Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca The special AT-rich sequence-binding proteins 1 and 2 (Satb1/2) are nuclear matrix-associated proteins that exert multiple functions by influencing the structural organization of chromatin and interacting with several co-activators and co-repressors of transcription. Satb1 was first cloned from a cDNA library of the human cell line and found in thymocytes i dynamics were registered. At the stage of data analysis, genotyping was carried out, the distribution of experimental data in accordance with the genotype of animals, while at the previous stages, blinded experiments were carried out.Newborn mice were used to obtain neuroglial cell cultures of the cerebral cortex. The tails of these mice were frozen and stored for subsequent genotyping. An experimental strategy is shown in For genotyping, the tail cuts were dissolved in 0.3 mL of lysis buffer  at 55 °C for 2–10 h. The non-lysed tissue was removed by centrifuging the samples for 10 min at 10,000 rpm. The DNA was precipitated by adding an equal volume of isopropanol, then mixed and centrifuged . The precipitated DNA was washed twice in 80% ethanol, air-dried, and dissolved in 50 μL of sterile distilled water. All PCR reactions were carried out in a volume of 20 μL.The following primers were used to determine the amplified product. Satb1-floxed allele and wild-type allele: 5′-GCATGTTTGTCTGTGTGCC-3′; 5′-CAGAAACAGTCTGGAGGGAGG-3 ′. Amplification program was as follows: 95 °C, 3 min; 95 °C, 30 s; 55 °C, 30 s; 72 °C, 30 s; 72 °C, 5 min, 30 cycles. The wild-type allele product is ∼120 bp, knockout ∼160 bp.Satb2-floxed allele and wild-type allele: 5′-CAAGAGAGCCATCCAACTGC-3′; 5′-AACCATCAGGCTGCCTCAACC-3′; 5′-CCAGACCGCGCGCCTGAAGA-3′. Amplification program was as follows: 95 °C, 3 min; 95 °C, 10 s; 54 °C, 15 s; 72 °C, 30 s; 72 °C, 5 min, 33 cycles. The wild-type allele product is ∼200 bp, knockout ∼450 bp.Primers for amplification of the NexCre allele and the wild-type allele: 5′-CCG CATAACCAGTGAAACAG-3′; 5′-GAGTCCTGGAATCAGTCTTTTC-3′; 5′-AGAATGTGGAGTAGGGTGAC-3′. Amplification program: 95 °C, 3 min; 95 °C, 20 s; 54 °C, 30 s; 72 °C, 60 s; 72 °C, 2 min, 35 cycles. The product of the wild-type allele is ∼750 bp, the NexCre allele is ∼500 bp.+/+ * NexCre/+), 6 Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb2-deficient (Satb2fl/+ * NexCre/+) mice, 6 Satb1-null mice (Satb1fl/fl * NexCre/+), and 8 Satb2-null mice (Satb2fl/fl * NexCre/+) mice, respectively were used. Mixed neuroglial cell cultures were prepared as described in detail previously i were performed by fluorescence microscopy using Fura-2/AM , a ratiometric fluorescence calcium indicator. Neurons were loaded with the probe dissolved in Hanks balanced salt solution (HBSS) composed of (mM): 156 NaCl, 3 KCl, 2 MgSO4, 1.25 KH2PO4, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4, at a final concentration of 5 μM at 37 °C for 40 min with subsequent 15 min washout. Coverslip containing the cells loaded with Fura-2 was then mounted in the experimental chamber. To measure free cytosolic Ca2+ concentration, we used the Carl Zeiss Cell Observer and an inverted motorized microscope Axiovert 200M with a high-speed monochrome CCD-camera AxioCam HSm with a high-speed light filter replacing system, Ludl MAC5000. Fura-2 excitation and registration were recorded, using a 21HE filter set  with excitation filters BP340/30 and BP387/15, beam splitter FT-409 and emission filter BP510/90, objective lens Plan-Neo fluar 10×/0.3, excitation light source HBO 103W/2. Calcium responses were shown as a ratio of fluorescence intensities of Fura-2 excitation at 340 and 380 nm. To discriminate neurons and astrocytes, short-term application of 35 mM KCl at the end of experiments was used. This method was described in detail in our previous work [2+-signal of neurons obtained from the cerebral cortex of control and Satb-knockout mice showed that the application of Ca2+ ionophore (Ionomycin) after inhibition of ATP synthesis (application FCCP and Oligomycin) induces Ca2+-signals differing in speed and amplitude in each individual neuron in experimental groups –Fmax-Fmin of Fura-2 fluorescence and an increase in Fura-2 fluorescence reflects a linear [Ca2+]i increase in response to receptor agonists. ImageJ 2002 software (RRID: SCR_003070) was used to analyze data.Therefore we determined the amplitudes of CaTo analyze the development of the cell network, the cell culture was loaded with a Calcein AM fluorescent probe, which allows visualizing the cell bodies and their outgrowths. Calcein AM was loaded in HBSS solution with 10 HEPES, pH 7.4, at a final concentration of 5 μM at 37 °C for 40 min with subsequent 15 min washout. To register the fluorescence of Calcein, we used the system based on an inverted motorized microscope Leica DMI6000B with a high-speed monochrome CCD-camera HAMAMATSU C9100. For excitation and registration of Calcein fluorescence, we used an L5 filter cube  with BP 480/40 excitation filters, 505 dichromatic mirror, and BP 527/30 emission filter.MagMAX mirVana Total RNA Isolation Kit  was used for the extraction of total RNA. The RNA quality was estimated by electrophoresis in the presence of 1 μg/mL ethidium bromide (2% agarose gel in Tris/Borate/EDTA buffer). The concentration of the extracted RNA was determined with a NanoDrop 1000c spectrophotometer. RevertAid H Minus First Strand cDNA Synthesis Kit  was used for reverse transcription of total RNA.fl/+ * NexCre/+-mice). Data were analyzed using Livak’s method i in the neurons of the cortical cultures in response to short-term (30 s) activation of glutamate receptors by selective agonists—NMDA, 5-Fluorowillardiine (FW), and Domoic acid (DA). 2+-signals of several hundred cells of the neurons derived from the cerebral cortex of control (Satb1+/+ * NexCre/+), Satb1fl/+ * NexCre/+, and Satb1fl/fl * NexCre/+ mice with Satb1 knockout in response to different NMDA concentrations in Mg2+-free medium. The basal level of [Ca2+]i in the neurons of the cerebral cortex obtained from different mice is 0.24 ± 0.04 a.u. and is not different in mice with Satb1-, Satb2- knockouts and control mice. Ca2+-signals are produced by the cortical neurons in Satb1fl/fl * NexCre/+- and Satb1fl/+ * NexCre/+-mice in response to 0.001 µM NMDA  increase of [Ca2+]i is 1.26 ± 0.04 µM (Hill coefficient (n) 1 ± 0.05) and 9.94 ± 0.06 µM (n = 0.97 ± 0.03) in neocortical neurons from Satb1fl/fl * NexCre/+ and Satb1fl/+ * NexCre/+ mice, respectively, and the EC50 value for the neurons from Satb1+/+ * NexCre/+-mice is—6.39 ± 0.09 µM (n = 0.98 ± 0.05)  is 0.254 and 0.153, respectively, on average, it is higher than control—0.139  C. On theol—0.139 .2+-signals produced by the cortical neurons after NMDA receptor activation. The high amplitude of Ca2+-signals in response to NMDA concentration of 0.001 µM is typical for neurons derived from the cortex of Satb2fl/fl * NexCre/+-mice