore, the bioinformatics analysis predicted potential targets and interaction networks of SS18L1 that may be implicated in the cellular processes of Schwann cells during peripheral nerve repair. Our findings combined with more comparative medicine researches will provide new insights into the biological roles of SS18L1 in peripheral nerve regeneration and provide a molecular pathological diagnostic model, as well as a potential therapeutic target for peripheral nerve repair in clinic.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Laboratory Animals center of Nantong University.TQ and HW conceived and designed the experiments. TQ, PQ, and YL performed the experiments and contributed reagents/materials/analysis tools. TQ and PQ analyzed the data. HW wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China (Grant No. 31730031), the Natural Science Foundation of Jiangsu Province (Grant No. BK20202013), the Priority Academic Program Development of Jiangsu Higher Education Institutions of China (PAPD), and Jiangsu Provincial Double-Innovation Doctor Program (grant no. JSSCBS20211095).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.