In vitro assays demonstrated that PCLU scaffolds could be colonized by cells through direct cell seeding and cell migration from outside to scaffold inside. Moreover, PCLU scaffolds could provide a suitable environment for stem cells proliferation in a 3D spatial arrangement, and allowed osteogenic differentiation under appropriate induction. In vivo results revealed the osteogenic properties of PCLU scaffolds through a drilled-hole femoral bone defect repair improvement in rats. Using histology and microtomography analysis, we showed that PCLU scaffolds fit well the bone cavity and were eventually entrapped between the newly formed trabeculae. Finally, no sign of inflammation or rejection was noticed. We envision that PCLU scaffolds can provide the clinicians with a substitute having appropriate characteristics for the treatment of bone defects.Bone loss can occur as a result of various pathologies, traumas and injuries and poor bone healing leads to functionally debilitating condition, loss of self-sufficiency and deterioration in life quality. Given the increasing incidence of facial trauma and the emergence of new procedural techniques, advanced scaffolds are currently developed as substitutes for bone tissue engineering. In this study, we investigated the capability of a chemically cross-linked ε-caprolactone-based poly(ester-urethane-urea) (PCLU) scaffold to support bone regeneration.  Most maxillary-craniofacial and long bone defects are the result of congenital deformities, diseases (bone loss after tumor resection), traumas  as well as war injuries (gunshots or explosive devices) . When thCurrent investigations aim at developing innovative bone substitutes that can provide reliable bone regeneration leading to functional and esthetic restoration. The ideal bone graft substitute should be biocompatible, bioresorbable, osteoconductive and osteoinductive. It should not present any risk of infectious disease contamination, be easy to use and readily available, as well as cost effective. A multitude of bone grafting materials are currently available on the market, including allografts, xenografts and alloplastic materials . If autoin vivo adverse responses during chemical gel curing. Furthermore, scaffold cellular colonization can be limited by the hydrogel degradation rate due to the lack of adequate pore size and interconnected porosity [Nowadays, the increase in facial trauma incidence, the emergence of new procedural techniques and the better understanding of bone-healing biology, urge the need to optimize biomaterials for bone defect care, and leads to the development of advanced scaffolds for bone tissue engineering. In this strategy, the challenging goal is to elaborate scaffolds allowing cell adhesion and colonization, as well as vascularization and bone maturation. Indeed, the diamond concept suggests that three parameters are mandatory for successful bone regeneration: osteoconductive scaffold, osteoinductive growth factors and osteogenic cells . In partporosity . A few sporosity . In partporosity . Moreoveporosity . Therefoin vitro compatibility of human mesenchymal stem cells (hMSC) toward a chemically cross-linked ε-caprolactone-based poly(ester-urethane-urea) (PCLU) scaffold [in vitro and in vivo bone regeneration. For this purpose, in vitro studies were carried out to prove the potential of PCLU scaffolds to be colonized by various cells using direct static or dynamic seeding, as well as from outside to inside cell migration; to act as three-dimensional (3D) frameworks for cell proliferation; and to allow osteogenic differentiation of hMSC. At last, the in vivo osteogenic properties of PCLU scaffolds were also assessed in a rat femoral bone defect model, providing a standardized environment for studies of induction and remodeling of new bone.In a previous study, we assessed the scaffold . PCLU sc® 80, dibutyltin dilaurate, collagenase, paraformaldehyde (PFA), 2-phospho-l-ascorbic acid, β-glycerophosphate, DAPI, alizarin red, Triton X100 and cetylpyridinium chloride were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was obtained from PANBiotech GmbH.All solvents were purchased from Fisher and used as received. Phosphate-buffered saline (PBS) solution, Dulbecco’s modified Eagle’s medium (DMEM), fungizone antimycotic (Fz), penicillin (Pen), streptomycin (Strep) and trypsin-EDTA were supplied by Gibco Life Technologies. Poly(ε-caprolactone) oligomers, hexamethylene diisocyanate, SpanHuman dermal fibroblasts were isolated, using standard procedures, from foreskins of 4-year-old children with informed consent of the parents. Fibroblasts were used for the experiments between passages 4 and 6. hMSC were obtained by plastic adhesion from bone marrow samples collected from hematologically healthy patients undergoing routine total hip replacement surgery. All samples were obtained after informed consent from donors from the Hôpital d’Instruction des Armées Percy .−1 with a resolution of 4 cm−1. The scaffold hydrophilicity/hydrophobicity was determined through water contact angle (WCA) measurement using a Digidrop Model DS GBX apparatus and Windrop++ software. The scaffold morphology was monitored using an environmental scanning electron microscope (ESEM TM3000—Hitachi) and by microcomputed tomography (micro-CT Quantum FX—Perkin Elmer). The scaffold elemental composition was determined by energy dispersive X-ray spectrometry using ESEM equipped with an EDX probe (Hitachi SwiftED3000).PCLU scaffolds were obtained through a poly-HIPE method and characterized by determination of their density, porosity and pore interconnectivity, as described previously . The chemwet). Vs is the molar volume of the swelling agent (toluene), χ is the Flory polymer–solvent interaction parameter calculated from the solubility parameters using Bristow and Watson υ2s is the actual polymer volume fraction determined by The number average molecular weight between cross-links Vs is the molar volume of the solvent (toluene), R is the universal gas constant, T is the absolute temperature, δs is the solubility parameter of the solvent (toluene) and δp is the solubility parameter of the polymer network calculated using the group contribution method based on Van Krevelen’s molar attraction constants [mdry is the mass of the scaffold before swelling, mwet is the mass of the scaffold after swelling, ρs is the density of the swelling agent (toluene), ρnonporous is the density of the nonporous PCLU network prepared in the same conditions compared to the porous scaffold without the addition of water, ρporous is the density of the porous PCLU scaffold and P is the scaffold porosity.In onstants . In Equa−1 testing speed. The stress/strain relation was computer recorded and processed using Realview 3.0 software. The linear regression at the beginning of the stress/strain curve gave the effective modulus of elasticity E1* of the porous PCLU scaffold, while the linear regression at the end of the curve gave the elastic modulus of the corresponding nonporous material (Enonporous). The number average molecular weight between cross-links PCLU scaffold mechanical properties were determined by compression test using a universal machine. Scaffolds  were tested during compression in the axial direction to the foam rise with a 500 N force range, 7 mm displacement range and 5 mm minρ is the PCLU scaffold density (ρ = 159 kg m − 3), R is the universal gas constant and T is the absolute temperature.In E3* of the confined porous PCLU scaffold was determined through linear regression at the beginning of the stress/strain curve.Mechanical properties were also evaluated when PCLU scaffolds were confined in 16 mm-diameter hole and the effective modulus of elasticity −1)/Strep (100 µg ml−1)/Fz (2.5 µg ml−1), and incubated at 37°C for different periods of time. The pH of the degradation medium was monitored during the course of the study. At the end of each time point, scaffolds were removed from the medium and washed extensively in distilled water. Subsequently, scaffolds were air dried up to a constant mass. The mass loss was determined from the scaffold initial mass and their residual mass after drying. Mass and volumetric absorption ratios were determined from the mass of the wet scaffolds before drying and their residual mass after drying. Finally, PCLU scaffolds were characterized by FTIR-ATR analyses, ESEM, as well as through the determination of the porosity, WCA and number average molecular weight between cross-links.The degradation assay was carried out according to ISO 10993-13 guideline standards. PCLU scaffolds were conditioned and sterilized as described below. After sterilization, 0.1 g of PCLU scaffolds were immersed in 1 ml of degradation medium composed of DMEM/Pen  and the temperature at which to study the effects of degradation (37°C). Therefore, the PCLU scaffold lifetime at 37°C was estimated to be that of the PCLU scaffold lifetime at 90°C multiplied by the factor f.In PCLU scaffolds were immersed in sterile water for 1 h under a vacuum system, followed by a 4-h immersion after water change. Thereafter, scaffolds were immersed for 1 h in 70 vol.% ethanol under a vacuum system. Finally, scaffolds were rinsed in sterile water overnight and autoclaved in wet condition.in vitro studies, PCLU scaffolds were conditioned and sterilized as described above. Thereafter, scaffolds were incubated in DMEM/Pen (100 IU ml−1)/Strep (100 µg ml−1)/Fz (2.5 µg ml−1) for 12 h at 37°C in a humidiﬁed atmosphere of 5% CO2. The medium was removed before the use of the scaffold. Scaffolds were tested in triplicate in all experiments.For all 7 cells ml−1 in complete medium (DMEM/Pen (100 IU ml−1)/Strep (100 µg ml−1) supplemented with FBS (10%)) were seeded onto the scaffold, and incubated for 2 h at 37°C in a humidiﬁed atmosphere of 5% CO2. Then, each PCLU scaffold was carefully rinsed with PBS and incubated at 37°C for 10 min in a solution of trypsin (0.05%) and collagenase (0.025%). Thereafter, the supernatant was recovered and centrifuged, and the pellet of cells was resuspended. In order to take off all the cells from the PCLU scaffold, this operation is repeated three times. Then, the total number of cells was determined by counting with a Coulter Counter Z1 (Beckman) to assess cell adhesion efficiency onto the scaffolds.For static seeding experiments, PCLU scaffolds  were placed in individual wells of a conical bottom 96-well plate, and 10 μl of a cell suspension at 5 × 10® mini bioreactor flooded with a cell suspension at 3 × 105 cells ml−1 in complete medium, and equipped with a stirrer bar. The system was incubated for 2 h at 37°C in a humidiﬁed atmosphere of 5% CO2 under stirring at 350 rpm. Then, the scaffolds were carefully rinsed with PBS, and the cells were trypsinized and counted as described for the static seeding experiments. Some scaffolds were maintained up to 54 days in complete medium at 37°C in a humidiﬁed atmosphere of 5% CO2 under static conditions. Medium was changed once a week.For dynamic seeding experiments, two PCLU scaffolds  were set on a needle immersed in a Corning4 cells per well. Fibroblasts were cultured at 37°C in a humidiﬁed atmosphere of 5% CO2 until an apparent 80% confluence was reached. Thereafter, one PCLU scaffold  per well was carefully set down over the 80% confluent cell layer, and incubated in complete medium at 37°C in a humidiﬁed atmosphere of 5% CO2 up to 35 days. Medium was changed once a week. At 10, 25 and 35 days, the scaffolds were carefully rinsed with PBS, and the cells were detached and counted as described for static seeding experiments.For migration experiments, fibroblasts at passage 5 resuspended in complete medium were seeded onto 24-well plates at 4 × 102. Thereafter, the medium was removed and 30 μl of a cell suspension at 5 × 106 cells ml−1 were seeded on the scaffold and incubated for 3 h at 37°C in a humidiﬁed atmosphere of 5% CO2. Then, the wells were filled with 1 ml of either complete medium or osteogenic medium (complete medium containing 2-phospho-l-ascorbic acid and β-glycerophosphate) and incubated at 37°C in a humidiﬁed atmosphere of 5% CO2 up to 30 days. The medium was changed every 3 days. At 7, 14, 21 and 30 days, scaffolds were carefully rinsed with PBS. Scaffolds were tested in triplicate.PCLU scaffolds  were conditioned and sterilized as described above. After sterilization, PCLU scaffolds were placed in individual wells of a 24-well plate and incubated in complete medium for 24 h at 37°C in a humidiﬁed atmosphere of 5% COFor cell counting, each PCLU scaffold was incubated at 37°C for 10 min in 2 ml of a trypsin (0.05%) and collagenase (0.025%) solution. Thereafter, the supernatant was collected and centrifuged, and the pellet of cells was resuspended. In order to recover all the cells from the PCLU scaffold, this operation is repeated three times. Then, the total number of cells was determined by counting with a Coulter Counter Z1 (Beckman).Alizarin red staining was used to assess the MSC differentiation into mature osteoblasts. Scaffolds were immersed for 5 min in PBS/ethanol solution (50/50 vol.