with Satb2 knockout ; the EC50 value for Satb2-deficient-neurons is 6.19 ± 0.08 µM (n = 1.04 ± 0.02), which is close to 6.39 ± 0.09 µM (n = 0.98 ± 0.05), the value for neurons from control mice  and high (10 µM) NMDA concentrations were completely suppressed by the selective antagonist, D-AP5  (n = 1.14 ± 0.06), and 0.002 ± 0.0004 µM (n = 1.12 ± 0.03), respectively  and 0.092 ± 0.006 µM (n = 1.1 ± 0.02) for neurons from mice with Satb2fl/+ * NexCre/+ and Satb2fl/fl * NexCre/+ knockout; it is greater by a factor of 5 and 23, respectively than that for neurons from Satb2+/+ * NexCre/+-mice  FW concentration to the cortical neurons lead, on average, to an almost complete repetition of the amplitude and shape of Ca2+-responses both in control and in cells from Satb1-null and Satb2-null mice. Incubation of cortical neurons obtained from control mice with 100 μM 1-Naphthyl acetyl spermine , an antagonist of the calcium-conductive AMPA-receptors, leads to a decrease in the amplitude of the Ca2+-signal upon application of 10 μM FW to the level of neuronal Ca2+-responses derived from Satb-null mice   DA concentration are 0.181, 0.239, and 0.137, respectively. Herewith, the EC50 values are 0.32 ± 0.003 µM (n = 0.89 ± 0.12) for neurons from control mice, 0.27 ± 0.003 µM (n = 0.95 ± 0.06) for Satb1fl/fl * NexCre/+, and 0.39± 0.002 µM (n = 1.04 ± 0.03) for Satb1fl/+ * NexCre/+  taking into account that the amplitudes of Ca2+ signals are virtually the same   .2+-signals in response to KAR activation in cortical neurons from Satb1fl/+ * NexCre/+-mice. Simultaneously, we observe a decline in Ca2+-signals of the neurons from Satb2fl/fl * NexCre/+-mice when compared to those in Satb1fl/+ * NexCre/+ and control mice. For instance, Satb2 deletion causes a decrease in the amplitudes of Ca2+signals in response to DA in cortical neurons from Satb2fl/fl * NexCre/+ and Satb2fl/+ * NexCre/+ mice when compared to control mice and Satb1 knockout mice. These results suggest that deletions in these homologous transcription factors have a somewhat multifaceted effect.Taken together, deletion in Satb1 transcription factor leads to higher amplitudes of Ca2+-signals in response to excitatory glutamate receptor agonists persist against the background of selective receptor antagonists . This difference could be related to the influence of the Satb1 deletion on the development of neuroplasticity. It was shown that the development of the cerebral cortex in Satb1-null mice proceeded without significant disturbances. However, for pyramidal neurons in the postnatal period, a decrease in the density of dendritic spines, playing a key role in synaptic transmission and plasticity, was shown, as indicated by Balamotis et al. i only after GABA application (1 µM) (n = 1.12 ± 0.07) and 20.6 ± 0.06 µM (n = 0.89 ± 0.14), respectively, it is greater than  for neurons from control mice (In immature (3 DIV) neurons from mice with Satb2-deficient and Satb2-null neurons the amplitudes of Carol mice E.2+-signals are suppressed after GABA application, it occurs when low-amplitude signals are recorded in response to 300 µM of the agonist   F, which  ± 0.12) F.2+-signals after application of this agonist are typical for neurons from control mice (Satb1+/+ * NexCre/+). However, during the maturation of control neurons in the tissue sample and the development of the neural network, Ca2+-signals of the cells from control mice are almost completely suppressed after GABA application suggesting a switch of the GABAergic system to inhibitory. Oppositely, it is shown that Ca2+-signals are generated by Satb1-deficient and Satb1-null neurons after application of even micromolar GABA concentrations and the EC50 value decreases for GABA receptors.Therefore, Satb1 knockout affects the activity of the GABAergic system and the process of switching the system from excitatory to inhibitory resulting in a lower sensitivity of immature Satb1-deficient and Satb1-null cortical neurons to GABAR-GABA, the natural agonist, while high amplitudes of Ca+/+ * NexCre/+) (p ≤ 0.0001) increase in the number of necrotic cells was revealed—16 ± 5%  . TherefoexCre/+)  and averexCre/+) . At the exCre/+) ; and in exCre/+) , compare—16 ± 5% , while d—16 ± 5% . Only fo—16 ± 5% , and theIn the case of deletion of Satb2, no significant differences were found in the number of cells dying by one pathway or another by 3 DIV . Interes2+-signals are suppressed after GABA application; Ca2+-dynamics and the EC50 value of these neurons are similar to the cortical neurons from control mice. However, Satb2-null neurons demonstrate higher sensitivity to GABAR activation at the early stage of the neural network development, and the same dynamics is also seen in the late stages of cultivation when neurons produce Ca2+-signals with high amplitudes in response to 300 nM GABA, and the EC50 value reduces by less than two times as compared to that in early stages of cultivation.As for Satb2-deficient neurons, it was found that during maturation of these neurons, CaSatb1 is a risk factor for the development of neurodegenerative diseases, particularly Parkinson’s disease (PD) ,46. In a2+-signals of cortical neurons when NMDAR and AMPAR are activated, and a smaller deletion effect is shown for KAR. In the cultured cortical neurons of Satb-deficient and Satb-null mice with deletions of these transcription factors, the amplitudes of Ca2+-signals are higher when NMDAR is activated due to increased expression of genes encoding the receptor subunits. Simultaneously, Ca2+-signals from neurons in response to the AMPAR agonist treatment are suppressed due to increased expression of the GluA2-subunit responsible for the formation of AMPAR that cannot conduct Ca2+ ions. Here, we report that Satb1 and Satb2 are involved in the mechanisms of NMDA receptor neurotransmission. There is evidence that Satb1 and Satb2 can affect intracellular signaling of neurons via chromatin looping of the NMDA receptor locus GRIN2B46 i in the cortical neurons from mice with Satb1 and Satb2 deletions may have an antiepileptic effect and protect neuronal networks. It was found that PTZ (pentylenetetrazol) injections cause epileptiform seizures by activating the mamillo-thalamic tract and creating an imbalance between excitation and inhibition in the hippocampal neuronal network i in neurons through L-type voltage-gated Ca2+-channels [2+-channels of L-type—nifedipine [+-interneurons results in abnormal integration of these interneurons in the process of network development, which can lead to their damage and death [+-interneurons deprived of Satb1 may be a homeostatic compensation that results from the reduced excitatory drive that cells with deletion receive or occur due to incomplete maturation of these neurons [In addition, the transcription factor Satb1 regulates a number of early genes  receptor, as evidenced by a shift in the EC50 value for activation of these receptors.


From the Sm1−xBaCo2O5+d oxide systems calcined at 1,100°C for 8 h, single phase  was maintained only in the case of the x = 0 composition. In the compositions of x = 0.05–0.10, BaCoO2.6 was mixed with the pattern of SBCO. In addition, in the composition of x = 0.15, it was confirmed that BaCoO2.6 and CoO phases coexisted with SBCO. In the compositions of Sm1−xBaCo2O5+d, the overall Area Specific Resistance (ASR) values decreased as the removal amount of Sm increased from x = 0–0.10; then, the values increased for compositions from x = 0.15. For example, the ASRs of SBCO_1, Sm0.95BaCo2O5+d (SBCO_0.95), Sm0.90BaCo2O5+d (SBCO_0.90), and Sm0.85BaCo2O5+d (SBCO_0.85) measured at 600°C were 0.301, 0.147, 0.119, and 0.179 Ω cm2, respectively. In particular, SBCO_0.90 was found to have an excellent ASR property of about 0.035 Ω cm2 at 700°C. Typical properties of the metal–insulator transition (MIT) electrical conductivity were shown in all measured compositions. The temperature at which MIT occurred increased as the non-stoichiometric composition increased.In this study, electrochemical properties of layered perovskites having non-stoichiometric compositions (Sm SBCO_0.90 showed the lowest value of 0.119 Ω·cm2 at 600°CArea specific resistance (ASR) of SmAs environmental problems such as global warming, caused by the indiscriminate use of fossil fuels since the beginning of the industrial revolution in the 19th century have recently emerged, interest in eco-friendly energy has been expanding Wigley, . AccordiFuel cells or fuel cell systems based on hydrogen energy have been emerging as alternative energy conversion methods to replace the energy generating from fossil fuel. Especially, among various fuel cell types, solid oxide fuel cells (SOFC) are the focus of attention recently. An SOFC is an energy converting device that produces water and generates electricity via the chemical reaction of hydrogen and oxygen Minh, .SOFCs are known to show higher values of energy efficiency and power density than those of other fuel cells. SOFCs, which are made of ceramics, are composed of a dense electrolyte and two porous electrodes (cathode and anode). The SOFC structure is simpler than those of other types of fuel cell. In addition, due to their high exchange current density and the kinetics characteristics that occur under relatively high temperature operating conditions, SOFCs do not require any precious or expensive catalysts for operation.While SOFCs have advantages of high efficiency at high temperature and a stable ceramic structure, their high-temperature operating conditions can rather act as a disadvantage, causing problems such as thermal degradation of ceramic materials, sealing issues, and rapid oxidation of metal materials used as interconnectors. To solve these problems, research on Intermediate Temperature-operating Solid Oxide Fuel Cells (IT-SOFC) that feature lowered operating temperature in the intermediate temperature range (500–750°C) has been actively conducted. However, the main obstacle to this lowered-temperature-range SOFCs is the decreased electrochemical activity of the two electrodes (cathode and anode), as well as lowered ionic conductivity and increased ohmic resistance of the electrolyte. Significantly, the major source of voltage loss in IT-SOFCs is the cathode materials. Therefore, to achieve high power densities at the reduced temperatures, it is necessary to develop cathode materials that show excellent electrochemical reaction and enhanced/advanced low polarization resistance Minh, ./A//B2O5+d chemical composition), various lanthanide atoms such as La, Pr, Nd, and Sm can be substituted into the A/-site; Ba can be replaced in the A//-site; and transition metal ions of Co, Mn, and Fe can be located at the B-site.Recently, to replace existing IT-SOFC cathode materials, many research groups have investigated new concept-based cathode materials with a chemical composition of layered perovskite, because this structure yields superior oxide ionic diffusivity, excellent oxygen surface exchange coefficients, high oxygen transport properties, and higher electronic conductivity  value of 0.23 Ω·cm2 at 650°C  showing non-stoichiometric compositions in which samarium (Sm) elements were stepwise removed at the A/-site of a SmBaCo2O5+d layered perovskite by using solid state reaction. Significantly, the electrochemical properties of Sm1−xBaCo2O5+d oxide systems were also investigated to apply this material as cathode material for direct application to IT-SOFCs.The goal of this research is to investigate the phase synthesis characteristics of Sm1−xBaCo2O5+d  oxide systems having layered perovskite structure were synthesized using Solid State Reaction (SSR). The starting materials were Sm2O3 , BaCO3 , and Co3O4 ; an electronic balance (WBA-320) was use for accurate weighing to the third decimal place.SmIn this study, two calcinations were performed for more accurate synthesis of single phase. The weighed materials were mixed and calcined for 6 h at 1,000°C in air. After the first calcination into uniform and small particles, wet ball milling was performed at room temperature for about 1 day to make the composite powder. The powder after ball milling was again calcined at a rate of 5°C/min in air, and secondary calcination was performed at 1,100°C for 8 h. Resulting materials after the two calcinations were pulverized to form fine powders. The compositions and abbreviations are summarized in 0.9Gd0.1O2−d  powders were used as the electrolyte of symmetrical half cells (Sm1−xBaCo2O5+d/CGO91/Sm1−xBaCo2O5+d). After weighing 2.5 g of CGO91, it was put into a metal mold and compression molding was performed by applying a force of 2 × 103 kg/m2. After that, sintering was performed at 1,450°C for 6 h in air. Cathode ink made using a-terpieal, Butvar(Sigma Aldrich), and acetone with cathode powder; composite ink was also prepared by mixing electrolyte powder and cathode material at a weight ratio of 50:50. Both sides of the sintered electrolyte support were printed' with the prepared cathode ink, and the printed cell was sintered at 1,000°C for 1 h in the air condition. After the sintering, surface area of both sides of cathode was measured and found to be 0.785 cm2. To confirm the electrical conductivity characteristics of the composites as identified through XRD analysis, a sample was prepared and the electrical conductivity was measured. After making a bar-type sample with dimensions of ~2.8 × 5.5 × 23 mm through compression molding, sample was sintered at 1,100°C for 3 h in air.Ce1−xBaCo2O5+d  oxide systems were examined using a field-emission scanning electron microscope (FESEM/Hitachi SU5000). To measure the electrical conductivity, a bar-type sample was prepared and the electrical conductivity was measured through the DC 4-probe method. A Keithley 2400 Source meter was used as measuring device; after connecting the sample with Pt-wire, the electrical conductivity was measured by increasing the temperature in steps of 50°C in a temperature range of 50–900°C. Additionally, the current range was measured from 0 to 1A through steps of 0.05A, and the limit of voltage was set to 20V.To understand the crystal structure of the cathode powder synthesized by SSR method, X-ray diffraction analysis was used. The experimental equipment was an X-ray Diffractometer (Model: SmartLab/Rigaku), operated using a Cu Kα filter under conditions of 9 kW, 45 kV, and 200 mA. A range of 10–140° (2θ) was employed with steps of 0.02°. Microstructural properties of Sm1−xBaCo2O5+d  oxide systems. According to existing literature, SmBaCo2O5+d does not react with Ce0.9Gd0.1O2−d (CGO91), so there is no problem to use it with CGO91  of the synthesized composition was calculated using the impedance results obtained through electrochemical analysis. Using the impedance results with ohmic resistance removed, ASR was calculated according to the difference between the low intercept and high intercept of the real X-axis. Impedance was measured using a multi-channel electrochemical analyzer .1−xBaCo2O5+d  calcined at 1,100°C for 8 h to investigate the synthesis characteristics of these oxide systems with respect to the non-stoichiometric compositions. In addition, indicators for BaCoO2.6 phase (▼) and CoO  indicate that the Sm1−xBaCo2O5+d oxide systems were synthesized as a single phase from the stoichiometric SmBaCo2O5+d (SBCO) composition in which Sm was not removed and the non-stoichiometric composition in which 0.01, 0.02, 0.03, and 0.04 mol% of Sm  were partially removed. However, when Sm was removed at 0.05 mol% or more, BaCoO2.6 phase appeared . Especially, when Sm was removed 0.15 mol%, CoO phase was additionally identified (SBCO_0.85).1−xBaCo2O5+d  oxide systems. Overall particle size decreases from (A) to (C), which results in porous microstructures as the non-stoichiometry composition increases. Especially, the particular microstructural characteristics can be seen in 2.6. In other words, it can be thought that a small amount of secondary phase particles was generated on the cathode surface. For the SBCO_0.90 composition, which further increased the non-stoichiometry, as can be seen in 2.6 increased via the increase of the non-stoichiometry.2 at 600°C. These ASR values are closely related to the composition because the lowest ASR value was observed for the SBCO_0.90 composition. However, in the composition of SBCO_0.85 with an increased non-stoichiometric condition, the ASR value measured at 600°C was 0.179 Ω·cm2, which is relatively higher than the ASR values of SBCO_0.95 and SBCO_0.90 measured at 600°C. That is, from the non-stoichiometric compositions of Sm1−xBaCo2O5+d, the characteristics of the percolation composition showing the lowest ASR value in the chemical composition of Sm0.90BaCo2O5+d can be found. This tendency can also be confirmed in the layered perovskite composition of LnBaCo2O5+d in which Pr was substituted into the A/-site of the layered perovskite , PrBaCo2O5+d (PBCO) and P0.92BaCo2O5+d (PBCO_0.92) exhibit ASR values of about 0.097 and 0.081 Ω·cm2 at 700°C  of Sm0.85BaCo2O5+d increased slightly compared to that of Sm0.90BaCo2O5+d. When comparing the activation values of the Sm1−xBaCo2O5+d oxide systems synthesized in this experiment with those of recently reported cathode materials showing excellent electrochemical reaction, it can be determined that Sm1−xBaCo2O5+d oxide systems have relatively lower activation energy  is directly related with the charge transfer (R1) when oxygen ions move across the interface between the cathode and the electrolyte. The resistance from the middle frequency  range is attributed to the resistance (R2) that occurs when oxygen ions move inside the bulk of the cathode. The lower frequency  arc is ascribed to oxygen dissociation and bulk or surface oxygen diffusion process (R3). Therefore, ASR results were analyzed by subdividing them into R1, R2, and R3.In general, the impedance results of SOFC cathode materials can be classified by the frequency range  applied in this experiment showed the same results. For example, R2 of SBCO_1 was calculated and found to be 0.2162 Ω·cm2, which is about 72% of the total ASR; however, R1 (0.074 Ω·cm2) and R3 (0.0104 Ω·cm2) occupied only 28% of the total resistance. This trend does not change with increasing degree of non-stoichiometry of compositions and the R2 values of SBCO_0.95 and SBCO_0.90 are 0.1112 and 0.0847 Ω·cm2, with proportions of about 75 and 71% of the total resistance. Therefore, it can be determined that the RDS of Sm1−xBaCo2O5+d  oxide systems tested is R2, which is the resistance when oxygen ions move in the cathode bulk. In addition, in 1, R2, and R3 ASR values decreased as the non-stoichiometry compositions increased. Especially for the SBCO_0.95 composition, ASR decreases rapidly. The reason for the rapid decrease in ASR results for SBCO_0.95 is the effect of BaCoO2.6 phase, as described in the above XRD and SEM results. In the case of the SBCO_0.90 composition, which showed increased concentration of BaCoO2.6, the ASR value was lower than that of SBCO_0.95.As can be seen from 0.6Zr0.4O3−δ (BZC-BC) in nanocomposite form, advanced electrochemical properties were reported when a secondary phase of BaCo0.96Zr0.04O3−δ transformed into BaZr0.82Co0.18O3−δ main phase. For example, single-phase BaZr0.80Co0.20O3−δ (BZC2) showed a power density of 33 mW·cm−2 at 750°C. However, the BCZ-BC oxide system containing the secondary phase (BaCo0.96Zr0.04O3−δ) showed a value of about 1,530 mW·cm−2, an increase of 46 times compared to the case of using single phase at the same temperature; this implies that the composition BZC-BC including secondary phase exhibited excellent properties. In addition, in the case of multi-phase cathode materials mixed with various compositions containing Ba and Co based oxide, it was reported that BaCoO3−x generates a large number of oxygen vacancies that promote the oxygen reduction reaction (ORR) by assisting the movement of reduced oxygen ions  oxide systems measured at 600°C. ASR values decrease as the effect of degree of non-stoichiometry composition increased. However, in the SBCO_0.85 composition, the R2 value increased sharply and the total ASR increased. From these results, it can be determined that the CoO phase found in the composition of SBCO_0.85, shown in 2 at 700 and 750°C; it showed a slight difference of only 0.004 Ω·cm2 compared to the ASR value of SBCO_0.90. In the general case of composite cathodes, ASRs measured from the composite cathode tend to decrease. However, a large amount of CGO91 could rather interfere with the movement of oxygen ions in the bulk and on the surface of the cathode, and so composite did not significantly affect the reduction of resistance of the composite cathode applied in this experiment.Additionally, electrochemical analysis was carried out using composite cathodes prepared by mixing CGO91 powder and SBCO_0.90. For the fabrication of the composite cathodes, the synthesized SBCO_0.90 was mixed with CGO91 at a weight ratio of 1:1. The summarized ASR results of the composite cathodes are given in /-site of the layered perovskite changes. However, the thermal expansion problem of the SOFC cathode operating at high temperature range can be solved when the composite cathode concept is applied. In other words, thermal stability can be secured by lowering the thermal expansion coefficient (TEC). Co-based materials have high TEC of 20–25 × 10−6K−1 when SOFC is operated in high temperature operating temperature range; however, CGO91 has a relatively low TEC of 12–13.1 × 10−6K−1  applied in this study were 0.083, 0.095, 0.088, 0.074, 0.075, 0.041, 0.035, and 0.045 Ω·cm2, respectively, at 700°C. All compositions can be sufficiently used as cathode materials at 700°C because these ASR results are all 0.15 Ω·Cm2 or less  oxide systems are summarized in 2O5+d , SBCO showed a maximum electrical conductivity value (570 S/cm) in the range of 200–250°C; then, the electrical conductivity started to decrease from 250°C, indicating Metal Insulator Transition (MIT) behavior  oxide systems, are summarized in x ≤ 0.05). That is, the electrical conductivity increases when a relatively small amount of Sm is removed in the range of Sm1−xBaCo2O5+d . Especially the SBCO_0.95 composition exhibited electrical conductivity values of about 917 and 732 S/cm at 500 and 600°C and showed the highest electrical conductivity values among the measured compositions. However, when the non-stoichiometric composition increased from x = 0.05 in the Sm1−xBaCo2O5+d  oxide systems, the electrical conductivity value decreased. It can be concluded that this is because a large amount of BaCoO2.6 phase activates the conductivity of ions but inhibits the transfer of charge carriers  oxide systems. In addition, the conductivity characteristics found in the non-stoichiometric compositions exceeding x = 0.04 can be determined by the influence of the secondary phase. However, the detailed influence of the BaCoO2.6 composition for electrical conductivity in SmBaCo2O5+d oxide systems is not discussed now and will be reported later.Therefore, it can be seen that a percolation phenomenon that affects the electrical conductivity according to the non-stoichiometry is found in the Sm/-site increases, the electrical conductivity behavior conditions in this experiment changed. In addition, the lowest conductivity value in the IT range was 169 S/cm, a value exceeding 100 S/cm. This is a value that exceeds the standard that must be satisfied for material to be used as cathode for SOFC; therefore, all compositions can be suitable for use as cathode material for IT-SOFC   oxide systems synthesized by SSR, single phase composition was confirmed for Sm0.96BaCo2O5+d (SBCO_0.96). However, secondary phase of BaCoO2.6 was found for compositions of Sm0.95BaCo2O5+d, (SBCO_0.95) in Sm1−xBaCo2O5+d oxide systems. Additional phase of CoO appeared for the SBCO_0.85 composition.The electrochemical properties of materials having non-stoichiometric compositions in which Sm of the A2 at 650°C. The Sm0.90BaCo2O5+d (SBCO_0.90) showed the lowest ASR characteristics, with values of 0.119, 0.061, and 0.035 Ω·cm2 at 600, 650, and 700°C. Additionally, the ASRs of the composite cathodes prepared by mixing with an electrolyte using CGO91 and SBCO_0.90 were 0.139 and 0.056 Ω·cm2 at 600 and 650°C. All compositions exhibit electrical conductivity values of more than 100 S/cm over the temperature range (500–750°C) for IT-SOFC. It was also confirmed that the behavior conditions of MIT change as the non-stoichiometry composition of the A/-site increases. SBCO_0.90 composition, which had the best electrochemical properties, showed the maximum (568 S/cm) and minimum (387 S/cm) electrical conductivity values in the temperature range of 500–900°C.All of the synthesized compositions showed excellent electrochemical properties, with ASR values <0.15 Ω·cmThe original contributions presented in the study are included in the article/CK was responsible for conducting all experiments, analyzing experimental data, and writing papers. SW teaches experimental methods and informs questions about results. KS, S-WB, HK, and WC helped to write the manuscript. KS provided additional help in thesis publication. Finally, JK coordinated the overall experimental design, direction, and schedule. In addition, he reviewed the paper and gave direction. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Using the non-hydrolysable label, Dil, TNFα could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), or apolipoprotein (apoE) depletion. Moreover, the TNFα-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. The effects of TNFα included an LDLR cell surface increase of 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNFα-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNFα on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-κB, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on Transwell inserts, TNFα did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNFα promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation.Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNFα) in modulating the low-density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNFα promoted an up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased  TNFα was originally described as a tumor-selective hemorrhagic factor ,2. HowevThe pro-atherogenic arm of TNFα is in part due to its ability to enhance monocyte entry into the intima through induction of adhesion molecules on arterial endothelial cells. In atherosclerotic lesions, macrophages  and smooThe serum concentration of TNFα rises with atherosclerotic severity . Thus, iUnlike macrophages ,46 and sIn this report, the effect of TNFα on the pro-atherogenic lipoprotein, LDL ,58 is in125I-LDL binding was performed. 125I-LDL surface releasable 125I-LDL at 4 °C, after the cells were pre-treated at 37 °C. The amount released increased in the presence of 20 folds unlabeled LDL. This indicates that the 125I-LDL bound to some native LDL receptor. Further, cell-associated 125I-LDL was also higher on TNFα-treated pHAECs , are stably fixed within LDL. To evaluate the ability of pHAECs to retain LDL hydrolysable [3H]CE or the non-hydrolysable Dil, the cells were treated with increasing concentration of TNFα in the presence of [3H]CE-LDL or Dil-LDL. As shown in 3H-cholesterol accumulation derived from [3H]CE-LDL was increased by about 2 folds with increasing TNFα concentration. On the other hand, Dil accumulation increased by about 50 folds  is the major protein in LDL . HoweverHaving found evidence that TNFα promotes cholesterol accumulation in pHAECs through the endocytosis of native LDL, I next investigated what members of the LDLR family are responsible. As can be seen in HDL3 critically depends on non-LDLR mechanisms for uptake, especially in the absence of apoE . Thus, tHaving determined that LDLR is responsible for TNFα-induced LDL accumulation in pHAECs, I next tested whether the total and surface LDLR is changed by TNFα. As can be seen in To evaluate the consequence of the increase in cell surface LDLR induced by TNFα on the association and entry of LDLR antibody, immunofluorescence studies were performed. As can be seen in TNFα signaling cascade involves multiple mediators, among which are the transcription factor, NF-κB, and apoptosis signal-regulating kinase 1 (ASK1), an upstream mediator of JNK and p38 , that fa125I-LDL detected in the BL medium was either non-intact  of getting rid of LDL-derived cholesterol. TNFα induced about 25 folds intracellular Dil-LDL compared to -inulin transport. The cell culture conditions were as indicated above. Arterial endothelial cells have been reported to form their own basement membrane with continued culture CE and/or 0.5 mg Dil in 10 µL DMSO aliquots under sterile conditions. After covering with foil, the mixture was incubated at 37 °C for 21 h. The lipoproteins were repurified as described above, after adjusting to their respective densities with solid KBr. Combined Dil, [3H]CE-LDL radioactivity was 8.1 dpm/µg protein.The generation of Dil-labeled lipoproteins was obtained essentially as previously described , with mi125I for 10 min at room temperature in glass vials. The transformation was stopped with 50 mM each of unlabeled sodium iodide and sulfite, and 100 µM BHT in DMSO (0.01%). The labeled LDL was washed with desalting columns, then passed through centrifugal filters, 3 kD MWCO, against dialysis buffer.About 2 mg LDL protein in PBS and 10 iodination beads were incubated with Na125I-LDL was added to 15 µg/mL for 1 h at 37 °C. The cells were then washed two times with PBS at room temperature, then chilled on ice. Afterwards, serum-free medium at 4 °C with 0 (buffer) or 300 µg/mL LDL was added. Following additional incubation at 4 °C for 1 h, the radioactivity released to the medium was taken as surface-releasable 125I-LDL.Confluent pHAECs on plastic dishes were cultured with 0 or 5 ng/mL TNFα in 15% serum for 48 h. The treatment continued with serum-free medium in the continued presence of TNFα for 3 h to deplete surface-bound LDL. Subsequently, 4 in PBS under sterile conditions. After adding BHT in DMSO (0.005%) to 1 µM and Na2EDTA to 10 mM, the mixture was dialyzed against dialysis buffer (under Lipoprotein purification), and finally filtered through 0.2 µm membrane under sterile conditions.LDL was oxidized essentially as described . Briefly2EDTA, respectively. After heating at 55 °C for 1 h, the mixture was centrifuged at 16,000× g/30 s. Fluorescence  reading in the supernatant was determined. Malonaldehyde bis- was used as a malondialdehyde (MDA) precursor for determining malondialdehyde standards. Care should be taken when performing this procedure, as volatile products are generated at 100 °C. Standards up to 5 nmol/μL were used. oxLDL was measured at 54 nmol MDA equivalents/mg protein.A 2-thiobarbituric assay to estimate the extent of LDL oxidation was determined essentially as previously described , with soNormal mouse IgG agarose or apoE3 agarose beads were washed 4× with dialysis buffer under sterile conditions. Subsequently, Dil-HDL3 or Dil-LDL was added to the beads in dialysis buffer. Following 24 h incubation at 4 °C, the unbound lipoproteins were sterile filtered through 0.2 µm membranes.Serum media from confluent pHAECs were replaced with 400 U/mL sodium heparin in mHBSS on ice for 5 min, washed 2× with the the same buffer, then 1× with ice cold mHBSS. After 1× wash with ice cold 1× PBS, the cells were treated with EZ Link Sulfo NHS Biotin in 1× PBS for 30 min at 4 °C. Unreacted biotin reagent was quenched with 100 mM glycine in PBS at 4 °C for 5 min. Following 1× wash with ice cold mHBSS, the cells were lysed on the plates with lysis buffer as indicated above under Western blotting on ice for 30 min, followed by sonication at 2 setting for 10 s on ice. Aliquots of the supernatant was mixed with 1 volume of 2× urea loading buffer without heating. The rest of the supernatant was mixed with strepavidin mag sepharose . After 10× dilution with the incubation buffer containing 0.5% each of protease inhibitor and phosphate inhibitor cocktails, the suspension was swirled for 12 h at 4 °C. The beads were washed 4× with precipitation buffer, then mHBSS at 4 °C. After 1× urea loading buffer (1:1 volume of lysis buffer and 2× urea loading buffer), with 1% protease and phosphatase inhibitors, respectively, the samples were heated at 95 °C for 5 min to solubilize the bound biotinylated proteins.125I-LDL was separated from non-intact LDL using dextran sulfate, Mn2+ procedure essentially as previously described [g/5 min at 4 °C. The pellet was redissolved with 20 mg/mL dextran sulfate before scintillation counting.Intact escribed . To the Confluent pHAECs in serum medium were incubated with 0 or 100 ng/mL TNFα for 24 h. Subsequently, the media were replaced with serum-free medium containing 10 µg/mL normal goat IgG or LDLR Ab (R&D Systems) in the continued presence of 0 or 100 ng/mL TNFα for 0, 5, 30, or 120 min at 37 °C. Afterwards, the cells were chilled on ice and further incubated at 4 °C for 1 h. Then the cells were washed 2× with ice-cold mHBSS, fixed with ice-cold methanol at −20 °C for 10 min, washed once with mHBSS at room temperature, then blocked with 50% human serum in mHBSS, 0.1% sodium azide (blocking buffer) for 1 h at room temperature. Afterwards, it was replaced with 4 µg/mL red antigoat antibody in blocking buffer. Following 30 min incubation at room temperature, the medium was replaced with mHBSS, washed 3× with blocking buffer (10 min each at room temperature), fixed with 4% paraformaldehyde in PBS, followed by fluorescence microscopy.Fluorescence microscopy was performed using Keyence phase-contrast fluorescence microscope. Images were analyzed using CellProfiler . Confocan = number of independent experiments performed on separate days. Most experiments were performed with an average of 3 or greater replicates. Analysis of variance, followed by Tukey post-hoc testing was done by using statpages.info website.Data are reported as averages ± standard deviation.


Staphylococcus aureus is a bacterium carried by 30–70% of the general population. It has been shown to produce functional amyloids, called phenol soluble modulins (PSMαs). Here, we studied the kinetics of α-syn aggregation under quiescent conditions in the presence or absence of four different PSMα peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure α-syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSMα peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSMα peptides displayed distinct effects on the kinetics of α-syn aggregation. Kinetic analyses of the data suggest that all four peptides catalyze α-syn aggregation through heterogeneous primary nucleation. The immunogold electron microscopic analyses showed that the aggregates were fibrillar and composed of α-syn. In addition of the co-aggregated materials to a cell model expressing the A53T α-syn variant fused to GFP was found to catalyze α-syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.Aggregated α-synuclein (α-syn) is the main constituent of Lewy bodies, which are a pathological hallmark of Parkinson’s disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric α-syn. Braak’s seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role of the microbiome in the initiation of pathology of PD.  Aggregated alpha-synuclein (α-syn) is a pathological hallmark of Parkinson’s disease (PD), multiple system atrophy and Lewy body dementia. The majority of synucleinopathies are of idiopathic nature and the environment is thought to play a causative role. In Braak’s landmark studies, α-syn inclusions were identified in the dorsal motor nucleus of the vagus at very early stages of PD. It was hypothesized that PD pathology could be initiated peripherally, either in the gastrointestinal or the olfactory system, where it would spread to the brain ,2. SeverE. coli) [Staphylococcus aureus.Both the olfactory cavities and the gastrointestinal tract harbor large numbers of microorganisms that are known to prime and modulate the immune system as well as directly modulate α-syn aggregation. Both LPS and bacterial chaperones have been shown to affect the kinetics of aggregation and toxicity of α-syn ,11,12. CE. coli) . SeveralE. coli) . One sucS. aureus is the bacterium responsible for the majority of skin and soft-tissue infections [S. aureus produces amyloid proteins called phenol soluble modulins (PSMs), which participate in biofilm formation. The α type of the PSMs comprise four different hydrophobic 20–25 amino-acid long peptides, often formylated at the N-terminal. The pH modulation of their aggregation mechanisms was recently reported (41) with PSMα1 found to aggregate through a mechanism dominated by secondary nucleation at neutral pH. Other factors also modulate PSM fibrillation, such as the sulfated polysaccharide heparin [S. aureus is stringently regulated and is associated with virulence. PSMα peptides are secreted by S. aureus at high concentrations, with high nanomolar concentrations acting as chemoattractants for neutrophils and micromolar concentrations causing cytolysis [S. aureus phagocytosis by neutrophils promoting phagosome lysis and bacterial escape [fections  as well fections ,19,20,21 heparin . The proytolysis ,24. Theil escape . PSMα pel escape .S. aureus have been shown to directly activate sensory neurons of the skin and increase firing in cultured dorsal root ganglia (DRG) neurons by disrupting membranes and forming pores, allowing cation influx and depolarization [PSMα peptides produced by rization ,29. In crization . Nociceprization  and Braarization . Lewy borization  and neurrization . PSMα peIn the present study, we hypothesized that these PSMα peptides could modulate α-syn aggregation. We monitored the aggregation kinetics of α-syn in vitro upon the addition of different PSMαs at a range of concentrations. The PSMαs induced rapid aggregation of monomeric α-syn into fibrils, which could induce seeding and phosphorylation in human embryonic kidney (HEK) cells expressing the A53T variant of α-syn fused to green fluorescent protein (GFP).We investigated whether the PSMαs could induce α-syn aggregation at physiologically relevant concentrations, i.e., in the 1–100 µM range. To study the effects on the kinetics under conditions where secondary nucleation strongly dominates over primary nucleation (pH 5.5 and low ionic strength), or conditions where secondary nucleation is less dominant (pH 7.5 and moderate ionic strength), the experiments were repeated in two different buffers. The PSMα peptides may interact with α-syn in the cytoplasm of sensory neurons or in the extracellular space, which further motivates the inclusion of the neutral pH condition.We first monitored the aggregation kinetics of 25 μM α-syn under quiescent conditions in the presence or absence of different PSMα peptides in 10 mM Tris, 50 mM NaCl, pH 7.6. The addition of each of the four PSMα peptides induced a rapid increase in ThT fluorescence, which was not observed for α-syn incubated on its own or with DMSO at the same concentrations as used with the peptides . All fouIn mildly acidic buffer, 10 mM MES, pH 5.5 with no additional salt, all four PSMα peptides significantly shortened the lag phase for α-syn aggregation to less than 20 h, and the ThT curves plateaued before 100 h even at low μM PSMα concentrations . At the To verify the catalytic effect of the peptides on α-syn aggregation, additional experiments were performed at higher α-syn monomer concentrations, at higher pH (7.4) and higher salt concentration (150 mM) in the presence of polystyrene surfaces, known to catalyze primary nucleation of α-syn. The PSMα peptides were again shown to accelerate the increase in ThT fluorescence .v/v, equal to 1.25 M) used in the previous experiments. The effects of the PSMα2 concentrations with additional DMSO on α-syn were similarly examined. The kinetic curves obtained with this PSMα2 dilutions series show similar trends as the PSMα2 dilutions without supplemented DMSO (varying DMSO concentrations)  .To ensure that the effects on α-syn aggregation observed were due to the peptides, and not to DMSO or impurities, one aliquot of PSMα2 was purified with size exclusion chromatography before the kinetics experiments. Different fractions of the central peak were added to freshly purified α-syn monomer in 10 mM Tris, 50 mM NaCl, pH 7.6 and compared to PSMα2 dissolved in DMSO. The results indicate similar effects of the purified PSMα2 fractions (without any DMSO) compared to the original DMSO-dissolved peptide .Next, we wanted to elucidate the mechanisms underlying the PSMα-catalyzed α-syn aggregation. As a first step, we examined the effects of the different PSMα concentrations on the rate of α-syn aggregation. The time at which ThT intensity reached half the intensity of the plateau was calculated for each concentration of the four different PSMα peptides and plotted against the concentration of the peptides . At pH 7When the interactions between α-syn and the different PSMα peptides were probed by peptide arrays, α-syn did not show any signal above cutoff for the arrays with PSMα1 or PSMα4 peptides. However, α-syn exhibited a strong signal, indicative of high affinity binding to the PSMα2 sequences IIAGIIKFIK, GIIKFIKGLI, and the PSMα3 sequence FVAKLFKFFK .Mass spectrometry was used to gain insight into whether the PSMα peptides formed low molecular weight heteromolecular aggregates with α-syn. Different PSMα-α-syn samples after reaching the plateau phase in the aggregation process were separated by SDS PAGE and the <10, 37, and 120 kDa bands were excised, subjected to in-gel digestion and analyzed with mass spectrometry. PSMα sequences were detected only in the lowest Mw bands (<10 kDa). All other bands showed the presence of α-syn but not PSMα . We thus+k2), while the combined rate constant for primary pathway (k+kn) was allowed to take different values at the different PSMα concentrations  with the following sequences:PSMα1: Formyl-MGIIAGIIKVIKSLIEQFTGKPSMα2: Formyl-MGIIAGIIKFIKGLIEKFTGKPSMα3: Formyl-MEFVAKLFKFFKDLLGKFLGNNPSMα4: Formyl-MAIVGTIIKIIKAIIDIFAKFor the experiments described in The lyophilized peptides were stored at −80 °C and used without further purification in all experiments except one. For one experiment, PSMα2 was further purified using size exclusion chromatography. The peptide was dissolved at 1.25 mg/mL in 6 M GuHCl, 10 mM MES and purified using a Superdex Peptide 10/300 GL column in 10 mM Tris, 50 mM NaCl, pH 7.6, with absorbance measurements at 214 nm and 256 nm.−1 cm−1 was used to calculate the concentration. Purified α-syn monomer was kept on ice to prevent aggregation.In order to obtain reproducible data, monomeric α-syn was isolated immediately prior to each experiment. Purified α-syn was lyophilized, dissolved in 6 M GuHCl,10 mM MES, pH 5.5 and purified using size exclusion chromatography into 10 mM MES, pH 5.5 or 10 mM Tris, 50 mM NaCl, pH 7.6 at the start of every experiment. Buffer solutions were filtered and degassed prior to each run. The purification was monitored by UV absorbance at 280 nm and only the central monomer peak collected. The absorbance of the collected α-syn fraction was measured at 280 nm and an extinction coefficient of 5960 Mv/v. Complementary α-syn aggregation experiments without PSMα peptides were performed at the same DMSO concentrations.The PSMα peptides were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 2 mg/mL and then diluted in buffer. The final DMSO concentration ranged from 0.1 to 8.9% 3. Replicates without ThT were also prepared for additions to cell cultures. For the first round of experiments, the α-syn concentration was kept constant at 25 μM and PSMαs added at 5 different final concentrations: 1, 3, 9, 26 and 78 μM. Plates were incubated under quiescent conditions at 37 °C and the fluorescence was measured for up to 400 h (excitation filter 440 nm and emission filter 480 nm). Each experiment was repeated 1–3 times. These experiments were performed in 10 mM Tris, 50 mM NaCl, 20 μM ThT, 0.01% NaN3, pH 7.6 or in 10 mM MES, 20 μM ThT, 0.01% NaN3, pH 5.5. Data from one experiment, representative of all, are shown in the results section and To follow the aggregation process, samples were aliquoted into 96-well black Corning polystyrene half-area microtiter plates with PEGylated surface (Corning 3881) in the presence of 20 μM ThT and 0.01% NaN3.For the second round of experiments, the PSMα concentrations were kept constant at 8.6 μM and α-syn added at concentrations of 1, 3, 9, 26, 78 and 116 μM. These experiments were performed in 10 mM Tris, 50 mM NaCl, pH 7.6 in the presence of 20 μM ThT and 0.01% NaNv/v). In these plates, the aggregation of α-syn was triggered by heterogeneous primary nucleation at the polystyrene surface [To verify the findings, kinetic experiments were also performed at different solution and surface conditions. For these experiments, described in  surface . The ThTThe data sets from each experiment were analyzed using Amylofit . The ThTTM overnight for optimal band detection. Gel bands of interest were excised and subjected to in-gel digestion. Gel pieces (1 × 1 mm2) were washed twice in 50 mM ammonium bicarbonate (NH4HCO3)/50% acetonitrile (ACN), followed by dehydrated in 100% ACN. Digestion was performed by adding 50 mM NH4HCO3 with 12 ng/µL sequencing-grade modified trypsin , incubation on ice for 4 h before overnight incubation at 37 °C. The next day trifluoroacetic acid (TFA) was added to a final concentration of 0.5% and the peptide containing solution above the gel pieces was withdrawn and used for further analysis. Mass spectrometry analysis was performed in reflector positive mode on an Autoflex Speed MALDI TOF/TOF mass spectrometer . All peptide mass spectra were externally calibrated using Bruker Peptide Calibration Standard II.The samples were analyzed by SDS-PAGE using Novex 10–20% Tricine precast gels and Tris/Tricine SDS Running buffer . 20 µL of each sample were mixed with 5 µL of gel loading buffer . 10 µL of each mixture were loaded, and the gel was run at 70 V for 15 min, followed by 120 V for 1 h. The gel was stained with InstantBlueTo probe interactions between α-syn and the PSMαs, 48 different 10-residue peptides corresponding to different parts of the PSM sequences were immobilized on a microarray chip and incubated with Alexa-fluor488-labelled α-syn. In this array, each new peptide constituted a 10-residue window of a given PSM sequence shifted forward by 1 residue compared to the preceding peptide, giving a 9-residue overlap. w/v) skim milk in PBS with 0.1 % Tween-20 incubated overnight at 4 °C and washed three times with 25mM TBS with 0.1% Tween-20 (TSB-T). Subsequently it was incubated with Alexa-fluor488-labelled α-syn (diluted to 0.5 mg/mL in PBS) for 4 h at room temperature. The microarray was washed 3 times with TSB-T, air-dried in the dark and scanned using a Typhoon Trio scanner . Dot intensities in the scanned image were quantified using ImageJ. As part of this procedure, the microarray was first blocked in a solution containing 3%  to prevent nonspecific binding and the specimens then incubated with 1:200 syn211 (sc-12767) for 1 h. Samples were again washed in distilled H2O, incubated with 1:20 10 nm gold-anti-mouse antibody , washed, fixed first with glutaraldehyde (Ted Pella 18427) and then uranyl acetate . The samples then were examined under a transmission electron microscope (TEM) .Samples were diluted 1:500 in sterile phosphate buffered saline  and 5 µL diluted sample added onto carbon-coated grids (made in-house). After washing in distilled HHEK 293T cells were stably transduced with the A53T mutant of α-syn fused to GFP and were maintained in DMEM GlutaMAX  supplemented with 10% fetal bovine serum (Gibco 10270-106) and 1% penicillin-streptomycin (Gibco 15140-122) and split every three days.2.For live-imaging experiments, cells were split 1:20,000 into collagen G-coated  black, clear bottom 96-well plates. Samples of α-syn and PSMα incubated separately or together were sonicated at 100% amplitude for 3 min at 50% duty cycle . 10 µL of each sample was added in 4 or 3 replicates into 100 µL medium. The plates were imaged for 48h with 1h intervals using a Nikon Ti-E microscope with 95% humidity 5% COAfter 48 h, the cells were fixed with phosphate-buffered paraformaldehyde pH 7.4 by slow addition into the cell medium to a final concentration of 2% for 20 min. The cells were then washed and kept at 4 °C. Cells were then permeabilized and blocked with 5% normal donkey serum (NDS) 0.25% Tween 20 in PBS. Primary antibody pS129 α-syn  was added at 1:1000 overnight and the plates kept at 4 °C. The cells were washed and incubated with 1:600 Cy3 conjugated anti-rabbit antibody (Jackson Immunolabs 711-165-152), washed and then imaged again. For each well, 25 (5 × 5 square) images were acquired with a 20× objective.Quantification of the number of GFP-aggregates and pS129 α-syn inclusions was performed using Cell Profiler. In brief, cell nuclei were identified as primary objects by segmentation by diameter sizes and counted in the GFP channel with a global thresholding strategy. In the GFP channel and the Cy-3 channel, aggregates and phosphorylated aggregates, respectively, were identified as secondary objects by segmentation by diameter size and counted using an Otsu adaptive thresholding strategy, which separates foreground from background, optimally used when percentage of the image covered by foreground varies substantially from image to image. The number of aggregates and cells were quantified per well. The number of aggregates and phosphorylated aggregates per 100 cells were determined per well and averages from 4 replicates were plotted for each condition.


P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRβ′s tyrosine kinase activity and directly promotes IRβ interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRβ) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol Obesity, which has reached alarming levels worldwide, is associated with an increased risk of metabolic abnormalities including type 2 diabetes (T2D). T2D accounts for more than 90% of diabetes cases, while type 1 diabetes (T1D) accounts for about 5 to 10% of diabetes. Insulin deficiency in T1D as well as insulin resistance and insufficient insulin secretion from impaired pancreatic β cells in T2D result in increased glucose production in the liver, which is the major cause of hyperglycemia in diabetic patients , 4. Seve972 motif that helps the recognition and binding of the phosphotyrosine binding (PTB) domain in the insulin receptor substrate (IRS) proteins to the IR (P3 (PIP3) in cellular membranes and subsequent activation of downstream mediator AKT (Insulin binds to the extracellular α-subunit of the insulin receptor (IR), causing transphosphorylation of integral membrane β-subunit of IR by its intrinsic tyrosine kinase activity , 8. Phoso the IR . This seo the IR , adds ano the IR . IRS cano the IR , 14, 15.o the IR , 17, wheo the IR . The phoator AKT , 20, 21.ator AKT , suggestObesity and metabolic disorders are associated with increased blood bacterial lipopolysaccharide (LPS) level and its initiated low-grade of inflammation , and actA). In genetically obese db/db mice, there are drastic increases in total, cytosolic, and nuclear P300 protein levels in the liver compared with age-matched heterozygous lean mice (B–E). Consistently, immunostained liver sections by validated P300-specific antibody . Next, we examined P300 protein levels in two normal human liver samples and three obese patients and found that obese patients had significantly elevated liver P300 protein levels and large amounts of P300 in the cytoplasm of hepatocytes, contrasting with the mostly nuclear localization of this protein in normal humans (G and H). In agreement with our previous report (I and J) and in Hepa1-6 cells (A and B). In HFD-fed mice, treatment with A-485 for 2 weeks improved the hyperglycemia .Our previous report showed that high-fat-diet (HFD) feeding led to increased P300 protein levels in the liver . We valiFig. 1antibody  showed is report , inhibit-6 cells , A and BA and B). To test the effects of P300 acetyltransferase activity on insulin signaling, we treated Hepa1-6 cells with P300 acetyltransferase-specific inhibitor C646 and control inactive compound C37 (A and B), along with their mobility shift that may be caused by their tyrosine phosphorylation (C and D). Treatment with C646 significantly augmented AKT and GSK phosphorylation (C and D). However, the tyrosine phosphorylation at Y972 and total tyrosine phosphorylation of IRβ remained unchanged after treatment with C646 , indicating that C646-mediated activation of insulin signaling is not through augmenting the binding of IRS1 and 2 to the NPE(p)Y972 motif in IRβ . In both cytosolic and membrane fractions, C646 treatment increased the tyrosine phosphorylation levels in positions that correspond to the IRS1 and 2 proteins in the immunoblot (highlighted with the green square) (E). Of particular interest, C646 treatment significantly increased membrane-located IRS1 and 2  (F–H). C646 treatment caused the mobility shift of IRS1 and 2 in both cytosolic and membrane fractions, suggesting that the tyrosine residues of IRS1 and 2 were phosphorylated in both cellular compartments. To further prove that C646 can facilitate the membrane translocation of IRS1 and 2, primary hepatocytes from HFD-fed mice were prepared and subjected to serum starvation for 1 h before the addition of inhibitor C646. Indeed, C646 treatment increased the membrane located IRS1 and 2 in the absence of insulin treatment (I and J and H and I).To determine how C646 activates cellular insulin signaling in the absence of insulin, we prepared cytosolic and membrane fractions from Hepa1-6 cells treated with control inactive compound C37, or inhibitor C646, in the absence of both insulin and FBS treatment because the membrane translocation of IRS1 and 2 is required for the activation of insulin signaling  E. Of par%, IRS2) , F–H. C610 GC/IRS/mouse). The protein levels of expressed wild-type IRS1 and 2, and their mutant proteins were comparable with their corresponding endogenous protein levels in the liver of HFD-fed mice. At 19 days after the viral injections, liver tissues were collected. There were significantly increased liver AKT and GSK phosphorylation in mice with the expression of mutated KR proteins of IRS1 and 2 compared with mice with the expression of wild-type IRS1 and 2 proteins (A and B). Additionally, there were significantly higher levels of membrane located IRS1 and 2 proteins along with reduction of cytosolic IRS1 and 2 proteins in the liver of mice with the expression of mutated KR proteins of IRS1 and 2 (C and D). These results verified that acetylation of IRS1 and 2 has a negative impact on their membrane translocation.We found previously that seven lysine residues in IRS1 and 15 lysine residues in IRS2 can be acetylated and generated adenoviral expression vectors, in which these acetylated lysine residues in IRS1 and 2 were substituted with arginine, as mimics of nonacetylated lysine (KR mutants) . To examproteins , A and BS1 and 2 , C and DA–H and E and F). To test whether IR is required for C646 stimulated membrane translocation of IRS1 and 2, we generated liver-specific IR knockout (L-IRKO) mice by injection of AAV-TBG-Cre to HFD-fed floxed IR mice (A) and treated these mice with inhibitor C646 (20 nmol/g/day) via intraperitoneal injection for 2 weeks (E); in contrast, C646 treatment facilitated IRS1 and 2 membrane localization (F). In cytosolic and membrane fractions prepared from these L-IRKO mice (B), C646 treatment significantly increased the protein levels of both IRS1 and 2 in the membrane fractions, while significantly reducing protein levels of both IRS1 and 2 in the cytosolic fractions compared with their protein levels of vehicle-treated L-IRKO mice (G–J). These results indicate that IR is not required for C646 stimulated membrane translocation of IRS1 and 2. However, C646 was unable to improve insulin signaling in the liver of mice without IR (data not shown).C646 treatment activated insulin signaling and increased membrane located IRS1 and 2, while the tyrosine phosphorylation at Y972 and total tyrosine phosphorylation of IRβ remained unchanged A–H and , E and F IR mice A and tre 2 weeks . In the  2 weeks E; in conlization F. In cytRKO mice B, C646 tRKO mice , G–J. ThE–J), we reasoned that IRS acetylation may have a negative impact on IRS binding to phosphatidylinositolP2 (PIP2), which is also the substrate of immediate downstream mediator of PI3K. To test whether IRS acetylation can reduce their bindings to PIP2 in the membrane as a mechanism of the impairment of insulin signaling by P300, we conducted in vitro binding assays using acetylated IRS1 and 2. First, we used P300 to acetylate purified IRS1 and 2 proteins (K) in the presence or absence of acetyl-CoA (groups without the addition of acetyl-CoA served as controls) (L and N). Second, specific antibodies against IRS1 and 2 were used to immunoprecipitate acetylated or nonacetylated IRS1 and 2 to remove P300. Then, PIP2-fluorescein was added to the immunoprecipitates, and IRS-bound PIP2 was determined. We found that IRS acetylation by P300 reduced ∼70% bindings of PIP2 (M and O). Furthermore, the substitution of all identified acetylated lysine residues with arginine (pan KR mutation) in IRS1 or 2 abolished P300’s negative effect on the binding of PIP2 to IRS1 and 2 protein (Q–T). These results suggest that IRS acetylation may inhibit their membrane translocation through reduction of binding to PIP2. Since the acetylation of IRS1 and 2 could not affect their tyrosine phosphorylation by IRβ (C and D); therefore, P300 impairment of insulin signaling is mainly through the prevention of the membrane translocation of IRS1 and 2.Membrane translocation and binding to inositol compounds in the membrane can affect IRS′ function. PH domain in IRS can recognize and bind to membrane phospholipids , 14, 15.proteins K in the ontrols) , L and N of PIP2 , M and O protein , