Remyelination of the peripheral and central nervous systems  is a prerequisite for functional recovery after lesion. However, this process is not always optimal and becomes inefficient in the course of multiple sclerosis. Here we show that, when acetylated, eukaryotic elongation factor 1A1 (eEF1A1) negatively regulates PNS and CNS remyelination. Acetylated eEF1A1 (Ac-eEF1A1) translocates into the nucleus of myelinating cells where it binds to Sox10, a key transcription factor for PNS and CNS myelination and remyelination, to drag Sox10 out of the nucleus. We show that the lysine acetyltransferase Tip60 acetylates eEF1A1, whereas the histone deacetylase HDAC2 deacetylates eEF1A1. Promoting eEF1A1 deacetylation maintains the activation of Sox10 target genes and increases PNS and CNS remyelination efficiency. Taken together, these data identify a major mechanism of Sox10 regulation, which appears promising for future translational studies on PNS and CNS remyelination. The molecular mechanisms regulating remyelination are unclear. Here, the authors show that promoting deacetylation of eEF1A1 prevents the translocation of Sox10 outside the nucleus, contributing to maintaining the expression of Sox10 target genes and increasing remyelination efficiency. Without myelin, the nervous system is poorly functional and sensitive to degeneration, thus myelin is critical for the function of the nervous system and the maintenance of its integrity. Different disorders in humans can lead to demyelination, hypomyelination, or dysmyelination. Demyelination occurs after traumatic lesions, which can regenerate in the PNS, but not in the CNS4. In the CNS, the most frequent demyelinating disease is an immune degenerative disease called multiple sclerosis that progresses through successive demyelinating lesions leading to permanent loss of function when remyelination fails7. In particular, the efficiency of CNS remyelination decreases dramatically with age, mainly due to a differentiation defect of OL precursor cells (OPCs) into mature myelinating OLs8. Other myelin disorders are inherited, such as subtypes of Charcot-Marie-Tooth disease11, a large group of peripheral neuropathies, and the group of leukodystrophies in the CNS12, or occur after an infection such as the Guillain-Barré syndrome13. Loss of myelin can also be secondary to diabetes, toxic agents, or aging. In summary, many pathological states can lead to myelin deficiency and thus to severe neurological disabilities. To optimize functional recovery and prevent irreversible loss of function, it is of utmost importance to identify treatments that enhance remyelination after lesion15.Many axons of the peripheral and central nervous systems  are myelinated. Two types of myelinating cells, Schwann cells (SCs) in the PNS and oligodendrocytes (OLs) in the CNS, build a thick myelin sheath rich in lipids around axons, which provides axonal insulation and saltatory nerve conduction18. In addition, post-translational modifications including acetylation and methylation have been recently shown to hold critical functions in regulating transcription factor activity and gene expression during myelination and remyelination21. For instance, in OLs, the highly homologous histone deacetylases HDAC1 and HDAC2 (HDAC1/2) are required for differentiation and myelination22. Interestingly, HDAC1, HDAC3, and HDAC10 can promote nuclear localization of the transcription factor Olig1 by deacetylation and thereby OL maturation23. Methylation marks, such as trimethylation of histone H3 lysine 4 (H3K4me3), are also involved in the myelination process. Indeed, H3K4me3, an activating methylation mark, accumulates at enhancers of promyelinating factors such as Myrf and Olig2 and of myelin genes to promote OL myelination25. In the PNS, HDAC4, together with HDAC3, deacetylate and silence the promoter of cJun, an inducer of SC demyelination, to promote myelination26, however, the loss of HDAC3 leads to hypermyelination in adult mice and promotes remyelination after PNS lesion28, suggesting a dual function for HDAC3 in the myelination and remyelination processes. The PRC2 complex, which adds repressive H3K27me3 marks, also possesses a dual function: it is necessary to prevent hypermyelination, whereas it also maintains myelin integrity by repressing injury-related genes29. Among the transcription factors that regulate myelination and remyelination, Sox10 holds major functions in both PNS and CNS32. Indeed, Sox10 activates the transcription of promyelinating and myelin genes such as Oct6, Krox20, and Myelin protein zero (P0) in SCs35, and Myrf and Myelin basic protein (Mbp) in OLs37. The mechanisms that control the activity and expression of Sox10 remain however partially understood. We previously showed that HDAC1/2 act as co-factors of Sox10 in SCs and are thereby critical for SC development, myelination, and remyelination41. In the context of remyelination, HDAC2 allows the recruitment of a multifunctional protein complex containing Sox10 and the two H3K9 demethylases KDM3A and JMJD2C to Oct6 and Krox20 genes to de-repress these Sox10 target genes and allow their activation by Sox1041. In these previous studies, the direct deacetylation target of HDAC1/2 that enables the formation of this complex and thereby Sox10 activity remained however elusive. Here we show that Sox10 nuclear localization and expression are negatively regulated by eEF1A1, thereby controlling Sox10 activity. EEF1A (two highly homologous proteins eEF1A1 and eEF1A2 coded by two different genes) is described as a major translation elongation factor, however, several studies have identified additional functions for this factor42. Indeed, eEF1A has also been involved in the nuclear export of tRNAs, transcriptional regulation, proteolysis, apoptosis, oncogenesis, and viral propagation44. We demonstrate that the regulation of Sox10 by eEF1A1 is controlled by acetylation. Indeed, Ac-eEF1A1 translocates to the nucleus to interact with Sox10 and drag it out of the nucleus. We show here that HDAC2 deacetylates eEF1A1 in the nucleus, which induces its return to the cytoplasm and maintains Sox10 on its target genes. In addition, we report that theophylline, a known activator of HDAC2, decreases the levels of Ac-eEF1A1, increases the expression of Sox10 and of Sox10 target genes, and enhances the remyelination process in the PNS after a traumatic lesion and in the CNS after a demyelinating lesion in young adult and old mice.To enhance remyelination, it is crucial to understand the mechanisms controlling this process. PNS and CNS myelination mechanisms have been extensively studied and several transcription factors are known to be involvedKrox2041, another major promyelinating factor46, to induce the remyelination process, which is almost complete at 60dpl. We previously showed that HDAC1 and HDAC2, which can compensate for the loss of each other, are necessary for Sox10 functions in the PNS, and that HDAC2 is primarily involved in this process41. To identify direct targets of HDAC1/2, we collected at 1dpl sciatic nerves of adult mice treated with the HDAC1/2 inhibitor mocetinostat or its vehicle and analyzed the levels of acetylated proteins by mass spectrometry41. Interestingly, analysis by Protein Match Score Summation identified eEF1A1 as a putative HDAC1/2 deacetylation target  with mice expressing floxed Hdac1 and Hdac2 . We found strongly increased levels of Ac-eEF1A in SCs of postnatal day (P)4 Dhh-Cre;Hdac1fl/fl;Hdac2fl/fl knockout (dKO) nerves as compared to Dhh-Cre-negative control littermates  in adult mice Fig. , a well-tes Fig. , while t49. We found by subcellular fractionation that each mutation leads to increased nuclear localization of eEF1A1 , Sox10-MIC , and Sox10-HMG (containing only the HMG domain51) . We show that eEF1A1 and Stat3 interact with sumoylated Tip60 in SCs  increases HDAC2 and Sox10 levels in primary SCs, whereas 1 µM CGS 15943 (antagonist of adenosine receptors) or 500 µM IBMX (inhibitor of phosphodiesterases) or 10 µM Rolipram (inhibitor of type IV phosphodiesterases) either have no effect or decrease HDAC2 and/or Sox10 levels  and P0 (at the intron 1) at the SC redifferentiation stage to induce remyelination  mice . Consistent with our findings in SCs, K273Q mutant was localized in both nuclear and cytoplasmic compartments of virtually all transfected cells, while wild-type eEF1A1 was either localized exclusively in the cytoplasm or in both nucleus and cytoplasm of Oli-neu cells cultured under proliferating conditions . We show that a short treatment with theophylline from 10 to 14dpdl leads to a strong increase of HDAC2, Sox10 and MBP expression Fig.  and to itio Fig. . These d38-41, but the direct deacetylation mechanism underlying this function remained unclear. We show here that HDAC1/2 deacetylate eEF1A1, which prevents interaction of eEF1A1 with Sox10 and re-localization of Sox10 to the cytoplasm, thereby allowing Sox10 to activate its target genes and enabling remyelination. We found that after a sciatic nerve crush lesion, Tip60 is upregulated and acetylates eEF1A1 in the SC cytoplasm, in a Stat3-dependent manner. Consistently, Stat3 has been previously shown to interact with cytoplasmic Tip6054 and with eEF1A153 in other cells. We show here that Stat3 promotes the interaction of Tip60 with eEF1A1 and eEF1A1 acetylation. eEF1A1 is a major translation elongation factor, which is mostly found in the cytoplasmic compartment. However, eEF1A1 has been shown to have non-canonical functions and to be localized in the nuclear compartment in some cases44. EEF1A1 can be submitted to various post-translational modifications, including phosphorylation, acetylation, methylation, ubiquitination49. Phosphorylation, which is the most studied post-translational modification of eEF1A1, has been shown to regulate eEF1A1 activity42. In contrast, the function of eEF1A1 acetylation is not or poorly described. We show here that acetylation of eEF1A1 induces its re-localization to the nuclear compartment of SCs. There are 20 putative acetylation sites in eEF1A149. We studied the functions of the three acetylation sites K41, K179, and K273 because they have been several times verified experimentally. In addition, K273 is found only in eEF1A1 and not in eEF1A249. Our results indicate that acetylation at any of these sites promotes nuclear localization of eEF1A1 and the decrease of Sox10 levels in SCs, and suggest that K179 and K273 are key acetylation sites for these functions. By using mocetinostat, a specific inhibitor of HDAC1/2, or by ablating HDAC1/2 specifically in SCs or in OLs, we show that HDAC1/2 are necessary to deacetylate eEF1A1 in SCs and OLs, and to maintain high Sox10 expression in the nuclear compartment of these cells.Demyelination occurs in various cases in humans. In many cases, the remyelination process is not efficient enough and the loss of myelin leads to permanent loss of function. For this reason, it is crucial to better understand how remyelination is controlled in order to identify strategies that improve remyelination. In this study, we have focused our analyses on the remyelination process that occurs in the PNS after a traumatic lesion and in the CNS after a demyelinating lesion. In particular, we identified a mechanism that controls the activity and expression of Sox10, a major transcription factor of PNS and CNS myelination and remyelination. We previously demonstrated that HDAC1/2 act as co-factors of Sox1065. We show here that while theophylline increases the levels of HDAC2 and Sox10 in SCs, compounds that specifically inhibit phosphodiesterases or antagonize adenosine receptors either have no effect on HDAC2 and Sox10 levels or decrease the levels of HDAC2 and Sox10, indicating that theophylline does not increase Sox10 levels through its effect on phosphodiesterases or adenosine receptors but is highly likely to act on Sox10 through HDAC2. Interestingly, a short treatment with theophylline in mice increased HDAC2 expression, decreased Ac-eEF1A1 levels, increased the expression of Sox10 and its target genes and improved remyelination efficiency in both PNS and