%), and then for 5 min in ethanol at −20°C. Thereafter, scaffolds were rinsed three times in distilled water, and cut in small pieces which were incubated in a 2% alizarin red solution. After 15 min, the solution was disposed and scaffolds were rinsed five times in distilled water. Afterwards, scaffolds were incubated in PBS for 15 min and the alizarin red fixed to the calcium-mineralized scaffold was detached by the action of a cetylpyridinium chloride solution (10%) for 15 min under stirring. The optical density of the recovered solution was read at 562 nm with a UVM 340 spectrophotometer.For ESEM imaging, scaffolds were fixed in PFA (4%), rinsed in PBS and stored in ethanol 70 vol.% up to observation. Images were carried out using a Hitachi TM3000 ESEM operating at 5 kV and equipped with a Peltier stage operating at −4°C.For DAPI staining, scaffolds were fixed in PFA (4%), rinsed in PBS and permeabilized with 1% triton X100 in PBS. Thereafter, scaffolds were immersed for 5 min in the dark in a DAPI solution (1/200). Scaffolds were then rinsed with PBS. Stained scaffolds were imaged on a Axioplan fluorescence microscope (Zeiss) connected to a Camedia CS550 color video camera (Olympus).All animal treatments and procedures were approved by the Institutional Animal Care and Research Advisory Committee of IRBA in accordance with French law and international guidelines. Adult male Lewis rats (Janvier Labs) weighing 220–250 g were individually housed under standard temperature conditions (22°C ± 1°C) and hygrometry (45–60%) with a 12-h to 12-h light-dark cycle (lights on between 7 a.m. and 7 p.m.). Rats had free access to water and standard laboratory feed.Sterilized PCLU scaffolds  were incubated in complete medium 1 day before the bone defect surgery. All implantations were conducted under aseptic conditions.−1 and 0.5 mg kg−1 of body weight, respectively). Skin incision and blunt dissection of the quadriceps were performed on the right shaved hind limb to expose the metaphyseal area of the distal femur. Defects were achieved by drilling a 3.0-mm diameter hole through the anterolateral cortical bone into the metaphyseal cancellous bone marrow, under continuous irrigation with saline. Scaffolds were carefully dried by absorption with a sterile compress at the time of implantation. Half rat population  were implanted with PCLU scaffolds into osseous cavities, while the other half rat population remained nonimplanted . Muscles and skin were sutured (Vicryl® 4/0) and rats were allowed to recover from anesthesia after an intramuscular injection of atipamezole (1 mg kg−1 of body weight). Analgesia was provided through subcutaneous injections of buprenorphine hydrochloride (50 µg kg−1 of body weight) 2 h after surgery and twice a day over three consecutive days. At 7, 15 and 30 days post-surgery, rats were sacrificed under sodium pentobarbital anesthesia .At day 0, 18 rats were anesthetized by intraperitoneal injection of ketamine hydrochloride and medetomidine (60 mg kg® WDF (Sysmex reference N° AA-325-279) staining reagent kit and a rat-specific analysis software allowing blood cell sorting and counting.During rat sacrifice, blood was collected by intracardiac puncture with an EDTA-coated syringe (80 µl of 1.6% EDTA-K3 for every 800 µl blood samples) for blood cell count. A complete peripheral blood cell count was carried out using a Sysmex XNL-550 hematology analyser equipped with FluorocellAfter rat sacrifice, femurs were collected and fixed in Burkhardt’s solution  and scanTo assess bone repair efficiency in injured femurs, two regions of interest (ROI) were delineated by freehand drawing by the same investigator with a view to measure the closing of the cortical defect and the filling of the medullary cavity (lesion area) with trabecular new bone. The first ROI was a 3.0-mm diameter circle  positioned around the edges of the cortical defect. The surface of newly formed bone dedicated to the cortical defect closing was measured and compared with the surface of the initial hole defect . The second ROI was bordered by the defect boundaries in the medullary cavity, encompassing regions containing newly mineralized bone and was defined as the BV/TV parameter (bone volume/tissue volume ratio).Following micro-CT imaging, the same sets of fixed femurs were dehydrated in methanol and processed for undecalcified histology in methylmethacrylate resin. Serial 6-µm thick longitudinal bone sections were prepared using a microtome equipped with a tungsten carbide blade (Leica 2055). Sections were stained with Masson–Goldner’s trichrome to identify bone structures, fibrous tissue and bone marrow cells. The PCLU scaffold was detected with a Sudan black B staining. Stained sections were imaged on a DMRB microscope (Leica) connected to a DXC930P color video camera (Sony).in vitro and in vivo studies, values are expressed as mean ± standard deviation (SD).For measured parameters of PCLU scaffolds and for the One major target in bone tissue engineering is to develop porous scaffolds with appropriate architecture since pore size, porosity and pore structure are crucial for scaffold osteoconductive properties . Further2, respectively [E1* of the porous PCLU scaffold was found to be 161 ± 14 kPa and the number average molecular weight between cross-links E1* and calculated using −1) was consistent with the value found by the swelling experiments (−1). Secondly, PCLU scaffold mechanical properties were studied when the scaffold was confined in a hole to mimic in vivo experiments. Indeed, PCLU scaffold is subjected to compression stresses in all 3D directions when implanted into bone defect cavity. As expected, compressing in triaxial stress mode of compression requires larger stresses than in uniaxial stress mode of compression. Therefore, the effective modulus of elasticity E3* was higher with a value of 321 kPa. Altogether, PCLU scaffolds always exhibited moduli of elasticity above 40 kPa and therefore seemed appropriate to induce hMSC differentiation into osteogenesis lineage.The mechanical behavior of the scaffold is also of importance, since cells regulate their metabolic activity and their response  depending on the scaffold rigidity and spatial structure , 33. Forectively . As a coin vivo conditions are more severe than in vitro experiments leading to a decrease of the scaffold lifetime [First of all, an important challenge in the development of polymeric scaffolds is to ensure effective sterilization of the biomaterial without causing drastic polymer degradation and modification of the porous structure. Regarding PU-based biomaterials, it is known that traditional and advanced sterilization methods can lead to polymer hydrolysis, oxidation and chain scission. Therefore, the sterilization process can elicit modifications of the biomaterial properties, and release of carcinogen 4,4′-methylenedianiline and analogs when PUs are synthesized with aromatic diisocyanate , 36. Morlifetime . For inslifetime . Furtherlifetime .E1* after conditioning and sterilization = 243 kPa). Once again, there was a good correlation between the number average molecular weight between cross-links found by the swelling experiments (−1) and the one derived from E1* and calculated from −1). As expected, the effective modulus of elasticity was higher when the scaffold was confined in a hole during the compression test (E3* after conditioning and sterilization = 400 kPa).In our study, PCLU scaffolds were synthesized with (1) ε-caprolactone-based oligomers, which are known to be slowly hydrolyzed in low-concentrated caproic acid since it is rapidly metabolized, and (2) aliphatic diisocyanate, which is degraded in nontoxic amine , 41. Forin vivo tissue engineering application for bone regeneration. Indeed, no significant chemical modification was noticed since scaffold mass  adhered to the scaffold thanks to its moderate wettability, since fibroblasts have a maximum of adhesion on substrates having WCA around 60°–100° [6 ± 2.0 × 105) adhered on the scaffold with the dynamic seeding, but a less concentrated cell suspension was used and the scaffold bigger size limited the cell infiltration. The dynamic seeding highlighted that the scaffold structure was adequate for the penetration of the cell suspension, and therefore achieving a whole PCLU scaffold volume colonization with a spatially homogeneous distribution (6 ± 3.5 × 105) colonized the PCLU scaffold volume providing a reasonable proof that the mechanical signals and 3D environment of the soft scaffold were suitable enough for colonization by mobilized external cells  was noticed when cells were cultured in the osteogenic differentiation medium which is similar to what is observed for a classical 2D culture onto tissue culture plate . In addiin vivo models can be used to evaluate the bone regeneration process, the bone–biomaterial interaction and pathophysiological evidence of ossification pathway modulation. In rat cavitary defect model, a 3-mm surgical cavity is drilled on the external face of the femoral distal head leading to a defect that does not heal without intervention. On the one hand, this model is able to produce bone morphogenetic factors, growth and differentiation factors that induce early bone repair mechanisms; on the other hand, chondrogenesis, osteogenesis and angiogenesis are affected. Therefore, the cavitary model is commonly used to investigate both the role and osteogenic properties of different biomaterials on bone healing [Bone healing is a complex process involving release of cytokines, chemokines and growth factors; induction of signaling pathways with activation of thousands of genes; as well as progenitor cell proliferation and differentiation. All these mechanisms occur in a highly organized spatial and temporal process that eventually leads to bone regeneration resulting in an overall increase in volume of new skeletal tissues. Specific  healing .in vivo experiments evidenced PCLU scaffold biocompatibility since neither complication, nor clinical sign of inflammatory response or scaffold rejection were noticed when scaffolds were implanted in cavitary defects up to 1 month. Indeed, the levels of circulating red blood cells and platelets were similar in the implanted rat group versus the nonimplanted one  (PCLU) scaffold with a multiscale and interconnected porosity by a simple and controlled manufacturing process using an emulsion technique.


An energy density of 68.1 W h kg−1 and a high maximum power density of 558.5 W kg−1 in 6 M KOH electrolytes were recorded for the optimum sample. The NG samples showed an appropriate cyclic stability with capacitance retention of 87.7% after 5000 cycles at 4 A g−1 with high charge/discharge duration. Thus, the prepared NG herein is considered to be promising, cheap material used in energy storage applications and the method used is cost-effective and environmentally friendly method for mass production of NG in addition to opening up opportunities to process waste materials for a wide range of applications.3D sponge nitrogen doped graphene (NG) was prepared economically from waste polyethylene-terephthalate (PET) bottles mixed with urea at different temperatures using green approach via a novel one-step method. The effect of temperature and the amount of urea on the formation of NG was investigated. Cyclic voltammetry and impedance spectroscopy measurements, revealed that nitrogen fixation, which affects the structure and morphology of prepared materials improve the charge propagation and ion diffusion. The prepared materials show outstanding performance as a supercapacitor electrode material, with the specific capacitance going up to 405 F g Electrochemical capacitors (ECs) are considered as one of the most promising energy storage technologies responding to the increasing demand of the clean energy related technologies and applications such as portable/mobile electronics, electrical vehicles, or other storage systems based on sources like windmills and solar cells.1.The graphene-based materials are promising for applications in supercapacitors and other energy storage devices and there are many researches on the refinement of their structures and development of effective low cost synthesis techniques as well as possible clarification of their electrochemical performance4. Among a variety of nitrogen sources, urea has a high nitrogen content (46 wt %), and can react with oxygen-containing groups. Hence, urea is a typical precursor that has been used widely as a nitrogen source in experiments on graphene7.Chemical doping has been exceedingly used to enhance capacitor property of graphene. Doping of graphene with nitrogen is of much interest, since it improves the overall conductivity and supercapacitive properties because introducing N atoms intrinsically improve the electronic properties of graphene in particular the performance as supercapacitor electrodes8. However, the extremely low yield and high cost of these methods limit their application only to fundamental studies. Later, it was found out that GO can be used as a platform for N incorporation via thermal annealing with NH3 or nitrogen plasma treatment but these methods involve either toxic precursors or need sophisticated equipment5. Many researchers aim to develop one-step simple preparation way for inquiring nitrogen doping into graphene and most of their efforts have used aqueous mixture of graphene oxide (GO) and nitrogen source as starting the materials. For instance, Hao et al. prepared novel cross-linking three-dimensional (3D) graphene layers utilizing gas-foaming technology by using colloidal solution of exfoliated GO and NH4Cl solution; the obtained material showed a capacitance of 231.2 F g−1 at a current density of 1 A g−1 in 1 M H2SO49. Chen et al. produced 3D graphitic carbon nitride functionalized graphene networks by the hydrothermal reduction of GO sheets and exfoliated carbonized melamine in their mixed aqueous dispersions at 180 °C for 12 h. The obtained material displayed 264 F g−1 at a current density of 0.4 A g−1 in 0.1 M LiClO410. Xiao et al. inserted nitrogen into graphene using nitric acid through thermal treatment at 500 °C, which showed a capacitance value of 370 F g−1 at 1 A g−1 in 6 M NaOH11. Zhang et al. produced nitrogen doped graphene using nitric acid and a high temperature co-processing method at 500 °C for 4 h in a quartz tube furnace with a high specific surface area (436.7 m2 g−1) and a high specific capacitance 12.There are numerous methods to synthesize nitrogen doped graphene, which can be categorized into two kinds of methods: direct synthesis such as chemical vapor deposition (CVD), arc-discharge, segregation growth, solvothermal methods, and post treatment such as hydrazine hydrate, thermal and plasma treatments+ ions induce the hydrogenation of dangling bonds on the formed graphene layer14, and hence, forming a strong C–H bond. Bonding configuration changes from sp2 to sp3 and less oxygen functional groups are present in grapheme, that in turn enhances the capacitive behavior because the oxygen functional groups would impede the migration of electrolyte ions into micropores and as a result reduced the effective pore volume of the graphenic materials. Both raw materials are low cost and are manufactured in large quantities. The great challenge is the conversion of waste PET bottles using a one step thermal decomposition method into supercapacitor electrode material through simple and cost effective method. and that was demonstrated by using only urea. In this method, graphene gets simultaneously doped with nitrogen.Therefore, developing a low-cost, scalable, and eco-friendly method is still of great interest. Herein, the nitrogen (N) doping was used to improve the capacitance performance of graphene through a novel synthesis using PET waste bottles and urea. Urea has two amine groups with nitrogen content about 46 wt% joined by a carbonyl (C=O) functional group. Therefore, it is a good candidate to be used as nitrogen precursor. Moreover, the presence of H6. Urea totally decomposes into volatile products with a total weight loss close to 600 °C. While, for PET, the degradation started at 360 °C and achieve maximum degradation rate at 480 °C. The curve exhibits one major mass loss which is attributed to thermal decomposition and the residue at 800 °C is 0.9 wt%. This is obviously associated with the segment corresponding to the carboxylic acid and the benzene ring appearing in the chemical unit of PET15. Compared to PET and urea curves, the curve of PET/urea is coalesce of their two curves. A mass loss of 60% took place in the first step at the temperature range of 150–240 °C, which is in good agreement with urea decomposition to biuret. This process was followed by two steps which ended at 480 °C similar to PET curve. During the thermal decomposition process, the out-gassing as CO, CO2, NO2, and H2O molecules could introduce force to expand the graphene layers and help the formation of porous frameworks16. That is confirmed by SEM and TEM images. Meanwhile, the embedded nitrogen atoms in the carbon lattice cause the formation of NG frameworks. In order to prove the presence of nitrogen doped in graphene structure, an elemental analysis was conducted on the dried product with XPS and FTIR. The mass fractions of carbon, oxygen, and nitrogen for all samples are presented in Supplementary Table Thermogravimetric analysis (TGA) is used to investigate the behaviors of PET and urea in the thermal decomposition process. Supplementary Fig. −1 corresponding to the stretching vibration mode of -OH and a characteristic peaks appearing at 1532 cm−1 correspond to the skeletal vibration of C=C (sp2 hybridization)10. It is noticeable from the spectrum of undoped graphene samples (5 G and 6 G) that the intensities of characteristic peaks decreased by rising the temperature of PET thermal decomposition and this indicates that the quality of produced graphene increases with increasing the treatment temperature due to decreasing amount of oxygenous groups. However, for NG samples, the spectra show marked changes in the 1000–1700 cm−1 region, particularly in the C–O stretching bands, in addition to the C=N stretching vibration modes which appeared at 1631 cm−1 and that confirm the success introducing of N atoms into graphene lattice. Complementary information on nitrogen functional groups were obtained by X-ray photoelectron spectroscopy (XPS) analysis as shown in Fig. 16. The XPS spectra of the graphene sample derived from PET bottles waste at 800 °C (sample 6 G) confirmed that carbon and oxygen were the only present atoms and nitrogen signal could be clearly observed in the XPS spectra of NG samples. For NG samples  was observed and this demonstrate that C atoms were arranged in a honeycomb lattice and the fitted peaks centered at 285.55, 286.7, 287.85, and 288.95 eV are assigned to C=N, C-N, C=O, and O=C-O, respectively. It is worth noting that a small amount of oxygen containing functional groups was in samples because the presence of oxygen functional groups on the surface of graphenic materials reduced its surface conductivity and prevented ions from entering into the pore17. But they have a great influence on the capacitive performance18 because it improves the hydrophilicity of the surface which enhancing the ions diffusion of the aqueous electrolyte and reduced the mass transfer resistance on the surface of electrode; subsequently make it easier for ions to form electrochemical double layer and as a result the capacitance will be increased18.As shown in Fig. 1. The second way for N-doping is that carbon nitride, produced by the decomposition of the N precursors, acts as an intermediate for the formation of N dopant in the NG. This way had a low handout here because the yield of carbon nitride from urea was very low and FTIR spectroscopy had confirmed the absence of the signature peak for carbon nitride at ~810 cm−1 indicating that it is unlikely that carbon nitride is involved in this process. Moreover, it was notable from XPS high-resolution N1s spectra for sample 1NG and 4NG  to 2.514 at.% (sample 4NG).However, the high-resolution N1s spectra of the N-doped graphene in Fig. As shown in Fig. 13 with distinct hysteresis loops at the range of relative pressures (P/P0) from 0.45 to 1.0 which can result from the coexistence of micropores and mesopores. BET surface areas of all samples are illustrated in Supplementary Table 21. The pore size distributions of N doped samples are shown in Supplementary Information Fig. 13.Nitrogen adsorption–desorption isotherm curves for NG samples as shown in Fig. 22.XRD patterns, illustrated in Supplementary Fig. −1 correspond to the D, G, and 2D bands of graphene-based materials, respectively23. The former D band originates from the defects in sp2 lattice structure (amorphous nature), the latter G band corresponds to the first order scattering of sp2 (graphitic nature) domains while 2D bands originate from two phonon double resonance processes and are related to the band structure of graphene layers24. Raman spectrum of NG shows a shoulder to G peak, known as D’ which generally appears when there is lattice defect or doping of another atom and that confirm N-doping20. The 2D band for NG samples is hardly detectable in the spectra due to significant structural defects induced by thermal synthesis and intercalation of N atoms in addition to further increase in layers leads to a significant decrease of the relative intensity of the lower frequency 2D peaks20. As shown in Fig. D/IG ratio of graphene blank samples is slightly lower than NG samples, demonstrating that N-doping increases the defects of graphene lattice and structural distortion. In addition from Supplementary Table However, Fig. 3 hybridization caused by structural distortions within the graphene network which induced by the thermal synthesis and embedded N atoms. Moreover, selected area electron diffraction pattern  were displayed in Fig. 27.The unique 3D structure combines high surface area in addition to nitrogen doping content leading to the prominent capacitive and cycling performance of these supercapacitor electrode materials using two similar electrode cell. In Fig. −1 in 6 M KOH, and no distinct IR drops appear in any curves, indicating excellent electrochemical reversibility, good rate capability and outstanding carrier transport within the electrodes for the electrochemical double layer capacitors (EDLCs). Furthermore, the capacitance of the 4NG electrode is four times higher than that of graphene electrode. The detailed GCD curves of the 4NG sample at various current densities are shown in Fig. −1 in 6 M KOH at current densities of 1, 2, 4, and 6 A g−1, respectively. However, the unchanged curves with linear and symmetrical shapes at various current densities are observed from this test. As a result it can be revealed that, the NG possesses considerable capacitive performance in 6 M KOH and that ascribed to the combination of microporous in the NG sheet and the mesoporous between the NG sheet in 3D structure with high active area which facilitates ion transport, thus contributing synergistically to the high rate performance.To assess the capacitive property, a two-electrode (2E) cell was used to conduct the galvanostatic charge–discharge (GCD) tests. As shown in Fig. −1. The 4NG electrode with the point power density of 558.5 Wkg−1 displays maximum energy density of 68.1 Wh kg−1 at a current density of 1 A g−1, which is over three times higher than that of 6 G (29.6 Wh Kg−1). The results illustrated in Supplementary Table The energy density (E) and power density (P) are calculated according to Equations  and 3)  3) from 4 than other prepared samples which exhibit straight lines at the low frequency region and a small depressed semicircle in the high-frequency region. Moreover, more vertical line was observed for N doped electrodes than undoped electrodes, which suggested high electrochemical activity of N doped graphene towards supercapacitors. However, the obtained semi-circle represents the charge transfer impedance at the interface of the current collector/electrode material while, the vertical line represents ion diffusion in the structure of electrode materials and the slope of this line is related to the speed of EDL formation28.The supercapacitive properties of all prepared samples as electrodes in 6 M KOH were studied through Electrochemical Impedance Spectroscopy (EIS). Nyquist plots as shown in Fig. s corresponds to the resistance of the electrolyte is 0.003 Ω, Rct is the charge transfer resistance (0.005 Ω), Cdl is the double layer capacitance (4-E6 Fg−1) and W represents the diffusion Warburg impedance (0.9) which is very low comparable to other prepared electrodes as illustrated in Supplementary Information Table 29 and the relaxation time for the 4NG electrode was found to be (335 ms). Therefore, it is confident to say that, 4NG has got faster frequency response capability, with shorter ionic transport paths that in turn reduced internal resistance and increase the electrical conductivity30.As shown in the inset of Fig. −1 using the galvano static charge/discharge technique. The capacitance retentions of 4NG were 87.7% of their initial capacitances, retained after 5000 charge/discharge cycles . The closed stainless steel reactor was placed inside the center of an electric furnace and the experiments were repeated at two different temperatures 600 °C and 800 °C with a rate of 10 °C min−1 under nitrogen gas flow. The composition of the prepared materials was studied using Fourier transform infrared (FTIR)  and X-ray photoelectron spectroscopy (XPS) Phi 5300 ESCA system  with the Mg (Kα) radiation (X-ray energy 1253.6 eV). The XPS samples were prepared by dropping 100 μL of NG aqueous dispersions (1.0 mg/mL) onto silicon substrates followed by air drying. Elemental analysis of the NG samples was measured using vario-Micro CHN Elemental analyzer (Germany).The thermal properties of raw PET, urea and a mixture of PET and urea with 1:1 wt. ratio were investigated by TGA using a thermogravimetric analyzer (Shimadzu TGA-50) from 20 °C to 800 °C at a rate of 10 °C min2) adsorption-desorption isotherms were recorded with Shimadzu, Micromeritics ASAP 2010 Instrument) cooled by liquid N2. Prior to recording sorption isotherm, the sample was degassed at 120 °C under vacuum for 12 h.Nitrogen (NThe morphology of the prepared samples was explored using scanning electron microscopy (SEM), JEOL  instrument with an operating voltage of 15 kV, and transmission electron microscopy (TEM), JEOL  under an accelerating voltage of 200 kV.−1 were employed to analyze the crystal structure of prepared materials.Raman spectroscopy  with a 514.5 nm excitation wavelength and X-ray diffraction  and dried at 60 °C overnight. The mass of active materials in the active surface was around 1–1.2 mg cm−2.To prepare the working electrode, the prepared active materials (90 wt %), carbon black (5 wt %), polyvinylidinefloride (5 wt %), and 1-methyl 2-pyrrolidinone as a solvent were mixed forming slurry. The slurry was then coated onto nickel sheet (1 cm−1 to 100 mV S−1. The charge–discharge measurements were performed at different current densities (from 0.5 to 6 Ag−1). Cyclic stability tests were conducted at a current density of 4 A g−1. The specific gravimetric capacitance of the supercapacitor was determined from the galvanostatic cycles by means of the formula:31.The electrochemical measurements were performed in a two-similar electrode cell using 6 M potassium hydroxide (KOH) electrolyte solutions, nickel sheet as current collector and Whatman fiber glass filter paper (GF/D) as a separator. The electrochemical characterization was performed at room temperature using a computer-controlled Potentiostat  involving cyclic voltammetry (CV), galvanostatic charge/discharge (GCD), and electrochemical impedance spectroscopy (EIS). The CV studies were performed between − 0.2 to 1 V at increasing sweep rates from 5 mV S32. Electrochemical Impedance Spectroscopy (EIS) tests were recorded in a frequency range from 0.001 to 100 kHz with a sinusoidal signal of 5 mV.Energy density (E) and power density (P) were calculated using the formulas,Supplementary Information


E is one of the 13 sigma factors encoded by the Mycobacterium tuberculosis chromosome, and its involvement in stress response and virulence has been extensively characterized. Several sigma factors are post-translationally regulated by proteins named anti-sigma factors, which prevent their binding to RNA polymerase. Rv1222 (RseA), whose gene lays immediately downstream sigE, has been proposed in the past as the σE-specific anti sigma factor. However, its role as anti-sigma factor was recently challenged and a new mechanism of action was hypothesized predicting RseA binding to RNA polymerase and DNA to slow down RNA transcription in a not specific way. In this manuscript, using specific M. tuberculosis mutants, we showed that by changing the levels of RseA expression, M. tuberculosis growth rate does not change (as hypothesized in case of non-specific decrease of RNA transcription) and has an impact only on the transcription level of genes whose transcriptional control is under σE, supporting a direct role of RseA as a specific anti-σE factor.σ M. tuberculosis faces many different stressful environments; therefore, a rapid adaptation to changing conditions is key for this microorganism. Among the different strategies that can be employed by the bacterium to survive and adapt itself to a hostile environment, specific transcriptional regulation mediated by sigma factors plays a relevant role. Sigma (σ) factors are small interchangeable subunits of the RNA polymerase (RNAP) holoenzyme required to initiate transcription and determine the promoter specificity of the enzyme through the recognition of specific −35 and −10 consensus sequences. They can undergo different regulatory mechanisms at transcriptional, post-transcriptional, translational and post-translational levels1. Post-translational regulation is usually due to anti-σ factors, which are proteins that specifically bind to σ factors preventing their interaction with the RNA core enzyme. Specific environmental signals can then cause disruption of such an interaction, leading to the release of the σ factor2. The genome of M. tuberculosis encodes 13 σ factors3 that represent the highest number of σ factors per Mb among obligate pathogens4.During the infectious process, E is an alternative σ factor of M. tuberculosis belonging to the extra cellular function (ECF) category whose role is of prime importance for M. tuberculosis physiology and virulence10. In a previous work we have shown that σE-mediated transcription is inhibited upon interaction with Rv1222, a protein encoded by the gene immediately downstream of sigE11. Amino acid sequence analysis of Rv1222 revealed a HXXXCXXC motif that is typical of the zinc-associated anti-sigma factor (ZAS) family13. Additionally, σE and Rv1222 interaction was shown to be redox dependent, and σE could also be released from Rv1222 as a result of a PknB-dependent phosphorylation of Rv1222 under surface stress conditions14. Taken together, all these observations led to the conclusion that Rv1222 is a regulator of sigma E factor and therefore was named RseA.σE factor15. They found that the interaction between σE and Rv1222 does not inhibit open-complex formation mediated by RNAP. Rv1222 was shown to interact with M. tuberculosis RNAP core enzyme but not DNA polymerase and bind non-specifically to DNA through its C-terminal region. In in vitro transcription experiments Rv1222 was able to inhibit transcription regardless the source of RNAP, for inhibition occurred by using holo RNAP or core RNAP coming either from M. tuberculosis, Escherichia coli or Bacillus subtilis. Additionally, they observed a growth rate reduction in both Mycobacterium smegmatis and E. coli upon inducible expression of Rv1222 that reversed when the C-terminal region of Rv1222 was removed. Such a growth defect was accompanied with a reduction of the amount of total RNA. Based on these results, they proposed a novel mechanism of transcriptional regulation according to which Rv1222 inhibits transcription by anchoring RNA polymerase onto DNA and slowing down its movement along DNA during elongation.However, in a recent paper Rudra and colleagues have called into question the role of Rv1222 as an anti-σM. tuberculosis physiology.In this paper, we have performed a series of experiments using a genetic approach to better characterize the role of Rv1222 in E. coli strain DH5α was grown in Luria broth (Difco) at 37 °C. When required, antibiotics were added at the following concentrations: kanamycin 50 µg/ml, hygromycin 150 μg/ml.M. tuberculosis H37Rv was grown in either Middlebrook 7H9 liquid medium or Middlebrook 7H10 solid medium (Difco) supplemented with 0.05% Tween 80 and ADN . M. tuberculosis liquid cultures were grown in roller bottles at 37 °C. Plates were incubated at 37 °C in sealed plastic bags. When required, antibiotics were added at the following concentrations: kanamycin 20 µg/ml; streptomycin 20 μg/ml; and hygromycin 50 µg/ml.All the strains tested in this work are listed in Table M. tuberculosis was handled and cultivated in a biosafety level 3 (BL3) laboratory.E. coli DH5α as the initial host. DNA restriction and modifying enzymes were obtained from New England Biolabs and used according to the manufacturer’s recommendations. Primers used are shown in Table All recombinant DNA techniques were performed according to standard procedures using 16. Results were normalized to the amount of sigA mRNA17. RNA samples that had not been reverse transcribed were included in all experiments to exclude significant DNA contamination. For each sample, melting curves were used to confirm the purity of the amplification products. Experiments were performed at least twice, starting from independent biological samples. Primers used for quantitative real-time PCR are described in Table RNA extraction and quantitative reverse transcription real-time PCR (RT-PCR) were performed using Sybr Green Master Mix (Applied Biosystems) as previously describedΔsigE_Δrv1222 deletion mutant was constructed in M. tuberculosis according to a published method18. Two DNA regions, one upstream sigE and one downstream rv1222 were amplified by PCR and sequentially cloned into p1NIL as ScaI/HindIII and HindIII/PacI fragments respectively  was amplified by RP1413 and RP1414 while the downstream region (1037 bp) was amplified by RP1415 and RP1416. A lacZ-sacB-hyg cassette from pGOAL19 was then introduced as a PacI fragment in the resulting vector to obtain the final suicide plasmid pSA23.1 that was electroporated into M. tuberculosis19. Transformants were selected on plates containing both kanamycin and hygromycin. The occurrence of single crossover was confirmed by PCR (data not shown). One mutant with the correct integration of pSA23.1 was grown in the absence of any drug to allow a second homologous recombination20. Recombinants were isolated as white colonies on plates containing sucrose and X-gal and the occurrence of a double crossover leading to sigE and rv1222 deletion was confirmed by PCR screening with the couple of primers RP1520/RP1521 (couple A) and RP1522/RP1523 (couple B)  Fig. . These psigE, a 1309 bp fragment containing the whole sigE gene and 535 bp upstream with the sigE promoter11 was amplified by RP1917 and RP1603, and then cloned into the integrative vector able to stably integrate into the single phage attB site in the M. tuberculosis genome21. The resulting plasmid, named pFRA250, was then electroporated into TB340 and transformants were selected on Km plates. The resulting strain was named TB514. To complement TB340 with both sigE and rv1222, a 1488 bp fragment containing the chromosomal region that includes the sigE promoter, sigE and rv1222 was amplified by RP1917 and RP1918 and then cloned into pMV306. The resulting plasmid was named pFRA242 and was introduced into TB340 by electroporation. Transformants were selected on Km plates. The resulting strain was named TB508.To complement TB340 with rv1222 was amplified by primers RP1942bis and RP1943 and then cloned into the replicative plasmid pMV261 under the Phsp60 transcriptional control. The resulting plasmid, named pLCM1 was electroporated into H37Rv and TB340 to obtain TB515 and TB516 respectively.15 proposed that overexpression of Rv1222 leads to a general decrease of RNA transcription and a consequent reduced growth rate, therefore hypothesizing that deletion of this gene  would result in an increase of M. tuberculosis growth rate. To challenge this hypothesis we compared the growth of H37Rv, TB340 (missing both rv1222 and sigE) and TB514 . As clearly shown in Fig. rv1222) grew with the same rate, whereas the double mutant grew slightly slower confirming the importance of σE on the growth of M. tuberculosis9. The same data could be observed when bacteria were grown on solid media  and TB515 . As shown in Fig. rv1222 indeed slightly decreased M. tuberculosis growth rate whose profile was comparable to that of ST28. This could be explained by two different reasons: either (i) a decreased RNA transcription rate which would lead to a decreased growth rate, as stated by Rudra and coll.15, or (ii) a reduced activity of σE due to the inhibitory action on σE carried out by an increased amount of Rv1222 that consequently would implicate a growth rate reduction as observed for the sigE mutant. To discriminate between these two hypotheses, we overexpressed Rv1222 in TB340, which is devoid of both sigE and rv1222 (TB516). If overexpression of Rv1222 directs a reduction of the bacterial growth rate, we would expect an additional slowdown in the growth of TB516. However, as shown in Fig. E activity, leading consequently to a slightly reduced growth rate.Rudra and colldia Fig. . The delE-specific anti-sigma factor, we would expect to detect an upregulation of those genes whose expression is regulated by σE when Rv1222 is absent. To investigate this hypothesis, we focused our attention on sigB and pspA. While sigB basal level of expression is almost totally σE dependent, pspA expression is known to be σE dependent only under stress conditions activating σE 9. Indeed, in a genetic background missing σE (TB340 and ST28) the expression of sigB and pspA was repressed compared to the wild type, of about 10 times and 2 times respectively , an essential gene required for the biosynthesis of phosphatidyl-myoinositol mannosides22 whose regulation is not controlled by σE. As shown in Fig. pimA expression level did not undergo any significant variation in all of the genotypic backgrounds tested. Taken together these data indicate that the absence of Rv1222 specifically increases the activity of σE.If Rv1222 acted as a σE activity, we compared the expression levels of a panel of genes in H37Rv and TB515 (H37Rv overexpressing rv1222). As shown in Fig. rv1222 was induced sevenfold in TB515 compared to the wild type. The expression of sigH, sigL, and pimA, whose transcription does not depend on σE 24, was not affected by rv1222 overexpression, whereas we could observe a reduction of sigB mRNA level, consistent with the role of Rv1222 as an anti-σE factor, as the upregulation of rv1222 would reduce the activity of σE that is responsible for the basal transcriptional level of sigB.To further confirm that Rv1222 specifically modulates σ15 recently challenged the hypothesis that Rv1222 represents a specific anti-σE factor and proposed a different mechanism of action through a series of in vitro experiments, according to which Rv1222 binds to both RNAP and DNA and reduces transcription rate. To corroborate this hypothesis the authors showed that overexpression of this protein in E. coli and M. smegmatis led to a reduction of total mRNA production and consequently of their growth rate, hence the proposal that the deletion of rv1222 might increase mycobacterial growth rate. In this manuscript, we described a series of genetic and physiologic experiments challenging this hypothesis.Rudra and coll.M. tuberculosis mutants, we first showed that an M. tuberculosis strain devoid of rv1222 does not exhibit an increase in growth rate. Then we showed that a sevenfold increase in rv1222 expression slowed down M. tuberculosis growth only in a genetic background where active σE was present. In fact, such a growth profile was similar to that observed for a sigE mutant, suggesting that such a decrease relied on an inhibitory action on σE activity caused by an increased amount of anti-sigma rather than on a direct, σE-independent, activity of Rv1222 on RNAP. This was also confirmed by RT-PCR experiments showing that Rv1222 overexpression led to a repression of sigB 9, but not of pimA, sigL or sigH, whose expression does not depend on σE 24. We were not able to explain the apparent discrepancy of our data with those shown by Rudra and coll., however in their experiments they used expression systems based on acetamide- or IPTG- inducible promoters (in M. smegmatis and E. coli respectively). These systems are known to produce large amounts of proteins26. To perform our experiments, we preferred to use a promoter that expresses the gene at “more physiological” levels (at the best of our knowledge no data reporting rv1222 inductions higher than 2.2 folds have been published). Perhaps the amount of Rv1222 obtained upon induction by Rudra and coll.15 was far above the physiological levels thus resulting in non-specific toxicity of the protein. Unfortunately, they did not report the fold induction obtained in their experiment to address this hypothesis.Using different 15 hypothesized that Rv1222 down-modulate transcription in a non-specific manner. If this were true, its overexpression or deletion of its structural gene should affect most of the M. tuberculosis gene transcription levels.As already mentioned, Rudra and coll.E-dependent gene sigB, but not of the σE-independent genes sigL, sigH, and pimA. Additionally, partial complementation of the repression of the σE-dependent genes pspA and sigB was obtained in the double sigE-rv1222 mutant upon reintroduction of both sigE and rv1222, whereas the reintroduction of the sole sigE in the double mutant led to increased expression of pspA and sigB, despite a reduced amount of sigE mRNA detected in this genetic background as compared to the wild-type. On the other hand, the mRNA level of the sigE independent gene pimA remained the same in all the tested strains. These observations suggested that Rv1222 is required to modulate σE activity specifically. The reduction of sigE expression in a genetic background devoid of rv1222 could be explained by the presence of some still unknown feedback control mechanisms able to down-modulate its expression to compensate the increased activity of σE caused by the loss of post-translational control.However, after overexpressing Rv1222 we could observe transcriptional repression of the σE and are in contrast with those published by Rudra and coll. These authors showed that Rv1222 couldn’t inhibit open complex formation in an EMSA assay. We noted however that a control without σE was lacking in their experiments in order to ensure that the shift observed was not due to non-specific binding of RNA polymerase to the probed DNA fragment. In addition, we also noted that these authors used high concentrations of RNA polymerase in their in vitro experiments, a condition that may well induce non-specific interactions of this protein with DNA. There is no doubt however, that Rv1222 inhibits in vitro transcription, regardless of what sigma factor is used, because it binds to both RNA polymerase and DNA through its C-terminal region, however our data strongly suggest that the situation is completely different in vivo, where Rv1222 is able to inhibit only σE-mediated transcription. In conclusion, our in vivo genetic analyses support a direct role for Rv1222 (RseA) as a specific anti-SigE factor and are in line with our previously published in vitro experiments14.Taken together these data strongly support the role of Rv1222 as an anti-sigma factor regulating σTable S1 and Figure S1


Stable isotope analysis of single amino acids (AA) is usually applied in food web studies for tracing biosynthetic origins of AA carbon backbones and establishing trophic positions of consumers, but the method is also showing promise for characterizing quantity and quality of dietary lipids and carbohydrates.δ13C values of glycolytic AA, i.e., AA carbon backbones sourced from the glycolytic pathway, we compared Atlantic salmon  from a feeding experiment with and without dietary inclusion of the red macroalga Palmaria palmata. The Control and experimental diets had similar relative proportions of macronutrients, but their ingredients differed; in the experimental treatment, 15% Palmaria inclusion substituted proteins from fishmeal and carbohydrates from corn starch.To investigate whether changes in high- and low-digestible carbohydrates affect 13C values of the glycolytic AA were highly sensitive to substitution of corn starch with Palmaria. The δ13C offsets of glycolytic AA between salmon and their diets were significantly greater in the Palmaria inclusion than Control treatment. This greater offset can be attributed to the different utilization of high- vs. low-digestible carbohydrate sources, i.e., corn starch vs. Palmaria, in the two treatments, and metabolic routing of dietary lipids. In addition, similar δ13C values of essential AA between treatments indicate similar nutrient assimilation efficiency for all terrestrial  and marine  derived protein sources. These results show that δ13CAA analysis is a promising tool for improving our understanding of how carnivorous fish utilize macronutrient and route metabolic intermediates to tissue.We found that  While stable carbon isotope analysis of AA is predominantly applied for tracing biosynthetic origins of AA , a numbe offsets . Some aq offsets . Metabolm health . Thus, pproteins .δ13C) variations of Ala are correlated to human sugar consumption   , it is uδ13CNEAA values by analyzing Atlantic salmon and their diets in a controlled feeding experiment  meal   and pyruvate (Ala)  because lipid derived intermediates would be routed to energy rather than tissue formation. We also analyzed the EAA to test whether EAA assimilation efficiency and protein preference would be similar for terrestrial  and marine  derived protein sources. Finally, to obtain the supporting information of the fate of dietary macromolecules, we also measured bulk carbon and nitrogen isotopes in the diet and fish.In this study we investigate how varying proportions of high- and low-digestible carbohydrates affect periment . The twoperiment , but witia) meal . While pt fibre) . Neithert fibre) . We hypote (Ala) . We focuP. palmata) macroalga was compared to the Control group fed on a basal diet formulation. The salmon were fed on the experimental diets for 14 weeks to ensure that at least half of the muscle carbon pool would reflect the new experimental diets (−1). The experimental group were fed alternative diet substituted with 15% of dried macroalgae into the diet by decreasing fishmeal by 5% and extruded corn starch by 10%, while the compositions of other ingredients remained unchanged  of salmon and diet at Leibniz-Laboratory for Radiometric Dating and Stable Isotope Research in Kiel, Germany. Freeze-dried fish fillet were homogenized and approximately 4 mg of each sample were analyzed for compound specific stable isotope analyses (v/v) for 30 s and subsequently discarded the solvent from the aqueous phase. The AA were derivatized to N-acetyl methyl esters following the protocols by δ13C values under the same conditions as the samples to calculate correction factors specific to each AA to account for carbon addition and fractionation during derivatization  notation: Isotope data are expressed in delta . The isotope ratios are expressed relative to international standards Vienna Pee Dee Belemnite (VPDB) for carbon. In regard to analytical uncertainty, the average reproducibility for the internal reference standard nor-leucine (Nle) was 0.3‰ (n = 3 for each batch) and the in-house AA standards ranged from 0.2‰ for Pro to 0.6‰ for Ala (n = 4–7 for each batch). We were able to analyze the following 15 AA: NEAA; alanine (Ala), asparagine/aspartic acid (Asx), glutamine/glutamic acid (Glx), glycine (Gly), proline (Pro), tyrosine (Tyr) and serine (Ser), and EAA; histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), threonine (Thr), and valine . See For the certain element E, the ratio of heavy (i) to light (j) isotope are measured in both sample and references . Since iδ15N and IAEA-CH-3, IAEA-CH6, IAEA-CH-7 for δ13C). Internal standard conducted for bulk δ13C and δ15N analyses during the sample sequence yielded 1σ = 0.2‰ and 0.2‰, respectively. Because lipids have more negative δ13C values than proteins, it is important to account for the large variability in lipid content, which can affect the δ13C values of bulk tissue and lead to false interpretation. Thus, the negative δ13C values are commonly corrected by extracting lipids from samples prior to isotope analyses, or applying a mathematical correction after isotope analyses based on sample C:N ratios  connected to a ThermoElectron DeltaPlus Advantage IRMS . The isoN ratios . We opteδ13CAA values of fish are based on triplicate fish analyses. Isotope offset between fish muscle (F) and diet (Δ13CF−D) were calculated for all amino acid and bulk isotopes of each treatment as Δ13CF−D = δ13CF − δ13CD, where the δ13CF and δ13CD represent the δ13C values of the fish  and diet , respectively. Assuming independent variables, the error term for Δ13CF−D are propagated according to δ13CAA values) are significantly different between groups (R function: summary.aov). The P-adjusted values from this ANOVA decomposition test was obtained using p. adjust function with FRD method  to decrease the probability of Type I error. The statistical tests results are presented in the All statistical analyses were performed using R version 3.4.3  Supplem. All valDF2+SDD2 . We usedD method . We did δ13CAA values of fishmeal were the highest for all AA except for Lys and Met, which were slightly lower than those of Palmaria by 1–2‰. In general, δ13C values (both AA and bulk) of marine sourced proteins  were much higher than the terrestrial protein sources  and narrowest for Ala and Tyr (∼6 ‰). For the EAA, the range was widest for Met (∼12 ‰) and narrowest for Phe (∼6 ‰). Among all AA in the individual ingredients, Ser, Gly and Thr were the most 13C-enriched in both treatments, whereas Tyr, Phe, and Leu were the most 13C-depleted . Likewise, individual AA δ13C and δ13Cbulk values of wheat gluten were similar to those of pea protein concentrate except for Gly and Ile   .13CF-D offset (Δ13CF−D) between fish and the compound diet for the individual NEAA. The Δ13CF−D values of all NEAA except Asx (∼0‰) showed much greater variability, ranging from Ala (−6.8‰) to Ser (5.9‰)  than those of the Control fed fish. The patterns observed for Δ13CF−D values of all NEAA also mirrored the patterns observed in the Δ13CF−D of bulk samples (There was a large variability in r (5.9‰)  and 2B.  samples  and 3B.δ13CEAA patterns of fish muscle largely to reflect that of the compound diets instead of individual protein components for both treatments . There was no apparent isotopic offset between salmon muscle (fish) and diets (Δ13CF−D) for EAA  of the glycolytic AA, Ser, Gly and Ala. In our study, the starch fraction of the diets  was the greatest variable that influenced Δ13CF−D because both lipid sources were kept consistent and proteins were functionally similar between the two compound diets on group  and 3B, l salmon  and 3B. hydrates , we posiPalmaria  increase13CF−D difference in Krebs AA between the two treatments further corroborating that Krebs AA are sourced more from proteins than lipid and carbohydrates. It is possible that the decrease in digestible starch content also increased gluconeogenesis, i.e., generation of glycolytic metabolites from AA oxidation. How this pathway affect the discrimination factor for the glycolytic AA in an obligate carnivore is unclear. A study with calorie restricted rats demonstrated that increased AA oxidation led to a Δ13CF−D decrease in proteins and their constituent NEAA and EAA  and no change for the EAA  are the same further suggesting no enhanced microbial activity in the Palmaria_inclusion experiment  would be needed to explain the isotope discrimination factor of Asx.Unlike the other NEAA, the Δ studies . The virlutarate . If salm13CF−D values of EAA between Atlantic salmon and their protein sources to investigate EAA assimilation of terrestrial and marine protein sources. For the EAA, δ13CEAA values between fish muscle and their compound diets are mostly identical in both treatments except for Met  during the feeding trial were comparable across the macroalgal inclusion diet and Control diet , which underlines that discrimination factors are determined by multiple factors. Our study highlights that one of those factors is metabolic utilization of dietary macromolecules; in our case how carbohydrate availability presumably affect lipid utilization. Other factors that could affect metabolic demands are infection, recoveries from injury, physical activities and digestive processes . By comparing treatments where corn meal was replaced by macroalga, we show that glycolytic AA are sensitive to the dietary carbohydrate sources and digestibility. The most parsimonious explanation for the more positive Δ13CF−D values of the glycolytic AA in the Palmaria treatment is a decreased sourcing of lipid derived intermediates to AA synthesis because Palmaria carbohydrates are more 13C depleted than corn starch. While more feeding studies are warranted for understanding metabolic routing of macronutrients, our findings show how characterization of δ13CAA variability can be used to trace relative contributions of dietary carbohydrates, proteins and lipids for de novo NEAA biosynthesis for carnivorous bony fish raised under normal feeding and husbandry conditions. Furthermore, our results indicate δ13CAA analysis can provide supplementary information on the gut microbiome’s role in carbohydrate digestion and metabolism. The wider adoption of compound specific isotope analysis, particularly for AA, can greatly improve our understanding of nutrient utilization during the fish growth of different aquaculture species and life stages (δ13CNEAA values in ecological studies.Our previous study  demonstre stages . Feeding10.7717/peerj.7701/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.7701/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.7701/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.7701/supp-4Supplemental Information 4Click here for additional data file.


Traumatic brain injury (TBI) is a risk factor for Alzheimer’s disease (AD), although the mechanisms contributing to this increased risk are unknown. Insulin resistance is an additional risk factor for AD whereby decreased insulin signaling increases synaptic sensitivity to amyloid beta (Aβ) and tau. Considering this, we used rats that underwent a lateral fluid percussion injury at acute and chronic time-points to investigate whether decreased insulin responsiveness in TBI animals is playing a role in synaptic vulnerability to AD pathology. We detected acute and chronic decreases in insulin responsiveness in isolated hippocampal synaptosomes after TBI. In addition to assessing both Aβ and tau binding on synaptosomes, we performed electrophysiology to assess the dysfunctional impact of Aβ and tau oligomers as well as the protective effect of insulin. While we saw no difference in binding or degree of LTP inhibition by either Aβ or tau oligomers between sham and TBI animals, we found that insulin treatment was able to block oligomer-induced LTP inhibition in sham but not in TBI animals. Since insulin treatment has been discussed as a therapy for AD, this gives valuable insight into therapeutic implications of treating AD patients based on one’s history of associated risk factors. In general, it is believed that synaptic dysfunction underlies the initiation and progression of the disease15. It has become increasingly evident that oligomeric aggregates of both amyloid beta (Aβ) and tau contribute to this synaptic dysfunction that precedes the cognitive decline seen in AD17. Notably, decreased insulin receptor function increases synaptic sensitivity to the binding of and dysfunction caused by Aβ18, and insulin and insulin-sensitizing therapy has been shown to be effective for cognition in mouse models of AD as well as in patients with mild cognitive impairment (MCI) or early AD22. Furthermore, AD patients exhibit insulin resistance and decreased insulin signaling response in the hippocampus24. This multitude of evidence strongly indicates the existence of an intimate relationship between synaptic insulin responsiveness and neuronal sensitivity to AD neuropathology. Several groups have reported hyperglycemia after TBI and found that uncontrolled blood glucose levels lead to a poorer outcome and recovery26. Previous reports have also found an increased mortality after head injury in people with T2DM27. While one group has reported acute decreased insulin signaling in the CNS after TBI28, no studies have investigated TBI-driven insulin resistance at the synapses, particularly in relation to synaptic vulnerability to Aβ and tau. Therefore, in the present work, we investigated changes in synaptic insulin responsiveness in the hippocampus after TBI in relation to an increased risk to AD pathology-driven synaptic dysfunction and protection by insulin treatment. Overall, our results indicate that, after TBI, hippocampal synapses become chronically insulin resistant and, contrary to sham animals, unresponsive to the beneficial effect of insulin therapy as a treatment against synaptic impairments due to Aβ and tau oligomers. Thus, this work further illustrates the importance of considering a prior history of associated risk factors and how these may impact the efficacy of particular treatments that are being investigated for AD in the general population.Alzheimer’s disease (AD) is a devastating neurodegenerative disorder for which there is no resolving therapeutic intervention. While the initial cause of this disease is still unknown, there are many innate and event-triggered factors that increase the risk of developing AD. For example, type 2 diabetes (T2DM)ex vivo insulin stimulation method described in the Materials and Methods section. Briefly, we isolated synaptosomes from sham and TBI animals at different times after injury (2 days post-injury (DPI), 7 DPI, 1 month post-injury (MPI), and 3 MPI), exposed them to insulin in the presence of ATP, and analyzed their extent of insulin receptor (IR) phosphorylation (i.e. activation) via Wes for both the ipsilateral  using the ral Fig.  and contral Fig. .Figure 1In the ipsilateral hippocampus, we found a significant decrease in synaptic insulin responsiveness by 7 DPI (p = 0.0041) Fig.  that wasIn the contralateral hippocampus, there was a significant decrease in IR response to insulin at 7 DPI (p = 0.0050) and 3 MPI (p = 0.0378) Fig. . InteresIn summary, we found a significant decrease in the synaptic insulin receptor’s response to insulin at 7 DPI, 1 MPI, and 3 MPI but not at 2 DPI. These data indicate that there are chronic deficits in synaptic insulin responsiveness in both the ipsilateral and contralateral hippocampi after lateral FPI.ex vivo Aβ oligomer binding on isolated synaptosomes from sham and TBI animals at 1 month and 3 months after injury and evaluated the maximum binding capacity (Bmax) and affinity (Kd) of Aβ binding with Scatchard plot analysis. We did not find any differences in Aβ binding at synapses in the ipsilateral hippocampi at either the 1 MPI  or 3 MPI  time points. We also did not find differences in Aβ binding at synapses in the contralateral hippocampi at the 1 MPI  time point  (for Bmax p = 0.0228) (for Kd p = 0.0390)  Fig. . These rex vivo tau binding on hippocampal synaptosomes isolated from both sham and TBI animals at 1 month and 3 months post-injury  and contralateral (p < 0.0001) hippocampi from the injured animals vs sham animals. At 3 MPI, we found this same significant reduction in the magnitude of LTP from untreated brain slices from the injured animals vs sham animals in the ipsilateral hippocampus (p < 0.0001).At 1 MPI, in slices treated with Aβ oligomers for 1 hour prior to recording, the magnitude of LTP was significantly lower in the TBI versus sham group in the ipsilateral hippocampus (p < 0.0001) Fig. . HoweverThe 200 nM insulin treatment during the Aβ oligomer-challenge was able to block LTP suppression in sham hippocampal slices at both time points in the ipsilateral (for both 1 MPI and 3 MPI p < 0.0001) Fig.  and contex vivo insulin stimulation analysis previously described and provides a valuable demonstration that even a significant administration of insulin cannot overcome this phenomenon.Collectively, these results suggest that TBI does not generally affect vulnerability of synapses to Aβ or tau oligomer-induced LTP impairments to a higher degree from that seen in sham animals at 1 or 3 months after injury. Insulin alone did not increase expression of LTP but rather significantly decreased LTP in both sham and TBI animal slices  to determine whether synaptic insulin responsiveness after TBI is dysregulated and (2) to investigate related changes in synaptic vulnerability to the dysfunctional impact of Aβ and tau oligomers.TBI increases the risk of developing AD later in lifeex vivo insulin stimulation method on isolated synaptosomes, we found the synaptic insulin receptor to have significantly decreased responsiveness as early as 7 days post-injury (DPI) after lateral FPI. This injury-promoted insulin resistance of hippocampal synapses became chronic as deficits were observed up to 3 months post-injury (MPI). While decreased insulin signaling in the CNS after mild TBI has previously been reported28, this is the first demonstration that insulin signaling is chronically impaired after a moderate TBI. Additionally, direct evaluation of the synaptic insulin receptor (IR), as performed here, bypasses assessment of the overall insulin signaling pathway and reveals that there is a chronic impairment at the level of the receptor.Using an In the ipsilateral hippocampus, we found a significant increase in synaptic IR levels at 3 MPI which could represent an attempted compensatory mechanism for the decreased insulin signaling here. However, these possible efforts were shown to be futile as we found that there was still a significantly decreased synaptic insulin response as well as decreased basal phosphorylation levels at this time point, indicating that there is a chronic alteration in the response of the receptor that cannot be overcome by upregulation of the receptor. On the other hand, we did not observe any changes in synaptic IR level at any of the time points for the contralateral hemisphere, possibly suggesting that any attempt to compensate for TBI-related synaptic insulin resistance is limited to the ipsilateral hemisphere.33. Specifically, SOCS3 protein expression can be induced by IL6 as well as IL10 and has been shown to negatively regulate insulin signaling34. Thus, SOCS3 was a good candidate to negatively regulate insulin signaling at early time-points, prior to the delayed (7 days) insulin resistance that we found. Furthermore, SOCS3 expression has been found to be upregulated in the CNS of AD patients35.In order to begin delving into the mechanisms responsible for driving the synaptic insulin resistance in TBI animals observed in our study, we determined levels of SOCS3. SOCS3 is a protein of the suppressor of cytokine signaling (SOCS) family that act as negative regulators of cytokine and growth factor signaling. We chose to look at SOCS3 due to its ability to be upregulated by inflammatory factors which have been found to be increased within hours after injury36 and the insulin resistance that we have reported here in hippocampal synapses after injury.In the ipsilateral hippocampus, we found a significantly increased level of SOCS3 at the synapse in TBI animals versus sham animals at 2 DPI, 7 DPI, and 1 MPI as well as a trend toward increased levels at 3 MPI. Since increased levels of SOCS3 occur as early as 2 DPI, prior to the onset of synaptic insulin resistance that was observed starting only at 7 DPI, increased SOCS3 inhibition of the insulin receptor could potentially be a driving factor in initiating the insulin resistance ensuing at the synapses after TBI. Furthermore, SOCS3 upregulation could be one molecular link between the chronic inflammation seen after TBI37. Although further studies are needed to establish the exact mechanisms of insulin resistance induction, this scenario would be consistent with prior reports describing opposing bilateral changes in proteins involved in TBI-related secondary injuries after a unilateral TBI, specifically in pathways involved in cell death, survival, and inflammatory response38.In the contralateral hemisphere, however, we did not find altered levels of SOCS3 at the synapse at any of the time points studied here. Since we still found synaptic insulin resistance beginning at 7 DPI in this hemisphere too, it is possible that the two hemispheres suffer insulin resistance after TBI driven by different mechanisms, with SOCS3 playing a significant role only in the ipsilateral hemisphere. This hypothesis is supported by previous findings that the gene expression level of IL-6 signal transducer (IL6ST), a protein involved in the complex for IL-6 signaling, was increased at an acute time point in the ipsilateral hemisphere and decreased in the contralateral hemisphere after a lateral, moderate TBIex vivo binding methodologies to assess synaptic resistance/vulnerability to extracellular Aβ and tau oligomer binding. Our data indicates that hippocampal synapses are not more susceptible to Aβ oligomer binding at the chronic time points of 1 month or 3 months after moderate FPI. Similar results were obtained using tau oligomers, whereby synapses were not more susceptible to tau oligomer binding at the chronic time points of 1 month or 3 months after TBI in either hemisphere.We next aimed to determine whether reduced synaptic insulin responsiveness in TBI animals was associated with altered synaptic vulnerability to extracellular Aβ and tau oligomers as well as oligomer-induced synaptic dysfunction. We used 39, we found basal LTP levels to be chronically impaired in the side of injury after lateral, moderate FPI through 3 MPI. Our electrophysiological results are also consistent with our ex vivo binding experiments, suggesting that synaptic vulnerability to Aβ and tau oligomers is not increased after TBI as we did not find an increase in oligomer-induced LTP impairments compared to sham animals at 1 or 3 MPI.We further investigated synaptic vulnerability to oligomers by determining functional vulnerability through oligomer-induced LTP inhibition. Consistent with previous reports40. This group suggested that this beneficial effect of insulin was due to a direct interaction of Aβ and insulin whereby insulin inhibits the formation of soluble Aβ oligomers and thus prevents oligomer-induced LTP impairment in a manner that is independent of insulin receptor activation. While these two proteins may indeed interact to hinder Aβ oligomer formation, our results would argue against this conclusion since the beneficial effect of insulin that we observed in slices from sham animals was not seen against either Aβ or tau in ipsilateral hippocampal slices from TBI animals at both 1 and 3 MPI. Insulin treatment also did not block Aβ impairment at either time point in the contralateral hippocampus. We did find that insulin treatment partially blocked LTP reduction due to tau oligomers in the contralateral hippocampus at both chronic time points, yet we do not have an explanation for this phenomenon. This differential finding of insulin treatment in slices from sham and TBI animals suggests that activation of the insulin receptor does, in fact, contribute to the functional protection afforded by insulin against oligomer toxicity.Insulin treatment of brain slices concurrent with either Aβ or tau blocked the LTP impairment induced by the oligomers in hippocampal slices from both the ipsilateral and contralateral hemispheres from sham animals. This is consistent with the literature showing that insulin inhibits Aβ-induced impairment of LTP using the same concentrations, animal species, and electrophysiological set-up used hereex vivo insulin stimulation analysis, we found chronically decreased insulin responsiveness of the synaptic insulin receptor in the hippocampus after moderate TBI that does not lead to increased synaptic susceptibility to Aβ or tau oligomers.In conclusion, from our electrophysiology data as well as our 41, some have pointed out that indirect pathways may influence cognition, and, thus, neuronal insulin signaling may not be needed for cognitive enhancement via insulin therapy41. Evidence such as increased regional cerebral blood flow and enhanced cognition in the insulin-resistant type 2 diabetes patients42 supports this notion. In an opposing conclusion from animal models of type 2 diabetes where brain insulin resistance has been confirmed, insulin therapy acting on the hippocampus was found to be unsuccessful in improving cognition43. This could suggest that once brain insulin resistance has developed, insulin treatment may not be sufficient to overcome resistance at the cellular level41. Our results would support this overall conclusion in a TBI model showing that brain insulin resistance would negate the efficacy of insulin as a therapy to provide protection against oligomer-induced synaptic dysfunction in injured animals as well as in AD patients with a history of TBI.While there is consensus that intranasal insulin treatment benefits cognition by acting on neuronal insulin receptors to overcome resistanceMale Sprague-Dawley rats were utilized for all of the experiments in this study. All rats were 2–4 months old (300–450 grams) at the time of surgery/injury. All experimental protocols involving animals in this study were approved by Institutional Animal Care and Use Committee of the University of Texas Medical Branch, and all experiments were performed in accordance with relevant guidelines and regulations. Animals were housed under USDA standards  at the UTMB vivarium. After the designated amount of time after surgery/injury, the rats were sacrificed by isoflurane overdose and decapitated. The brains were quickly removed, dissected into major regions and opposing hemispheres , snap frozen, and stored at −80 °C until ready for further analysis.Male Sprague-Dawley rats (300–450 grams) were anesthetized (4% isoflurane) and prepared for moderate or sham parasagittal fluid percussion injury (FPI). Rectal temperatures were monitored and maintained within a range of 37.5 ± 0.5 °C using an overhead lamp and a thermostatically controlled water blanket . Rats were placed in a stereotaxic apparatus, a midline incision of the skin was performed, and the skull was exposed. A craniotomy was performed using a 5 mm diameter Michele trephine at 1 mm lateral (right) to the sagittal suture, midway between the lambda and bregma. The bone chip was removed, leaving the dura intact. A modified 20-gauge needle hub was secured in place over the exposed dura with superglue and cemented into place with hygienic dental acrylic.TBI was administered by means of a fluid percussion injury device . The animal’s craniotomy hub site was directly connected to the transducer end of the injury device. The pendulum of the device was lifted to a specified height to correspond to the intensity of injury desired, moderate injury level for these studies. The pendulum was released and struck the back of the sterile saline-filled cylinder causing a direct brain deformation injury. The fluid pressure pulse and the righting reflex time were recorded. At 2 days, 7 days, 1 month, or 3 months after TBI or sham injury, the rats were euthanized and brain tissue collected as described above.The control sham animals used in this study underwent the craniotomy and connection to the fluid percussion injury device, just as the TBI animals, with the exception of the pendulum release.44. Homogenate was centrifuged at 1,200 × g RCF for 10 minutes at 4 °C. Supernatant was collected and centrifuged at 15,000 × g RCF for 20 minutes at 4 °C.Synaptosomal pellets was resuspended in 48 μL HEPES-buffered Krebs-like (HBK) buffer . The quality of the synaptosomes are routinely verified by Western blot and electron microscopy as previously reported45.Synaptosomes containing both pre- and post-synaptic components were isolated using SynPER  with 1% protease and phosphatase cocktail inhibitors from frozen tissue that had been snap frozen on dry ice and transferred to −80 °C as described in Comerota, 201747. In short, 100 mM ATP stock was added for final concentration of 8 mM to synaptosomes in HBK, and U-100 insulin was added for 200 nM final insulin concentration. Samples were vortexed and incubated for 15 minutes at 37 °C. Synaptosomes were pelleted and resuspended in 1X RIPA  plus 1% protease and phosphatase cocktail inhibitors to solubilize the proteins for Western blot and Wes detection. Samples were then stored at −80 °C.After isolation of synaptosomes, insulin stimulations were performed as previously describedhttps://www.proteinsimple.com/wes.html. Phosphorylation extent of the insulin receptors was analyzed using Wes  with specific antibodies against the phosphorylated form of the 1150/1151 tyrosine residue of the insulin receptor . The phosphorylated form was normalized against β-tubulin . Another Wes was run for the total amount of IR  which was normalized to β-tubulin as well. The ratio of normalized phosphorylated-IR over normalized total IR was used to assess the extent of insulin responsiveness.Wes is an automated, capillary-based electrophoresis system for the separation and analysis of proteins for quantitative data. Further information on the Wes instrumentation can be found 1–42 peptide was purchased from Department of Biophysics and Biochemistry, Harvard University, MA, and Aβ oligomers were prepared from lyophilized synthetic Aβ aliquots as previously described48. Briefly, 200 µL of 1,1,1,3,3,3- Hexafluro-2-propanol (HFP) was used to dissolve the Aβ lyophilized aliquots of 0.3 mg. 700 µL of DDI water was then added, and a cap with four holes was placed on the tube. The sample was magnetically stirred under a fume hood for 48 hours. The Aβ oligomers were aliquoted, frozen at −80 °C, and used within 3 months. For the flow cytometry analysis of Aβ oligomer-binding to synaptosomes, Aβ oligomers spiked with Flour 647 tagged Aβ  were utilized. These Aβ oligomers were prepared by adding 7 µL of the tagged Aβ to the HFP-Aβ mixture, prior to oligomer formation. Thus, the tagged Aβ is distributed throughout the oligomers so that a single oligomer can be composed of both tagged and untagged Aβ. The quality of the oligomeric preparations was routinely checked by Western blot and dot blot analysis using 6E10 and A-11 antibodies (Aβ oligomer specific).Human Aβex vivo binding challenge and evaluated using flow cytometry. An equal number of isolated synaptosomes per animal determined by flow cytometry were pooled for each experimental group. For each group, the pooled samples were then aliquoted into 10 separate tubes containing 10 million synaptosomes each. This was repeated 3 times for 3 separate curves. Each sample was incubated with Aβ oligomers tagged with HyLite Fluor 647 for the desired μM concentration, ranging from 0 μM to 20 μM, for 1 hour at room temp. Synaptosomes were then pelleted, washed 3 times with HBK buffer, and resuspended in PBS. Data was acquired by a Guava EasyCyte flow cytometer  and analyzed using Incyte software (EMD Millipore).Oligomeric Aβ to be employed in the same experiment was always used from aliquots of the same batch of Aβ. Hippocampal synaptosomes were treated with Aβ oligomers for an 49.Prepared recombinant tau oligomers were graciously given to us by Dr. Rakez Kayed’s laboratory. The tau oligomers were produced as previously describedex vivo binding challenge and evaluated using ELISA as previously performed in our lab50. Hippocampal synaptosomes from each animal were challenged and evaluated independently. Using flow cytometry, 10 million synaptosomes from each animal were aliquoted and challenged with 2 µM of tau oligomers for 1 hour at room temperature. The samples were then centrifuged and washed with HBK buffer 3 times to thoroughly remove any unbound tau oligomers. Synapse number was acquired using flow cytometry once again, and an equal amount of synaptosomes per sample were analyzed by tau5 ELISA.Synaptosomes were treated with tau oligomers for an Total tau levels were measured by ELISA analysis using the total tau antibody, tau5 (Biolegend Cat. # 806401). Samples were incubated on the ELISA plate at 4 °C overnight. Samples were discarded and each well was washed with Tris-buffered saline with low Tween 20 (0.01%) (TBS-low T) followed by blocking with 10% nonfat milk for 2 hours. After a second wash, the primary tau5 antibody (1:1000 in 5% nonfat milk in TBS-low T) was incubated in each well for 1 hour at room temperature. After a third wash, the plates were incubated for 1 hour at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody . Following the fourth wash, 3,3,5,5-tetramethylbenzidine  was added to each well. After 30 min of incubation in the dark, 1 M HCl was added to stop the reaction, and the plate was read at 450 nm for tau detection and quantification.2 with 5% CO2. For oligomer challenges, the slices were incubated for 1 hour at room temperature prior to recording with 200 nM Aβ oligomers, 50 nM tau oligomers, and/or 200 nM insulin. For slices treated with insulin, 200 nM insulin was also present in the aCSF recording solution. Evoked field excitatory post-synaptic potentials (fEPSPs) recordings were performed by stimulating the Schaffer collateral pathway using a stimulating electrode of 22 kΩ resistance placed in the CA3 region and recording in the CA1 region. LTP was induced using a high frequency stimulation protocol  as previously described51. Recordings were digitized with Digidata 1550B , collected using an Axon MultiClamp 700B differential amplifier (Molecular Devices), and analyzed using Clampex 10.6 software (Molecular Devices). Current stimulation was delivered through a digital stimulus isolation amplifier  and set to elicit an fEPSP approximately 30% of maximum for synaptic potentiation experiments using platinum-iridium tipped concentric bipolar electrodes . Baseline recordings were obtained by delivering single pulse stimulations at 20 second intervals. All data are represented as a percentage change from the initial average baseline fEPSP slope obtained for the 10 minutes prior to HFS.Animals were euthanized with deep isoflurane anesthesia, decapitated by guillotine, and brains were harvested and sliced using Compresstome VF-300  in NMDG-aCSF to obtain 450 μm transverse brain sections. Slices were allowed to recover for 10 minutes in NMDG-aCSF at 35 °C. Slices were then maintained at room temperature in a modified HEPES holding aCSF solution. Slices were recorded in standard recording aCSF. All solutions were aerated using 95% OThe bicinchoninic acid (BCA) assay method was used for protein estimation to prepare samples of equal protein concentration. Samples were prepared in 2-mercaptoethanol (2-ME) and boiled prior to loading. Thirty micrograms of protein were loaded with appropriate marker on 10% SDS-PAGE gels followed by transfer to Amersham Protran nitrocellulose transfer membrane  for 1 hour at 100 V. The membrane was blocked using Odyssey blocking buffer  for 1 hour at room temperature. Primary antibodies were diluted 1:1000 in 1X TBST and incubated with the membrane at 4 °C overnight for SOCS3  and 1 hour at room temperature for β-tubulin . The membrane was washed twice with 1X TBST for 15 minutes each and incubated with LI-COR secondary antibodies diluted at 1:10,000 in 1X TBST with 3% non-fat dry milk for 1 hour at room temperature. The membrane was again washed twice for 15 minutes each.Western blots were imaged using LI-COR Odyssey infrared imaging system , application software version 3.0.30. The density of each immunoreactive band was measured using ImageJ software.All data represented as mean ± SEM. All data were statistically analyzed using Graphpad Prism6. Unpaired t-tests were used to determine statistical significance between two groups for the insulin stimulations and oligomer binding experiments. Student’s t-tests were used in cases of equal variance and t-tests with Welch’s correction were used in cases of unequal variance. For the electrophysiology experiments, a one-way ANOVA with Bonferroni’s post hoc test was used to determine statistical significance between the LTP of each condition.Supplementary Figures and Table