CNS. Of major importance, theophylline also increased the efficiency of CNS remyelination in old mice, thereby suggesting a potential beneficial effect on the remyelination process in aged individuals, where remyelination is strongly impaired.In the aim of identifying a treatment that promotes this mechanism and thereby potentially improves remyelination efficiency, we tested the effect of theophylline treatment in vivo. Although theophylline inhibits phosphodiesterases and antagonizes adenosine receptors when used at high concentration, it is also a known potent activator of HDAC2, increasing HDAC2 activity and expression when used at low concentrationKrox20 MSE38, a critical enhancer for Krox20 expression. In addition, overexpression of HDAC2 alone leads to a 3-fold induction of the Krox20 MSE and co-overexpression of Sox10 and HDAC2 has a synergistic effect on the activation of the Krox20 MSE, with a 25-fold increase38. These data indicate that increased Sox10 levels in differentiated SCs can further increase the activation of its target gene Krox20 and that this is potentiated by simultaneous increase of HDAC2 expression. In the case of remyelination, Sox10 is upregulated at the remyelination stage to induce the remyelination program by the upregulation of Krox20 and of myelin proteins. We show here that theophylline treatment at 10dpl before remyelination starts allows to reach faster a high upregulation of Sox10, faster recruitment of Sox10 to its target genes Krox20 and P0, faster upregulation of Krox20 and P0, faster remyelination and faster functional recovery. We also showed that theophylline treatment does not change the density of SCs or of inflammatory cells nor the percentage of proliferating cells in the sciatic nerve after lesion. Upregulating Sox10 to a high level faster is thus very likely to induce faster remyelination and faster functional recovery. In addition, raising Sox10 to a higher expression level is also likely to help sustain the remyelination process for a longer time, as shown by the high expression of Krox20 and P0 at 30dpl and more efficient remyelination in theophylline-treated mice at 2 months post lesion. Thus, our previous work38 and current findings suggest that Sox10 levels and activity including the timing of Sox10 upregulation and the levels of nuclear Sox10 are rate-limiting for the speed and efficiency of remyelination, and that increasing Sox10 levels and activity in conjunction with HDAC2 appears as a very promising strategy to accelerate and improve remyelination after lesion.We previously showed that in differentiated primary SCs where Sox10 levels are already high and which express the major promyelinating factor Krox20 and the myelin protein P0, the overexpression of Sox10 results in a 5-fold induction of the In summary, this study identifies Ac-eEF1A1 as a critical negative regulator of the remyelination process and shows that theophylline, by activating HDAC2, promotes eEF1A1 deacetylation, increases Sox10 levels and activity and remyelination speed and efficiency after lesion of the PNS and CNS, thus appearing as a very promising compound to test in future translational studies to accelerate and promote remyelination after traumatic lesions or in the context of demyelinating disorders.p values were calculated in Microsoft Excel (Mac version 16.34) using two-tailed (black asterisks) or one-tailed (gray asterisks) Student’s t-tests. P values: *<0.05, **<0.01, ***<0.001, data are presented as mean values ± SEM. Individual data points are represented in the graphs by scatter points. Sample size was determined by the minimal number of animals or individual experiment required to obtain statistically significant results and increased in some cases to improve confidence in the results obtained. No animal or data point was excluded from the analysis.For each data set presented, experiments were performed at least three times independently or with at least three animals and Dhh promoter47 with mice expressing floxed Hdac1 and Hdac248. To induce ablation of HDAC1/2 in OLs of adult mice, mice expressing floxed Hdac1 and Hdac2 were crossed with mice expressing tamoxifen-inducible Cre recombinase under control of the OL (and SC)-specific Plp promoter67. To ablate HDAC1/2, mice received daily injections of 2 mg tamoxifen (Sigma) for five consecutive days. Genotypes were determined by PCR on genomic DNA.We ablated HDAC1/2 specifically in SCs by crossing mice expressing the Cre recombinase under control of the 41): An incision was made at the height of the hip and the sciatic nerve was exposed on one side. The nerve was crushed (5 ×10 sec with crush forceps: Ref. FST 00632-11). The wound was closed using Histoacryl Tissue Glue (BBraun). After the operation, mice were wrapped in paper towels until recovery from anaesthesia. Lysolecithin injections were carried out at T8 level as follows (by a procedure that was previously described68): to prevent dehydration, a single i.p. injection of 500 µl electrolyte solution with glucose  was administered preoperatively under anesthesia. A 1 cm long skin incision was made through the lower thoracic spine of the animal, followed by the separation of the paravertebral muscles from their insertion points at the processi spinosi over a length of ~3 mm to allow access to the spinal cord. The ligamentum flavum, which connects the dorsal lamella of two adjacent vertebrae, was then incised and the underlying dura mater was pierced with a needle at the injection site . Two microliters of lysolecithin  were injected focally into the dorsal funiculus and the ventral horn of the spinal cord with a glass capillary. We used a stereotactic three-way micromanipulator to hold the syringe and insert it into the spinal cord at a defined angle  and at a defined depth: the needle of the Hamilton syringe was slowly inserted into the spinal cord on the right of the central vein at T8 vertebrae level until resistance was observed. The needle was then withdrawn 0.1 mm, 1 µl of lysolecithin was injected within 30 s, then the experimenter waited 30 s, the needle was then moved up 0.5 mm, 1 µl of lysolecithin was injected again within 30 s, the experimenter waited 1 min, then the needle was withdrawn.For all surgical procedures, we used isoflurane  for anesthesia. For analgesia, 0.1 mg/kg/body weight buprenorphine  was administered by i.p. injection 1 h before surgery, a second time in the evening on the day of surgery and afterwards every 12 h during 3 days. The mice were placed on a heat pad during the entire procedure until waking up from anaesthesia. To prevent dehydration of the eyes, a carbomer liquid eye gel  was used preoperatively. Mice were shaved either at the height of the hip for sciatic nerve crush lesion or on their back for lysolecithin lesion of the spinal cord and the field of operation was cleaned and disinfected. Sciatic nerve crush lesions were carried out on 3 to 4-month old adult mice  as follows  and received only the animal number given at birth by the animal caretaker. Treatment allocation and collection/analysis of data were done by different persons. Treatments: 10 mg/kg mocetinostat (HDAC1/2 inhibitor) or 10 mg/kg theophylline or their respective vehicle were injected in the pelvic cavity after wound closure and treated again once a day for the indicated time in the figure legends. In mouse pups, 10 mg/kg theophylline or its vehicle were injected subcutaneously once a day for 3 days at P1, P2, and P3 and collection of tissues was carried out at P4. For analysis in the spinal cord of adult mice , 10 mg/kg mocetinostat or its vehicle were injected intrathecally once and animals were sacrificed 24 h later, or 10 mg/kg theophylline or its vehicle were injected intraperitoneally once a day for 4 days at 10, 11, 12, and 13dpdl. All mice used in this study were from mixed strains backcrossed at least 10 times to C57BL/6 J. Mice were housed in a standard mouse facility with controlled ventilation , temperature (22 ± 2°C) and humidity (65 ± 5%) in individually ventilated type II long cages (L 365 mm × B 207 mm × H 140 mm) containing sawdust bedding, a cardboard cylinder and 2 paper tissues on the floor, food pellet and water ad libitum. Light cycle: 12:12 h. This study complies with all relevant ethical regulations concerning animal use, which was approved by the Veterinary office of the Canton of Fribourg, Switzerland and the Veterinary office (Landesuntersuchungsamt) of Rheinland-Pfalz, Germany.Mice were placed four times on the Rotarod apparatus at a fixed speed of 15 revolutions per minute to test balance and motor coordination, at 14 and 30dpl. The duration of each trial was limited to 600 s, and trials were separated by a 30 min recovery period. Latency to fall from the rotating beam was recorded and the average of the three trials was used for quantification. Recovery of sensory function was tested at 14 and 30dpl by toe pinch test: each toe of the rear foot on the right side (lesioned side) was pinched with equal pressure applied by the same experimenter using flat tip forceps. Immediate withdrawal was recorded as functional sensitivity of the pinched toe. In case no toe exhibited sensitivity, the same test was applied to toes of the contralateral side (uninjured side), which always resulted in immediate withdrawal. All tests were carried out with the same experimental animals (10 theophylline-treated mice and 9 vehicle-treated mice).Mbp promoter-luciferase construct were kindly provided by Dr. Michael Wegner . K to Q or to R eEF1A1 mutants were generated by site-directed mutagenesis using pCMV6-AC-mouse eEF1A1-turbo GFP as template. Primers were as follows: K41Q forward, 5′- GGAATCGACAAGCGAACCATCGACAAGTTTGAGAAAGAGGCTGCTGAG - 3′, K41Q reverse, 5′- CTCAGCAGCCTCTTTCTCAAACTTGTCGATGGTTCGCTTGTCGATTCC - 3′, K41R forward, 5′- GGAATCGACAAGCGAACCATCGAAGAGTTTGAGAAAGAGGCTGCTGAG - 3′, K41R reverse, 5′- CTCAGCAGCCTCTTTCTCAAACTCTTCGATGGTTCGCTTGTCGATTCC - 3′, K179Q forward, 5′- CGTTAAGGAAGTCAGCACCTACATTCAGAAAATTGGCTACAACCCTGACACAG - 3′, K179Q reverse, 5′- CTGTGTCAGGGTTGTAGCCAATTTTCTGAATGTAGGTGCTGACTTCCTTAACG - 3′, K179R forward, 5′- CGTTAAGGAAGTCAGCACCTACATTAGGAAAATTGGCTACAACCCTGACACAG - 3′, K179R reverse, 5′- CTGTGTCAGGGTTGTAGCCAATTTTCCTAATGTAGGTGCTGACTTCCTTAACG - 3′, K273Q forward, 5′- GCCGAGTGGAGACTGGTGTTCTCCAACCTGGCATGGTGGTTACCTTTGC - 3′, K273Q reverse, 5′- GCAAAGGTAACCACCATGCCAGGTTGGAGAACACCAGTCTCCACTCGGC - 3′, K273R forward, 5′- GCCGAGTGGAGACTGGTGTTCTCAGACCTGGCATGGTGGTTACCTTTGC - 3′, K273R reverse, 5′- GCAAAGGTAACCACCATGCCAGGTCTGAGAACACCAGTCTCCACTCGGC - 3′. All constructs were verified by sequencing.Expression constructs: pCMV6-AC-mouse eEF1A1-turbo GFP (C-ter) and GFP backbone constructs were acquired from Origene. Control shRNA (PLKO) and Stat3 shRNA  were acquired from Sigma-Aldrich. Sox10 wild type and mutant constructs, 95, MIC and HMG and the 69): primary SCs were derived from P2 Wistar rat sciatic nerves and dissociated in 0.3 mg/ml collagenase type I (Sigma-Aldrich) and 2.5 mg/ml trypsin (Sigma-Aldrich) in DMEM (Invitrogen) at 37°C and 5% CO2/95% air for 1 h. After the addition of DMEM containing 10% FCS (Gibco), cells were centrifuged at 500 × g for 10 min, resuspended in DMEM containing 10% FCS, 1:500 penicillin/streptomycin (Invitrogen), and 10 μM cytosine arabinoside (Sigma-Aldrich), and plated on plastic dishes coated with poly-D-lysine (Sigma-Aldrich). After 24 h at 37 °C and 5% CO2/95% air, cells were washed and incubated at 37 °C and 5% CO2/95% air until they reached confluency in SC proliferating medium: DMEM containing 10% FCS (Gibco), 1:500 penicillin/streptomycin (Invitrogen), 4 µg/ml crude GGF , and 2 µM forskolin (Sigma). SCs were then purified by sequential immunopanning in plastic dishes coated with a Thy1.1 antibody70. Identity and purity were checked for each primary preparation by immunofluorescence of SC-specific markers . SCs were grown in proliferation medium: at 37°C and 5% CO2/95% air. SC de-differentiation culture protocol mimicking SC demyelination and conversion into repair cells that occur after a PNS lesion was as follows (previously described41): SCs were first growth-arrested in defined medium (DM58) for 8 to 15 h, then 1 mM dbcAMP (Sigma) was added to induce differentiation. Cells were incubated in this medium for another 3 days. The medium was then changed to DM only without dbcAMP, and RSCs were incubated in this medium for 3 days (differentiation mimicking adult SC stage). To induce de-differentiation, cells were then changed to proliferation medium and incubated in this medium for 1 to 3 days (exact duration stated in the figure legends). For some experiments, 10 µM MG132 (proteasome inhibitor), 5 µM TH1834 (Tip60 inhibitor), 0.6 µM mocetinostat (HDAC1/2 inhibitor), 1 µM theophylline (HDAC2 activator at this concentration), 1 µM CGS 15943 (adenosine receptor antagonist), 500 µM IBMX (phosphodiesterase inhibitor), 10 µM rolipram (type 4 phosphodiesterase inhibitor) or their vehicle were added on cells.Purified primary rat SC cultures were obtained as follows . Identity and purity were checked for each primary preparation by immunofluorescence of Sox10 and CC1.Primary OLs were isolated from P1 to P2 rat cortices, which were first diced, incubated with DNase I and Trypsin for 15 min at 37°C and 5% CO66  were cultured either in proliferating conditions in DMEM containing 1% horse serum (Gibco) and the following media components: 40 nM Biotin, 100 µg/ml Apo-transferrin, 16 µg/ml Putrescine, 6 ng/ml Progesterone, 4.8 ng/ml Sodium selenite, 5 µg/ml Insulin , 1:500 Penicillin-Streptomycin (Invitrogen), or in differentiating conditions in DMEM containing 100 µg/ml Apo-transferrin, 16 µg/ml Putrescine, 6 ng/ml Progesterone, 4.8 ng/ml Sodium selenite, 5 µg/ml Insulin, 40 ng/ml Triiodothyronine , 1:500 Penicillin-Streptomycin (Invitrogen) on poly-D-lysine (Sigma)-coated dishes.Oli-neu cellsHEK293 cells were obtained from ATCC and were cultured in DMEM containing 10% FCS (Gibco) and 1:500 penicillin/streptomycin (Invitrogen) on poly-D-lysine (Sigma)-coated dishes.Mycoplasma contamination was not tested, because of the low incidence of mycoplasma contamination in primary cells, and because mycoplasma contamination results in inefficiency of transfection, which we did not observe in our primary cells or in Oli-neu cells or in HEK293 cells.40, and embedded in Spurr’s resin (Electron Microscopy Sciences). Ultrathin sections (70-nm thick) were cut. G-ratio (diameter of axon: diameter of axon+myelin) analyses were done at 12 mm distal to the lesion site for sciatic nerves and in the lesion site for spinal cords . To delineate the lesion site in the spinal cord and identify remyelinated axons, the g-ratio was calculated. Axons with a g-ratio above or equal to 0.835 were classified as remyelinated axons. This g-ratio threshold value was defined after calculating the g-ratio of axons in non-lesioned mouse spinal cords, where axons with the thinnest myelin sheath had a g-ratio under or equal to 0.83. No contrasting reagent was applied. Images were acquired using a Philips CM 100 BIOTWIN equipped with a Morada side-mounted digital camera (Olympus).Mice were killed with 150 mg/kg pentobarbital i.p.  and sciatic nerves or spinal cords were fixed in situ (sciatic nerves) or by perfusion  with 4% paraformaldehyde (PFA) and 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Fixed tissues were post-fixed in 2% osmium tetroxide, dehydrated through a graded acetone seriesMouse sciatic nerves were fixed in situ with 4% PFA for 10 min, dissected, embedded in O.C.T. Compound , and frozen at −80°C. Sciatic nerve cryosections (5-μm thick) were first incubated with acetone for 10 min at −20°C, washed in PBS/0.1% Tween 20, blocked for 30 min at room temperature (RT) in blocking buffer (0.3% Triton X-100/ 10% Goat serum/ PBS), and incubated with primary antibodies overnight at 4°C in blocking buffer. Sections were then washed 3 times in blocking buffer and secondary antibodies were incubated for 1 h at RT in the dark. Sections were washed again, incubated with DAPI for 5 min at RT, washed and mounted in Citifluor (Agar Scientific).Cells were washed with PBS, fixed for 10 min with 4% PFA at RT, washed again in PBS, blocked, incubated with antibodies, DAPI, and mounted as described above.For immunofluorescence on spinal cords, mice were deeply anesthetized with a lethal dose of pentobarbital and perfused with 4% PFA after blood removal with heparin. Spinal cords were collected 2 mm above and below the lesion site, post-fixed in 4% PFA for 2 h at RT, incubated in 20% sucrose overnight at 4°C, embedded in O.C.T. compound, and frozen at -80°C. We used 5-μm cryosections. Sections were first submitted to antigen retrieval in citrate buffer  for 2 h at 65°C, washed, blocked, and incubated with antibodies, DAPI, and mounted as described above.Primary antibodies: eEF1A1 , eEF1A-pan (Acetyl-Lys41) , HDAC2 , Sox10 , Sox10-Nterm , CC1 , Olig2 , 20 S proteasome subunit , MBP , S100 , F4/80 , STAT3 . All secondary antibodies were from Jackson ImmunoResearch. Photos were acquired using a Leica TCS SP-II confocal microscope or a Visitron VisiScope spinning disk confocal system CSU-W1. Fiji (version 1.0) and Adobe Photoshop (CC 20.0.8 Release) were used to process images. Single optical sections or z-series projections (stated in the figure legends) are shown.29 with modifications: freshly dissected mouse sciatic nerves were minced in 1% formaldehyde for 10 min for crosslinking, which was then quenched for 10 min with glycine to a final concentration of 0.125 M. Samples were then sequentially lysed as follows: samples were first resuspended in ice cold lysis buffer 1 , rotated at 4°C for 10 min and centrifuged at 4°C at 1350 x g for 5 min. After discarding the supernatant, the same procedure was repeated in lysis buffer 2 . The pellet was then resuspended in lysis buffer 3 , incubated on ice for 10 min, homogenized and diluted by 2 in lysis buffer 3. Chromatin was fragmented by sonication with a Bioruptor (Diagenode) set at medium power, interval: 30 s on / 45 s off, time: 4 cycles of 10 min, rest of 2 min between cycles. Sonicated chromatin containing 75 µg of proteins and 3 µg of antibodies were used per IP. Sonicated chromatin was incubated overnight a 4 °C on a rotating wheel, and Magna ChIP™ Protein A + Protein G Magnetic Beads (Millipore) were added and incubated for 2 h at 4 °C. Magnetic beads were washed, immunoprecipitated samples were eluted in elution buffer , crosslinking was reversed at 65 °C for 8–16 h and after RNase and proteinase K treatment, the DNA was purified using a PCR clean-up kit (Macherey-Nagel). Quantitative real-time PCR analyses were performed with an ABI 7000 Sequence Detection System (Applied Biosystems) using FastStart SYBR Green Master (Sigma), according to the manufacturer’s recommendations. A dissociation step was added to verify the specificity of the products formed. Primers sequences were as follows: Krox20 MSE, forward, 5′-TTTCGTCTTTGGGCTCATTC-3′, reverse, 5′-AGCCCTTCACAAAGCTGAAA-3′, P0 intron 1, forward, 5′-GTGCTGGACTCGGCATCTTT-3′, reverse, 5′-CCAGGTTACCATGACTGGGG-3′. For Sox10 IP, we used 2 µg/IP of anti-Sox10 antibody  and as negative control IP we used 2 µg/IP of anti-GFP antibody .We used a previously described protocolFor all in vivo Western blots and immunoprecipitations, we have collected the injured sciatic nerve from the lesion site to around 12 mm distal to the lesion site . We have collected the same region of the contralateral nerve as internal control for each animal. After perineurium removal, sciatic nerves were frozen in liquid nitrogen, pulverized with a chilled mortar and pestle, lysed in radioimmunoprecipitation assay (RIPA) buffer  for 30 min on ice, and centrifuged to pellet debris. Supernatants were collected.Cells were washed once in PBS, lysed in RIPA buffer for 15 min on ice, and centrifuged to pellet debris. Sciatic nerves and cell lysates were submitted to SDS-PAGE and analyzed by Western blotting.72), Lamin A/C , KAT5/Tip60 , STAT3 , GFP .Primary antibodies used: eEF1A1 , eEF1A-pan (Acetyl-Lys41) , HDAC2 , Sox10 , Sox10 , GAPDH , P0 , Krox20  and goat anti-rabbit-HRP, and heavy chain-specific goat anti-chicken-HRP.For non-denaturing IPs, tissues were prepared and lysed as described above in the following RIPA buffer: 10 mM Tris/HCl, pH7.4, 150 mM NaCl, 50 mM NaF, 100 mM Na3VO4, 1 mM EDTA, 0.5% Nadeoxycholate, 0.5% NP40. For denaturing IPs, tissues were lysed in 10 mM Tris/HCl, pH7.4, 1% SDS, boiled, mixed with 9 volumes of RIPA buffer, reboiled, and centrifuged. Lysates were pre-cleared for 1 h with 30 µl protein A/G PLUS agarose beads (Santa-Cruz Biotechnology). One milliliter of cleared lysates was rotated overnight at 4° C with immunoprecipitating antibodies: 2 µg of Sox10 , eEF1A1 , EEF1A2 , GFP , Flag , SUMO-1  antibodies were used per nerve. We used rabbit anti-GFP or mouse anti-Flag antibodies as control IPs. Forty microliters of beads were added, and samples were rotated for 1 h at 4°C. Immunoprecipitates were pelleted, washed four times with RIPA buffer, eluted with 15 µl 0.1% formic acid and neutralized with 1.5 M Tris, pH 8. Six microliters of Laemmli buffer were added and samples were boiled for 10 min. Analysis was done by Western blot.Forty to fifty-percent confluent Oli-neu cells in 12-well plates were transfected either in proliferating or in differentiating medium with Fugene 6 (Promega) at a 3:1 ratio Fugene 6:DNA, according to the manufacturer’s recommendations with the following modification: the DNA was incubated in Optimem for 5 min at room temperature before addition of Fugene 6. The mix Fugene 6:DNA was then incubated for 25 min at room temperature before being added to the cells. Cells were lysed 24 h after transfection in 180 µl Reporter Lysis Buffer (Promega) and assayed for luciferase activity. Twenty microliters of lysate were subjected to 2 consecutive injections of each 25 µl luciferase substrate and values were recorded after an integration time of 30 s. Efficiency of transfection was evaluated by measuring beta-galactosidase activity of a co-transfected beta-gal construct (Promega). Oli-neu cells were co-transfected with 2 µg of the luciferase construct pGL2-Mbp promoter (kind gift from Dr M. Wegner) or empty pGL2 (Promega), 2 µg of the overexpressing construct pCMV6-AC-mouse eEF1A1-turbo GFP (C-ter) or GFP backbone (Origene) or pCMV6-AC-mouse eEF1A1K273Q-turbo GFP (C-ter), and 300 ng beta-gal construct. Luciferase activity was first normalized to beta-galactosidase activity and then normalized to the empty pGL2 luciferase control (transfected with the overexpressing construct at the same ratio Fugene 6:DNA). For beta-galactosidase activity, we followed the instructions of the manufacturer (Promega). Briefly, 50 µl Assay Buffer 2x was added to 50 µl lysate, and beta-galactosidase activity was recorded after a 15-20 min incubation time at 37°C. Endogenous beta-galactosidase activity (as determined from cells only transfected with overexpressing construct at the same ratio Fugene 6:DNA) was subtracted.41. The data of the present study results from a different analysis of the previous experiment by Protein Match Score Summation.The mass spectrometry experiment and method after IP of acetyl-lysine at 1dpl in mouse sciatic nerves treated with mocetinostat or vehicle has been previously publishedFor identification of eEF1A1 binding partners by mass spectrometry analysis, the collected beads were heated in SDS-PAGE loading buffer containing 1 mM DTT for 10 min at 75°C. After alkylation using 5.5 mM iodoacetamide for 10 min at room temperature the samples were centrifuged and the supernatants were loaded on 4-12% gradient gels  for protein separation. After staining, each gel lane was cut into 7 slices, the proteins were in-gel digested with trypsin (Promega) and the resulting peptide mixtures were processed on STAGE tips and analyzed by LC-MS/MS. The LC-MS measurements were performed on a QExactive Plus mass spectrometer coupled to an EasyLC 1000 nanoflow-HPLC. Peptides were separated on fused silica HPLC-column tip  to a length of 20 cm) using a gradient of A (0.1% formic acid in water) and B (0.1% formic acid in 80% acetonitrile in water). The mass spectrometer was operated in the data-dependent mode; after each MS scan  a maximum of ten MS/MS scans were performed using a normalized collision energy of 25%, a target value of 1000 and a resolution of 17500. The MS raw files were analyzed using MaxQuant Software version 1.4.1.2 for peak detection, quantification, and peptide identification using a full length UniProt mouse database  and common contaminants such as keratins and enzymes used for in-gel digestion as reference. Carbamidomethylcysteine was set as fixed modification and protein amino-terminal acetylation, lysine acetylation, and oxidation of methionine were set as variable modifications. The MS/MS tolerance was set to 20 ppm and three missed cleavages were allowed using trypsin/P as enzyme specificity. Peptide and protein FDR based on a forwards-reverse database were set to 0.01, minimum peptide length was set to 7, and minimum number of peptides for identification of proteins was set to one, which must be unique. The “match-between-run” option was used with a time window of 1 min.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Movie 1Reporting Summary


The synthesis of self-organized TiO2 NTs from Ti-6Al-4V alloy was carried out via one-step electrochemical anodization in a fluoride ethylene glycol containing electrolytes. The electrodeposition of the polymer electrolyte onto anatase TiO2 NTs was performed by cyclic voltammetry. The anodized Ti-6Al-4V alloys were characterized by scanning electron microscopy and X-ray diffraction. The electrochemical properties of the anodized Ti-6Al-4V alloys were investigated by cyclic voltammetry and chronopotentiometry techniques. The full-cell shows a high first-cycle Coulombic efficiency of 96.8% with a capacity retention of 97.4% after 50 cycles and delivers a stable discharge capacity of 63 μAh cm−2 μm−1 (119 mAh g−1) at a kinetic rate of C/10.All-solid-state batteries were fabricated by assembling a layer of self-organized TiO The selective dissolution of aluminium oxide (Al2O3) and vanadium oxides  might be occurred simultaneously at this stage. As a result, the balance of the metal oxidation and chemical dissolution of the oxide layer leads to the growth nanotubular structures. The dominant reaction mechanisms are proposed in Equations (7)–(9) as follows:The formation of TiO2 NTs is achieved by the cyclic voltammetry (CV) technique. The nanotubes synthesized in ethylene glycol electrolyte containing 20 wt% water are used for the electropolymerization of the polymer electrolyte into the nanotubes. The starting monomer is methyl ether methacrylate poly (ethylene glycol) . The upper potential limit was set according to the open circuit voltage of the system, which is around −0.35 V versus Ag/AgCl (3M KCl), and the lower limit was set to −1V versus Ag/AgCl (3M KCl) [2 NTs is based on the formation of intermediate hydrogen-free radicals [+-producing H2 is accompanied by the formation of hydrogen-free radicals that can react with the monomers, leading to the polymerization of methyl methacrylate (PMMA). 2 NTs for 25 cycles. Clearly, the absolute value of the cathodic current at −1 V versus Ag/AgCl (3M KCl) drops as the increment of cycles. The reason for the obvious fading can be explained by the successive growth of thin insulating polymer layers on the walls of TiO2 NTs.In the present study, the electrochemical deposition of the polymer electrolyte into TiO(3M KCl) . The staradicals . For cat2 NTs layer was confirmed by morphological analysis. 2 NTs after a different number of CV cycles such as 5, 10, and 25 cycles, confirming the conformal deposition of the polymer electrolyte. After 25 cycles, the nanotubes are filled and almost covered by a thin polymer film, following the surface rugosity of the surface. The polymer electrolyte must be conformally deposited onto the electrodes and form a continuous layer without any pinholes in order to be used as separator to prevent the short-circuiting of the electrodes.The electrodeposition of the polymer electrolyte into the TiO2 NTs and the polymer-coated TiO2 NTs were investigated as anode materials for Li-ion batteries. Herein, the synthesized TiO2 NTs with 20 wt% H2O has been used to study the charging and discharging mechanisms during the electrochemical cycling experiments. The samples were tested at scanning rates of 0.5 mV s–1 in the potential range between 1 and 3 V versus Li/Li+ at room temperature, as displayed in +, which is responsible for the electrode phase transformations. The redox peaks are well-defined and distinguishable (+ insertion (cathodic peak) and extraction (anodic peak) occur at the potential of 1.68 and 2.06 V versus Li/Li+, respectively. The slightly decrease of the current densities in the absolute cathodic and anodic peak indicates the small discharge capacity fading upon cycling. However, after first cycle, well-overlapped redox peaks are visible, indicating a good electrochemical stability of the electrode. An additional peak pair at a potential of approximately 2.62 V versus Li/Li+ with a low current density is also appeared due to the presence of an electrochemically active VO2 phase, but the VO2 phase does not significantly contribute to the storage performance of the electrode [2 NTs, showing the anodic peak at 2.08 V versus Li/Li+ and cathodic peak at 1.63 V versus Li/Li+  and 139.2 µAh cm−2 (328.5 mAh g−1) µm−1, respectively, corresponding to a Coulombic efficiency (CE) of 53.78%. Meanwhile, the bare TiO2 NTs shows flat plateaus of charge at 1.86 V versus Li/Li+ and discharge 1.74 V versus Li/Li+, a delivering charge capacity of 59 µAh cm−2 µm−1 (139.2 mAh g−1) and a discharge capacity of 132.5 µAh cm−2 µm−1 (312.7 mAh g−1) with a significantly lower CE of 44.5% compared to that of polymer-coated TiO2 NTs. The specific discharge capacity of the polymer-coated TiO2 NTs is higher compared to some previous studies. For instance, Fasakin et al. [2 NTs prepared from pristine anatase TiO2 nanoparticles via a low temperature modified stirring-hydrothermal technique delivered a capacity of 160 mAh g−1 at a specific current of 36 mA g−1. Auer et al. [2 NTs synthesized by a two-step anodization process, and the anatase TiO2 NTs exhibited a reversible capacity of 185 mAh g−1 (Li0.55TiO2) at a slow C-rate (C/20). In addition, Savva et al. [2 NTs via electrochemical anodization and subsequently annealed under N2 and water-vapor (WV) atmospheres. The obtained capacities for the N2 and WV-treated TiO2 NTs were 231.9 mAh g−1 and 230.6 mAh g−1 at C/20, respectively.n et al.  reporteda et al.  synthesi2 NTs layer exhibits a stable capacity of 41.8 µAh cm−2 µm−1 (98.6 mAh g−1) at C/5, 25.9 µAh cm−2 µm−1 (61.1 mAh g−1) at C/2, and 17.2 µAh cm−2 µm−1 (40.6 mAh g−1) at 1C. Obviously, there is a significant capacity increase for the polymer-coated TiO2 NTs compared to the bare TiO2 NTs for all C-rates. Polymer-coated TiO2 NTs layers deliver a stable capacity of 53.6 µAh cm−2 µm−1 (126.5 mAh g−1) at C/5, 36.9 µAh cm−2 µm−1 (87.1 mAh g−1) at C/2, and 27.4 µAh cm−2 µm−1 (64.7 mAh g−1) at 1C. The capacities can be recovered at C/10 for both samples. Herein, we note a large irreversible capacity at the first initial cycles for both samples, which may originate from the side reaction between the electrodes and Li+, as well as the presence of residual water at the surface of TiO2 NTs after electropolymerization. This phenomenon is in agreement with the previous reports [2 NTs can be ascribed to the lower interfacial resistance due to the better interfacial contact between the anode and polymer electrolyte; hence, it also enhances the diffusion of Li ions. Indeed, the Coulombic efficiency of the electrode is significantly improved close to 100% upon cycling  drop at the anode–electrolyte interface.Herein, a LiFePOndliness . Comparetructure . The elevoltages b. The ch2 NTs suggested it as a suitable anode material for application in full all-solid-state Li-ion batteries using the LiFePO4 cathode material, as schematically depicted in 2 NTs during the electropolymerization process serves as a continuous layer with an additional drop-cast polymer layer, with the total thickness of the polymer electrolyte layer being approximately 200 µm. As seen in 2 NTs/polymer/LiFePO4 full-cell by performing the cyclic voltammetry (CV) test is investigated. The CV curves of the full-cell recorded at the scanning rate of 0.5 mV s−1 exhibit well-defined redox peaks. The presence of the oxidation peaks at 1.93 V is attributed to the extraction of Li+ from the cathode and their subsequent insertion into the anode material. By contrast, reduction peaks at 1.21 V are assigned to the extraction of Li+ from the anode and their simultaneous insertion into the cathode according to the reactions given in Equations (10)–(12).The promising performances of the polymer-coated TiO2 NTs, the capacity values are reported versus the mass and the surface of the anode. 2 NTs/Polymer/LiFePO4 full-cell in the potential window of 0.5–2.8 V cycled at the C/10 rate. At the first cycle, the initial charge and discharge capacities recorded at the C/10 rate are 66 μAh cm−2 μm−1 (155.8 mAh g−1) and 63.9 μAh cm−2 μm−1 (150.8 mAh g−1), resulting in a high Coulombic efficiency (CE) of 96.8% and high capacity retention of 97.4% after 50 cycles at the C/10 rate. The discharge plateau at approximately 1.75  V and a charge plateau at approximately 1.50 V almost match with the two intensive peaks in the CV curves. By taking the middle point of two plateaus, we noted that the operating cell voltage of the TiO2 NTs/Polymer/LiFePO4 full-cell is approximately 1.6 V. An additional plateau observed at 0.75 V could be attributed to the insertion of Li+ from LiFePO4 to the VO2 structure. This result is in agreement with the galvanostatic cycling, resulting in half-cell TiO2 NTs on Ti-6Al-4V alloy where the plateau at 2.62 V corresponds to the VO2 phase.Since the limiting reactant material of the thin-film battery is TiO2 NTs/polymer/LiFePO4 full-cell recorded at multi-C rates for 50 cycles. The battery delivers capacity values of 63 μAh cm−2 μm−1 (119 mAh g−1) at C/10, 51 μAh cm−2 μm−1 (96.4 mAh g−1) at C/5, and 23 μAh cm−2 μm−1 (43.5 mAh g−1) at C/2, which are well recovered after the fast cycling rate (C/2). The high capacities for the TiO2 NTs/polymer/LiFePO4 full-cell are attributed to the enhanced surface area between the nanotubes and the gel polymer electrolyte, hence providing a robust and a high quality electrode–electrolyte interface for long charge–discharge cycles36. The better electrode–electrolyte contact established between the electrode and polymer electrolyte drastically facilitates the insertion and diffusion of Li+. Furthermore, calculated using a working voltage of approximately 1.6 V and taking into account the reversible areal capacity of the polymer-coated TiO2 NTs (69 μAh cm−2 μm−1), an areal energy density of 110.4 μWh cm−2 μm−1 and an areal power density of 11.04 μW cm−2 μm−1 at C/10 rate can be achieved.4F) with two different water contents (20 and 25 wt%). All anodization experiments were carried out under a constant voltage of 60 V using a generator (ISO-TECH IPS-603) for 3 h [The Ti–6Al–4V alloy  was cut into 1.2 cm × 1.2 cm and ultrasonically cleaned in acetone, 2-propanol, and methanol for 10 min each. Then, the treated foils were rinsed with ultrapure water and dried in compressed air. At room temperature, the anodization was performed in a two-electrode electrochemical cell with Ti-6Al-4V alloy as the working electrode and Pt foil as the counter electrode in ethylene glycol electrolyte containing 0.3 wt% ammonium fluoride  as the electrode. An aqueous solution of lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) and MMA-PEG monomer was mixed and stirred for 3 h; then, the solution was introduced into the cell. Prior to the electropolymerization, the solution was purged with N2 for 10 min to eliminate dissolved oxygen. Cyclic voltammetry tests were carried out in a cathodic current with a potential range of −0.35 to −1 V versus Ag/AgCl (3M KCl) at the scan rate of 10 mV s−1 for 5, 10, and 25 cycles. After the CVs, the sample was removed from the cell without rinsing and dried in the BUCHI vacuum dryer at 70 °C overnight to evaporate the residual water.The electropolymerization was performed by the cyclic voltammetry (CV) technique in a three-electrode configuration consisting of the layer of TiO2 NTs anode, there was no need to further mix with any additive due to their self-supported structure. For the positive electrode materials, the LiFePO4 powder was synthesized as previously reported [N-methyl-2-pyrrolidone (NMP) was added in the powder mixture to obtain a paste. The paste was spread on an Al disk (diameter of 8 mm) that was used as the current collector. The electrode was dried at 110 °C for 8 h.For the TiOreported . Each ma2 NTs and LiFePO4. After drying the polymer-coated TiO2 NTs electrode at 70 °C for 24 h, 7 µL of the MMA-PEG carrying LiTFSI salt was drop-casted at the polymer-coated TiO2 NTs surface. The sample was dried again under vacuum at 70 °C for 24 h to obtain a homogeneous polymer thin film. The cells consisting of a polymer-coated TiO2 NTs anode and an LiFePO4 cathode were assembled in an argon-filled glove box  with <0.5 ppm H2O and <0.5 ppm O2 atmosphere. The LiFePO4 composite cathode on the Al disk was pressed to polymer-coated TiO2 NTs and a polymer electrolyte dropcast. Two wires were connected to the backside of each current collector using silver conductive paste and wires.All-solid-state batteries were assembled using bare and polymer-coated TiO2 NTs was examined using a field-emission scanning electron microscope . The purity of TiO2 NTs was examined by X-ray diffraction (XRD) using a Diffractometer D5000  with CuKα1 (λ= 1.5406 Å) radiation. Then, they were analyzed by comparing with the JCDS-ICDD database  to check the purity of the samples.The surface morphology of the self-organized TiO2 NTs and polymer-coated TiO2 NTs having a surface area of 0.50 cm2 were used as electrodes without the use of any binders and conductive additives, assembling using standard two-electrode Swagelok cells. All cells assembly were conducted in a glove box that was filled with high-purity argon atmosphere in which the moisture and oxygen contents were less than 0.5 ppm. Cyclic voltammetry tests were carried out using a VMP3  in the potential window of 1–3 V versus Li/Li+ with a scan rate of 0.5 mV s−1 for the bare TiO2 NTs and polymer-coated TiO2 NTs. Galvanostatic charge–discharge cycles were performed with a VMP3 (Bio Logic) in the potential window between 1 and 3 V versus Li/Li+. The Swagelok test cells were assembled in an Argon-filled glove box. In the half-cell system, TiO2 NTs electrodes were assembled against metallic Li foil using the gel electrolyte of LiTFSI and MMA-PEG. The Li foil was cut in a circular shape with the diameter of 9 mm, and two circular sheets (10 mm in diameter) of the gel electrolyte in the Whatman paper were used as the separator. For the LiFePO4 electrodes, cyclic voltammetry tests were carried out in the potential window of 2–4.2 V versus Li/Li+ with a scan rate of 0.5 mV s−1 and galvanostatic charge–discharge cycles were performed in the potential window between 2 and 4.2 V versus Li/Li+.For the Swagelok test cells employing the gel electrolyte, the separators were prepared by soaking each circular sheet (diameter of 10 mm) of the Whatman glass microfiber with 80 µL of a mixture of 10 mL of 0.5 M LiTFSI with 2.5 g MMA-PEG. The separators were dried overnight at 70 °C in the BUCHI vacuum dryer. For the electrochemical performance tests, bare TiO2 NTs/polymer/LiFePO4 were cycled at different C-rates varying from C/10 to C/2 in the potential window of 0.5–2.8 V. The charge and discharge rates of a battery are expressed by the C-rate; it means a battery is charged and discharged relative to its maximum capacity. The rate nC means the current will charge and discharge the full capacity in 1/n hour.Full-cells consisting of the polymer-coated TiO2 nanotubes obtained by anodization of Ti-6Al-4V alloy. A vertically aligned and uniformly opened TiO2 NTs are distributed over the alloy substrate at 20 wt% H2O, and by adding 5 wt% H2O, the nanotubes have become less uniform. The electropolymerization of the MMA-PEG into TiO2 nanotubes was carried out at different CV cycles . After 25 cycles, the nanotubes are almost covered by a thin polymer film following the nanotubes surface rugosity. As a result, there is a significant increase in the capacity of the polymer-coated TiO2 NTs compared to the bare TiO2 NTs in all C-rates due to the improvement of the electrode-electrolyte interface. Therefore, polymer-coated TiO2 NTs layers deliver higher stable capacity of 53.6 µAh cm−2 µm−1 (126.5 mAh g−1) at C/5, 36.9 µAh cm−2 µm−1 (87.1 mAh g−1) at C/2, and 27.4 µAh cm−2 µm−1 (64.7 mAh g−1) at 1C compared to that of bare TiO2 NTs. The full-cell battery was composed of a polymer-coated TiO2 NTs as an anode, a thin layer of MMA-PEG polymer electrolyte, and a composite LiFePO4 cathode shows an operating voltage of 1.6 V with good electrochemical performance, not only a stable discharge capacity of 62.2 μAh cm−2 μm−1 (117.6 mAh g−1) after 50 cycles versus anode at C/10 but also a high capacity retention of 97.35% and a Coulombic efficiency of 96.78% beyond 50 cycles.In the present work, we showed that the water content in the electrolyte significantly affects the morphology of self-organized TiO


H1t is the major linker histone variant in pachytene spermatocytes, where it constitutes 50–60% of total H1. This linker histone variant was previously reported to localize in the nucleolar rDNA element in mouse spermatocytes. Our main aim was to determine the extra-nucleolar localization of this linker histone variant in pachytene spermatocytes.We generated H1t-specific antibodies in rabbits and validated its specificity by multiple assays like ELISA, western blot, etc. Genome-wide occupancy studies, as determined by ChIP-sequencing in P20 mouse testicular cells revealed that H1t did not closely associate with active gene promoters and open chromatin regions. Annotation of H1t-bound genomic regions revealed that H1t is depleted from DSB hotspots and TSS, but are predominantly associated with retrotransposable repeat elements like LINE and LTR in pachytene spermatocytes. These chromatin domains are repressed based on co-association of H1t observed with methylated CpGs and repressive histone marks like H3K9me3 and H4K20me3 in vivo. Mass spectrometric analysis of proteins associated with H1t-containing oligonucleosomes identified piRNA–PIWI pathway proteins, repeat repression-associated proteins and heterochromatin proteins confirming the association with repressed repeat-element genomic regions. We validated the interaction of key proteins with H1t-containing oligonucleosomes by use of ChIP-western blot assays. On the other hand, we observe majority of H1t peaks to be associated with the intergenic spacer of the rDNA element, also in association with SINE elements of the rDNA element. Thus, we have identified the genomic and chromatin features of both nucleolar and extranucleolar localization patterns of linker histone H1t in the context of pachytene spermatocytes.H1t-containing repeat-element LINE and LTR chromatin domains are associated with repressive marks like methylated CpGs, histone modifications H3K9me3 and H4K20me3, and heterochromatin proteins like HP1β, Trim28, PIWIL1, etc. Apart from localization of H1t at the rDNA element, we demonstrate the extranucleolar association of this linker histone variant at repeat-associated chromatin domains in pachytene spermatocytes. We hypothesize that H1t might induce local chromatin relaxation to recruit heterochromatin and repeat repression-associated protein factors necessary for TE (transposable element) repression, the final biological effect being formation of closed chromatin repressed structures. The chromatosome is a structural unit of chromatin, consisting of about 166 bp DNA wrapped around the histone octamer with histone H1 . DespiteMammalian spermatogenesis is an excellent model system to study the biological roles of linker histone variants, as various germ cell variants like H1t, HILS1 and H1T2 are expressed in a stage-specific manner. The testicular linker histone variant H1t is expressed from preleptotene spermatocytes till early round spermatids (in mouse) or till late-round spermatids (in humans) –12. EvenVarious defense mechanisms have evolved in germ cells of different species to prevent expression of the retrotransposable elements, thus limiting their mutagenic potential. DNA methylation at LINE and LTR retrotransposable elements can be dependent on piRNA expression or not, also termed as piRNA-dependent and piRNA-independent, respectively . DNA metRetrotranscripts originating from TE (transposable element) sequences in the nucleus are cleaved into sense and antisense piRNAs. The sense RNAs associate with GasZ-containing MILI–TDRD1 complexes within pi-bodies . On the Our present main aim was to determine the genome-wide occupancy of linker histone variant H1t in pachytene spermatocytes, which would give an idea about its association with specific chromatin domains and consequent biological functions. H1t is not exclusively expressed in testis, but also expressed in various cancer cells and mouse embryonic stem cells . Recentl2SO4) derived from P20 mouse testicular cells, wherein we observed the specific reactivity towards the variant histones  48,681 peaks of H1t occupancy (refer methods section for details). The chromosome-wide distribution of these H1t peaks is shown in Fig. Since H1t is not significantly enriched in TSS, active gene promoters, DSB hotspots, and ATAC-seq positive regions, the primary question remained to what genomic regions are H1t localized at. Upon initial annotation of the H1t-occupied genomic regions, we observed that H1t is majorly associated with intergenic and intronic regions Fig. a. On furIn addition to DNA methylation, repressive histone modifications like H3K9me3  and H4K2Linker histone variant H1t was previously demonstrated to be localized to the rDNA element of the nucleoli in spermatocytes by immunofluorescence assays . We confThe rDNA element harbors many repetitive elements, the dominant element being SINE . SINE elH1t being the major linker histone component of pachytene chromatin, we wanted to identify the proteins that co-associate with H1t-containing chromatin fragments. For this purpose, after carrying out the H1t ChIP, we performed mass spectrometric analysis of the eluted proteins, to identify the H1t-associated proteins in the chromatin context. The proteins were identified based on the enrichment of proteins observed in the H1t ChIP fraction compared to non-specific rabbit IgG ChIP fraction Fig. a. IntereFurther, we observed that the H1t-associated proteins could be identified into three classes—nucleolar-associated, repeat element and heterochromatin-associated and other important proteins Fig. b. We expIn addition to H3K9me3 and H4K20me3, there could be other histone marks that could be signatures of repeat-element nucleosomes. Uhrf1 has been shown to interact with PRMT5 (arginine methyltransferase) to catalyze the formation of H4R3me2s and H3R2me2s. These marks also act together with PIWI proteins to facilitate retrotransposon silencing in the germline . We founRecently, tremendous progress has been made towards understanding the molecular functions of linker histone H1 and its subtypes in developmental processes beyond their architectural roles in chromatin. The lack of studies on H1 function can be largely attributed to technical difficulties during the use of mass spectrometry approaches yielding reduced protein coverage and also the generation of subtype-specific antibodies. Raising variant-specific antibodies is a challenge. In this background, we have successfully generated H1t-specific antibodies in rabbit and validated its specificity by multiple assays.Arabidopsis thaliana, linker histone H1 and DNA methylation jointly regulate gene expression patterns by repressing TE in vivo [An important observation made in the present study is the predominant localization of linker histone variant H1t at repeat elements belonging to LINE and LTR. Even though repeat-element RNAs orchestrate gene expression patterns during early embryonic development , 49, par in vivo . Thus, iAn interesting question that arises here is what determines the localization of H1t to these specific genomic loci. We observed a combination of 12 motifs to be significant in the total H1t peaks as identified through motif analysis using MEME , LADs (lamin-associated domains), etc. –62. ReceNucleosomal retention occurs in repetitive sequences associated with intergenic and intronic regions in mammalian sperm , 67. ThiVery few studies have shed light on the biological roles of testicular linker histone variants in the context of differentiation of male germ cells. The present study is focussed towards understanding the genomic and chromatin features of H1t-occupied chromatin domains. We observe that linker histone H1t is not closely associated with TSS, active gene promoters, DSB hotspots and open chromatin regions. We have extensively characterized the nucleolar and extranucleolar localization features of linker histone variant H1t and found that H1t to be predominantly associated with repeat element chromatin domains in pachytene-enriched P20 mouse testicular cells. These chromatin domains are positive for methylated CpGs, repressive histone modifications like H3K9me3 and H4K20me3, protein marks like piRNA–PIWI pathway components, heterochromatin proteins, etc. We propose that H1t might induce local chromatin relaxation to recruit proteins effectors necessary for repression of these repeat elements.E. coli, and His-tagged proteins were purified using Ni–NTA purification method.The coding sequence (CDS) corresponding to 112–207 amino acid residues of H1t protein was cloned using specific primers (forward primer-ATAGAAGCTTGCAGCTTCAGGCAACGAC and reverse primer-ATATGCGGCCGCCTTTCTTCCTGCTGCCTTCC) into pET22b(+) vector using HindIII and NotI restriction sites. The expression vector was transformed in Rosetta strain of The recombinant protein  was injected into rabbits, and the 14-day cycle of antibody generation was followed. Immunoglobulins were purified by caprylic acid-based purification method. Antigen-affinity-based purification with the Sulfolink columns containing immobilized proteins was used to purify the H1t-specific antibodies. The H1t antibody was outsourced from the Abgenex company .The recombinant proteins were used at 200 ng per well. The pre-bleed and immune sera were used at 1:5000 dilution. Goat anti-rabbit HRP was used as the secondary antibody at 1:5000 dilution. TMB  was used as the substrate for color development. After 3 min of enzyme–substrate reaction, the plate was read at 450 nm.g for 10 min at 4 °C to pellet the residual chromatin. The proteins were extracted from the supernatant using 30% trichloroacetic acid (TCA). After incubation on ice for 30 min, the proteins were recovered by centrifugation at 12000g at 4 °C for 10 min. The protein pellets were sequentially washed once with cold acetone containing 0.05% HCl and twice with ice-cold acetone. The pellet obtained was dried, dissolved in water and stored in aliquots at − 20 °C.The linker histones along with other acid soluble nuclear proteins were extracted from rat or mouse testes by perchloric acid method , 74. Bri2 and 1 mM DTT, 1× protease inhibitor cocktail) and then incubated on rotator for 30 min at 4 °C. The nuclei were pelleted by centrifugation at 10,000g, 10 min, 4 °C. The pellet was resuspended in 0.4 N H2SO4, incubated on rotator for 30 min at 4 °C. Histones were then precipitated using 33% TCA  and the histone pellet was then washed with ice-cold acetone two times before resuspending in appropriate volume of water.Histones were extracted from the rodent testes using the published protocol . BrieflyMeiotic spreads were prepared according to the published protocol .g for 5 min at 4 °C. The pellet was resuspended in Buffer A , and incubated on ice for 5 min. Again, the contents were centrifuged at 500g for 5 min at 4 °C. The pellet was then resuspended in Buffer B , incubated on ice for 10 min, centrifuged again at 500g for 5 min at 4 °C. Finally, the pellet was resuspended in SDS lysis buffer . The contents were incubated on a rotating wheel for 30 min at 4 °C. Sonication was carried out 40 cycles, and chromatin fractions were diluted 1/10 times with the dilution buffer  before subjecting it to antibody binding overnight at 4 °C on a rotating wheel. Dynabeads were added the next day, washes were given with W1 , W2 , W3a , W3b , W4 , W5  wash buffers. The complexes were eluted from input, rabbit IgG control ChIP, and H1t ChIP samples using elution buffer  and then incubated at 65 °C overnight with intermittent shaking. The supernatants recovered from the tubes were subjected to RNase A and proteinase K digestions at 37 °C. DNA was then extracted from the elute fractions using the phenol–chloroform method. The input and ChIP DNA libraries were prepared using the NEBNext Ultra II DNA library preparation kit. The resulting libraries were quantified before getting sequenced on the Illumina HiSeqX system to generate 2X150 bp sequence reads.Chromatin immunoprecipitation (ChIP) was carried out using the published protocol . BrieflyAll the four samples (input controls two and H1t ChIP two samples—the raw Illumina sequenced files in FASTQ) were quality checked with FastQC (version 0.11.5). Each sample (two input controls and two H1t ChIP files) were then aligned to mouse genome version (GRCm38.p5) using Bowtie2  with aliVarious in-house Perl scripts and database comparison scripts at BioCOS Life Sciences were used to do the rest of the analysis like rDNA, Motif, and comparative methylation, etc. FASTA sequences corresponding to the rDNA element were obtained from NCBI . A FASTQhttps://www.r-project.org/)  database SRS557654). SRR1170580 is the Single end WT sample from SRS557654, is used for extraction of methylation locations and compare locations with H1t peaks. The reads were aligned using Bowtie2 onto the reference genome of mouse (GRCm38.p5)., The methylation analysis was done using bismark_v0.22.1  and the . SRR1170Motif analysis was carried out using MEME software . The parImmunoprecipitation of H1t-associated protein complexes was carried out, and the proteins were extracted from the beads using the elution buffer of the Pierce co-IP kit. The eluted proteins were resolved on 15% SDS gel and the gel was subjected to Coomassie staining. The stained wells corresponding to the IgG and IP lanes were outsourced for mass spectrometry to identify the interacting proteins. The detailed experimental protocol of the mass spectrometry was followed according to the published protocol . The lisThe proteins were resolved by gel electrophoresis and transferred onto the nitrocellulose membrane. The blots were blocked using blocking solution (5% skim milk in 1× TBS). The primary antibodies were made in 1% skim milk (prepared in 1× TBS) and added onto the blots and were left for overnight incubation in shaking conditions at 4 °C. Next day, two washes were given using 0.1% TBST (0.1% Tween-20 in 1× TBS) for 5 min each. The secondary antibodies prepared in 1% skim milk was added to the blots and left for shaking for 1-h incubation at room temperature. 2–3 washes were given with 0.1% TBST before developing the blot using Millipore Immobilon Forte Western HRP Substrate kit (Cat number-WBLUF0100) in the Biorad Chemidoc Touch imaging system.For stripping of proteins from the blot, 1X Millipore Reblot Plus Strong Antibody Stripping Solution (Cat number-2504) was added to the blot and left for shaking at room temperature for 20–25 min. The blot was developed using the ECL kit to check for any signal on the membrane. Two washes were then given with blocking solution (5% skim milk in 1× TBS) for 5 min each and then proceeded with incubation with the primary antibody solution.For the protein competition assays, H1t or H1.2 antibodies were preincubated with 10 µg of H1t C-terminal protein fragment before their addition to the blot.Additional file 1: Figure S1. A. Coomassie-stained gel showing the successful purification of His tagged C-terminal fragment of H1t. The purity of proteins was determined after elution using 100 mM, 200 mM, 300 mM, and 400 mM imidazole, wherein the protein fractions were obtained after elution using 300/400 mM concentration of imidazole. (B-C) Validation of specificity of the H1t antibody towards the recombinant H1t C-terminal protein fragment by ELISA using B. Immune sera and C. Purified antibody. The sera, as well as purified antibodies, showed reactivity against the H1t C-terminal protein fragment. The color code schemes have been indicated on the right of the figures. D. Immunoblotting using anti-H1t and anti-H1.2 antibodies probing against acid extracted histones from liver of 20-day old mouse. H1t is absent in the liver acid extracts, whereas the somatic H1.2 is found in the liver histone extracts. The ponceau stained blots and coomassie blue-stained SDS gel are given for reference.Additional file 2: Figure S2. A. Western blotting analysis of rat testicular perchloric acid extracts using H1t and H1.2 antibodies confirming the specificity of the H1t and H1.2 antibodies. The blots to the right are the immunoblotting results obtained after preincubation of the H1t and H1.2 antibodies with the recombinant H1t C-terminal antigen. B. Immunoblotting performed with H1t and H1.2 antibodies probed against rat testicular acid extracts. The blots to the left represent the immunoblotting pattern obtained against the rat testicular acid extracts. The blots to the right indicate the results obtained after performing the protein competition assay with the H1t C-terminal antigen. The reactivity of the H1t antibodies but not H1.2, was abolished upon preincubation with the recombinant H1t C-terminal protein fragment. Ponceau stained blots and Coomassie-stained gel are given for reference.Additional file 3: Figure S3. A. Immunostaining pattern of linker histone variant H1t across various stages of meiotic prophase I. Staining of anti-H1t and anti-Scp3 across leptotene , leptotene-zygotene , zygotene , and pachytene . B. Profile of DNA fragments obtained after 10, 20, 30, 35, and 40 cycles of sonication of P20 mouse testicular chromatin. 100-300 bp of fragment sizes were predominantly obtained after 40 cycles of sonication were used further for ChIP assays. Linker histone variant H1t is not associated with histone mark H3K4me3-containing chromatin domains- C. IP was carried out using the anti-H3K4me3 antibody where the H3K4me3 and H1t were probed by western blotting. D. Reciprocal IP using the anti-H1t antibody where H3K4me3 and H1t were detected by western blotting. The antibodies used for the western blotting are indicated in alpha alongside the blot. Ponceau stained blots are given for reference.Additional file 4: Figure S4. A. Peak to peak comparison of H1t ChIP-sequencing peaks with DSB hotspots, total H3K4me3 marks, Dmc1, TSS-associated H3K4me3, Hotspot-associated H3K4me3, PRDM9 and ATAC sequencing datasets. 99% of the H1t peaks overlap with methylated CpGs in the rDNA element. The y-axis represents the number of methylated H1t peaks weighted by the number of methylated bases, and the x-axis represents the individual H1t peaks that are aligned on the rDNA element. The various regions of the rDNA element have been labelled below the peak distribution maps.Additional file 5: Figure S5. A. Table showing the detailed comparison of H1t peaks and methylated CpGs in the extranucleolar (non rDNA) and nucleolar (rDNA) regions of the mouse genome. B. Venn Diagram showing the distribution of methylated H1t peaks in the rDNA and the extranucleolar regions of the mouse genome. C. Table of motifs identified of H1t bound genomic regions in pachytene spermatocytes using MEME software.Additional file 6. ChIP-sequencing peaks of H1t in P20 mouse testicular cells.Additional file 7. Annotation of H1t peaks using HOMER.Additional file 8. H1t-associated proteins obtained after mass spectrometry.Additional file 9. H1t and associated heterochromatin-related proteins.


Bacteroidetes, one of the main phyla in rumen and human gut microbiota, have been shown to encode polysaccharide utilization loci dedicated to the degradation of xylan. Here, we present the biochemical characterization of a xylanase encoded by a bacteroidetes strain isolated from the termite gut metagenome.Xylan is the major hemicellulosic polysaccharide in cereals and contributes to the recalcitrance of the plant cell wall toward degradation.  Pseudacanthotermes militaris termite gut metagenomic library revealed an array of xylan-degrading enzymes, including P. militaris 25 (Pm25), a multimodular glycoside hydrolase family 10 (GH10). Sequence analysis showed details of the unusual domain organization of this enzyme. It consists of one catalytic domain, which is intercalated by two carbohydrate binding modules (CBMs) from family 4. The genes upstream of the genes encoding Pm25 are susC-susD-unk, suggesting Pm25 is a Xyn10C-like enzyme belonging to a polysaccharide utilization locus. The majority of Xyn10C-like enzymes shared the same interrupted domain architecture and were vastly distributed in different xylan utilization loci found in gut Bacteroidetes, indicating the importance of this enzyme in glycan acquisition for gut microbiota. To understand its unusual multimodularity and the possible role of the CBMs, a detailed characterization of the full-length Pm25 and truncated variants was performed. Results revealed that the GH10 catalytic module is specific toward the hydrolysis of xylan. Ligand binding results indicate that the GH10 module and the CBMs act independently, whereas the tandem CBM4s act synergistically with each other and improve enzymatic activity when assayed on insoluble polysaccharides. In addition, we show that the UNK protein upstream of Pm25 is able to bind arabinoxylan. Altogether, these findings contribute to a better understanding of the potential role of Xyn10C-like proteins in xylan utilization systems of gut bacteria.The functional screening of a IMPORTANCE Xylan is the major hemicellulosic polysaccharide in cereals and contributes to the recalcitrance of the plant cell wall toward degradation. Members of the Bacteroidetes, one of the main phyla in rumen and human gut microbiota, have been shown to encode polysaccharide utilization loci dedicated to the degradation of xylan. Here, we present the biochemical characterization of a xylanase encoded by a Bacteroidetes strain isolated from the termite gut metagenome. This xylanase is a multimodular enzyme, the sequence of which is interrupted by the insertion of two CBMs from family 4. Our results show that this enzyme resembles homologues that were shown to be important for xylan degradation in rumen or human diet and show that the CBM insertion in the middle of the sequence seems to be a common feature in xylan utilization systems. This study shed light on our understanding of xylan degradation and plant cell wall deconstruction, which can be applied to several applications in food, feed, and bioeconomy. The nature of xylan backbone decorations varies depending on the species, the tissue, and the stage of development of the plant  that hydrolyze internal β-1,4 bonds, while decorations are removed by a variety of accessory enzymes, including α-l-arabinofuranosidases (EC 3.2.1.55), α-d-glucuronidases (EC 3.2.1.139), feruloyl esterases (EC 3.1.1.73), and acetyl xylan esterases (EC 3.1.1.72). Finally, β-d-xylosidases (EC 3.2.1.37) break down xylooligosaccharides, removing d-Xylp from the nonreducing end   protein fold in the Protein Data Bank (PDB) and which forms an active cleft able to accommodate up to seven xylosyl backbone units (http://pfam.xfam.org/), 20 to 30% of β-d-xylanases are multidomain proteins, comprising catalytic domains associated with accessory or helper domains, such as carbohydrate binding modules (CBMs). The latter have been attributed various roles, including the ability to target specific regions in substrates (Xylanases are mainly found in the glycoside hydrolase (GH) families 5, 8, 10, 11, 30, and 43 in the CAZy database , followed by xusE and xyn10C, the latter encoding a CBM-containing GH10 β-d-xylanase  encoding putative XusC/D-like proteins, unknown protein (UNK), GH10 containing an insertion of two CBM4s (GH10|CBM4), GH115, GH11, a putative transporter protein, GH10, and GH43 (P. militaris 25 (Pm25) here, presents an insertional modular structure homologous to Xyn10C protein Pm25 revealed a multimodular architecture composed of a signal peptide (residues 1 to 32), a GH10 catalytic domain (amino acid residues 66 to 151 and 514 to 753), and two putative tandem CBM4s  that constitute insertion domains 8 structure . Moreover, using this similarity, we could assign possible ligand binding roles to residues Y213, Q216, and Y257 in CBM4-1 and Y378, Q381, and Y422 in CBM4-2  present in the CAZy database , Bacteroidetes (14%), Proteobacteria (14%), and Firmicutes (13%).To identify database . ArchaeaPm25 is located in the bottom right cluster (Pm25_cluster), and all classified sequences (61 nodes) thereof are from Bacteroidetes.The SSN is centered on the main cluster, with other minor clusters scattered around. Pm25_cluster and main cluster, sequences in each cluster were aligned before the sequence logo was constructed  than in the main cluster (92 amino acids), which indicated interrupted catalytic domains in Pm25_cluster.To investigate the sequence differences between the structed . The −2  enzymes –43. ImpoPm25_cluster with PUL, each sequence was searched against PULDB (http://www.cazy.org/PULDB/) , suggesting that sequences in Pm25_cluster are Xyn10C-like proteins from the xylan utilization system in gut Bacteroidetes. The remaining sequences (16 out of 61) were not found in PUL, perhaps because of poor assignment.To verify the relationship of /PULDB/) . The majPm25 was determined using beechwood GX as the substrate. The purified enzyme retained greater than 80% of its activity in the pH range from 4.5 to 9.0  or 45°C (pH 9). Overall, optimal conditions for routine Pm25 assays were defined as pH 7.5 and 50°C.The optimal pH of 5 to 9.0 . The act5 to 9.0 . HoweverPm25 on various substrates revealed that it is 3- to 4-fold more active on arabinoxylans (30 U·mg−1) than on beechwood GX (7.4 U·mg−1), indicating that the latter is the least suitable substrate among those tested , which possess more O-3 substitutions (Pm25 (Table S3).Determining the hydrolytic activity of e tested . When coitutions , qualifiitutions  on diffePm25 revealed that the hydrolysis of X6 (after 60 min) produced a mixture of X2, X3, and X4 at a ratio of 1:2:1, reaching 74 μM  obtained when using p-nitrophenyl-β-d-xylotetraose (pNPX4) as the substrate was approximately 2.3-fold greater than that measured when using p-nitrophenyl-β-d-xylotriose (pNPX3)  (Pm25 devoid of CBMs)  . These d (pNPX2) . Furtherof CBMs)  to hydroPm25 E546A, M7 Pm25ΔCBMs E546A, M8 CBM4-1, M9 CBM4-2, and M10 CBM4-1-CBM4-2) were designed and expressed (dK) of M10 (CBM4-1 and -2 in tandem) was much lower than that of M8  and M9 , ranging from 3.2 to 9, 10.9 to 31.9, and 8.0 to 17.5 10−2 mg·ml−1, respectively, suggesting that there is cooperativity between the two CBMs. Comparing the behavior of the inactive mutants M1 and M7 with that of M10 revealed that the affinity of the inactive Pm25 M1 for xylans was similar to that exhibited by M10 and the CBM4-1/-2 tandem, whereas the dK values obtained for the inactive enzyme devoid of CBMs  were significantly higher. This suggests that xylan binding is largely driven by the two CBM4 domains. Comparing M8 and M9 revealed that M9 systematically exhibited lower (1.4 to 1.8 times) dK values for tests involving xylans, suggesting that its binding ability is stronger . The sigmoidal titration curves were used to calculate dK values . The dK values obtained when using M7, M8, and M9 were 1.5 ± 0.2 mM, 4.7 ± 0.4 mM, and 1.8 ± 0.1 mM, respectively. Recalling that M7 is devoid of CBM domains, it is noteworthy that this construction displayed the highest binding ability, with M9  exhibiting a similar ligand binding ability.The determination of the Pm25|E546A) was also able to bind to wheat bran despite its catalytic impotency . However, this was not the case for M7 , clearly demonstrating the role of the CBM in binding.Substrate depletion experiments performed using M1, M7, M8, and M9 on wheat bran revealed that the latter two constructions  exhibited binding ability. Similarly, M1  was investigated. The retardation of the UNK band suggested the UNK can bind to xylan .Pm25 and its variants M5 and M6 failed to reveal significant differences in d-Xyl and XOS release . This suggests that the Pm25 GH10 domain forms part of a distinct group and implies that the intercalated GH10 arrangement is robust from an evolutionary standpoint. Moreover, the biochemical data described here demonstrate that, despite its discontinuous organization, Pm25 is a fully functional xylanase.Unlike the vast majority of multimodular enzymes that display a sequential arrangement of their modules, the enzyme described here is characterized by a discontinuous organization that involves the insertion of two CBM domains into one GH10 xylanase domain. In this regard, it is significant that the SSN analysis performed using the amino acid sequence of M6 replacing Pm25 analog was identified in a rumen-based member of the Bacteroidetes phylum  adjacent to an susC-susD-(unk) cluster. This evidence of strong conservation is consistent with the fact that in their native host, the genes encoding Pm25 homologs are highly induced/expressed during growth on xylan  and BiXyn10A, which were identified in the human gut microbiome , which displays approximately 30% identity to Pm25 and whose structure is known (PDB entry 1US3). Like Pm25, CjXyn10C exhibits rather poor activity on XOS, ascribed to weak substrate binding in subsite −2 . Low-viscosity wheat arabinoxylan , acid-debranched wheat arabinoxylan , enzyme-debranched wheat arabinoxylan , rye arabinoxylan , galactomannan , xyloglucan (tamarind), arabinan (sugar beet), β-glucan , xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), xylohexaose (X6), p-nitrophenyl-β-d-xylopyranoside (pNPX), p-nitrophenyl-β-d-xylobiose (pNPX2), p-nitrophenyl-β-d-xylotriose (pNPX3), and p-nitrophenyl-β-d-xylotetraose (pNPX4) were all purchased from Megazyme . Cellulose nanocrystals (nanocellulose) from cotton linters were prepared as previously described  with accession number S0DFK9. Multiple-protein sequence alignment of CBM4s was done using Clustal Omega , and the alignment of the secondary structure elements of Pm25 with other structurally characterized GH10 family members was achieved using both Clustal Omega and ESPript 3 at http://espript.ibcp.fr/ESPript/ESPript/ , updated on 29 May 2020. To remove redundant sequences, the 4,936 sequences were winnowed down to 2,539 by a sequence identity cutoff of 0.9 (https://www.ebi.ac.uk/Tools/msa/mafft/), and sequence logos were constructed via WebLoGo (https://weblogo.berkeley.edu/logo.cgi).Amino acid sequences of GH10 family members were extracted from the CAZy database . The mutation of E546 to A in M6 yielded M7, while M8 and M9 were constructed by cloning the sequences encoding CBM4-1 and CBM4-2, respectively, into pET28a(+) expression vector. Likewise, the construct M10 is the pET32a(+) expression vector containing the sequence encoding both CBM4-1 and CBM4-2 cloned in frame with the thioredoxin tag using the In-Fusion cloning kit . The DNA sequence of UNK (UniProt ID S0DDM9) deprived of its signal peptide sequence, as identified by the SignalP 4.1 server  pLysS grown in ZYP autoinduction medium (E. coli Tuner(DE3) and cultured for 2 h at 37°C until the optical density at 600 nm (OD600) reached 0.6. At this point, isopropyl-β-d-thiogalactopyranoside  was added and growth was pursued at 16°C overnight. Cell pellets were collected by centrifugation, washed, and lysed using sonication , and the clarified cell lysates were applied to TALON metal affinity resin . After elution, protein purity was estimated by SDS-PAGE to be 95%. Protein concentrations were determined by measuring absorbance at 280 nm and applying the Beer-Lambert equation. Theoretical molar extinction coefficients were calculated using ProtParam online software  on 1% (mol/vol) beechwood GX at 37°C. The buffers used were 50 mM citrate buffer for pH 3.0 to 6.0, 50 mM phosphate buffer for pH 6.0 to 8.0, 20 mM bicine buffer for pH 8.0 to 9.0, and 20 mM glycine-NaOH for pH 9.0 to 11.0. Xylanase activity was determined by measuring the release of reducing sugars using the 3,5-dinitrosalicylic acid (DNS) assay (d-xylose series (0 to 1 mg/ml) was used to prepare a standard curve. The apparent optimal temperature was determined over the range of 21 to 90°C in 50 mM phosphate buffer (pH 7.5). Thermostability was monitored by preincubating the enzyme in the absence of substrate in 50 mM phosphate buffer (pH 7.5) at 45, 50, 55, and 60°C from 0 to 24 h. Residual enzyme activity in each case was then assayed as described above.The apparent optimal pH of S) assay , 65. ReaPm25 concentration of 0.4 μM and 0.08 μM to degrade beechwood GX and other soluble polysaccharides, respectively. Initial rates  were determined using a range of substrate concentrations (from 0.5 to 40 g/liter beechwood GX and 0.25 to 10 g/liter for other soluble substrates) under optimal conditions. The DNS assay was used to monitor reducing sugar release as described earlier. The kinetic parameters (kcat and apparent mK) were calculated using nonlinear regression in SigmaPlot 11.0 . One unit of xylanase activity was defined as the amount of enzyme that catalyzes the release of 1 μmol of d-Xyl equivalents per min.Enzyme kinetics were measured using a 4 to X6), reactions were performed using various concentrations (0.05 to 0.8 mM) and the optimal reaction conditions. Assays began upon the addition of enzyme, its final concentration being fixed to account for the nature of the substrate. Accordingly, 2.60, 0.26, and 0.026 μM enzyme were used for X4, X5, and X6, respectively. At regular intervals , aliquots were removed and immediately heated at 95°C for 10 min to stop the reaction. The hydrolyzed products were then analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an ICS 3000 dual device  equipped with Carbo-Pac PA-100 guard and analytical columns  as described before  at concentrations from 5 to 50 μM. Plotting the hydrolysis rate (micromolars per minute) versus oligosaccharide substrate concentration (micromolars) yielded a linear relationship, meaning that the catalytic constant kcat/mK could be calculated from the slope k using E] is the final concentration of enzyme.To study the hydrolysis of xylooligosaccharides (XOS) with a degree of polymerization of 4 to 6 , and R is the universal gas constant (8.314 J mol−1 K−1).The binding affinities of glycone subsites were calculated using Pm25 used was 6, 27, and 136 nM for pNPX4, pNPX3, and pNPX2, respectively. Similarly, the final concentration of M6 was 10 nM for pNPX4 and pNPX3 and 54 nM for pNPX2. The concentration range of substrate was 0.025, 0.05, and 0.1 mM for pNPX3 and pNPX4 and 0.5, 2, and 5 mM for pNPX2. All experiments were performed in duplicate. The plot of hydrolysis rate against pNP substrate concentration was linear, which indicated that substrate concentration was far below the mK. Therefore, the kcat/mK of reaction mixtures containing aryl β-xylosides was determined under optimum conditions using S0]) and at specific times ([tS]) were fitted to k = (kcat/mK) [Enzyme] and [Enzyme] is the final concentration of enzyme.To determine the catalytic parameters of reaction mixtures containing pNP-XOS, the final concentration of pNP  was determined experimentally by measuring the absorbance at 404 nm for a standard curve ranging from 0 to 0.12 mM pNP at pH 7.5 and 50°C.The molar extinction coefficient of dK was calculated as previously described , using 7.5% (mol/vol) acrylamide gels containing various amounts of polysaccharide . ADWAX, EDWAX, and LVWAX samples and beechwood GX were used at 0.006 to 0.06% (mol/vol), while other polysaccharides were used at 0.5% (mol/vol). Pure protein (6 μg) was migrated (10 mA/gel for about 1 h at room temperature) on gels in 25 mM Tris, 250 mM glycine buffer, pH 8.3. BSA (15 μg) was also included in the experiment as a negative, noninteracting control. Proteins were visualized by Coomassie blue staining. The dissociation constant escribed . In equaR0 is the relative protein migration distance compared to that of BSA in the control gel (without ligand). The variable r is the relative protein migration distance compared to that of BSA in ligand-containing gels. cR is the relative protein migration distance of complex between protein and ligand. c is the concentration of ligand. When R0 − r) as the ordinate and 1/c as the abscissa, a straight line was obtained. The intercept of the line on the abscissa provided a negative reciprocal value of the dissociation constant (−1/K). All experiments were performed in triplicate, and reported values are the means from three experiments.−1 to 0.3 mg·liter−1) in either buffer 1  or buffer 2 . Data analysis was performed with MO Affinity software (NanoTemper). The Hill equation was chosen to determine a value for the 50% effective concentration (EC50).Microscale thermophoresis (MST)  was carr6 containing either 0.075 μM M7, 0.13 μM M8, or 0.07 μM M9 in 50 mM phosphate buffer, pH 7, 0.05% Pluronic F-127 were loaded onto 16 standard capillaries. The initial fluorescence of all 16 samples was obtained by performing a capillary scan with LED power of 25% for M8 and 20% for M7 and M9. The dissociation constant dK was calculated by selecting the tab “Initial Fluorescence Analysis Set” in the Affinity Analysis software . Reactions were performed in 0.2-ml PCR tubes and incubated at 10°C for 2 h with agitation in an Eppendorf Thermomixer R at 1,400 rpm. For each reaction, the supernatant containing the unbound enzyme fraction was recovered after centrifugation using a benchtop microcentrifuge. The pelleted substrate was washed 3 times with reaction buffer. Finally, 20 μl of Laemmli sample buffer was added to the pellet and heated at 95°C for 10 min to denature the protein (bound fraction). All the fractions were verified by SDS-PAGE. BSA was used as a negative control.Pm25, M3, M4, M5, and M6 on either LVWAX (0.5% [mol/vol]) or wheat bran . The enzymes  were incubated with the respective substrate in 50 mM phosphate buffer (pH 7.5) and 1 mg/ml BSA. Enzymatic reaction mixtures were incubated at 37°C for either 24 h for LVWAX or 14 h for wheat bran, and aliquots were removed at regular time intervals and heated at 95°C for 10 min to terminate the reaction. Each sample was centrifuged at 20,000 × g for 5 min and quantified by HPAEC-PAD on a Dionex PA1 column equipped with a Carbo-Pac PA-1 guard and analytical columns . Separation of oligosaccharides was achieved by isocratic elution with 100 mM NaOH at a flow rate of 1 ml/min from 0 to 5 min, a gradient of 0 to 120 mM sodium acetate in 100 mM NaOH from 5 min to 25 min, and isocratic elution with 500 mM sodium acetate in 100 mM NaOH from 25 min to 35 min. The column then was reequilibrated with 100 mM NaOH for another 10 min. Calibration was achieved using d-Xyl and XOS  at concentrations from 5 to 100 μM.Product profiles were generated with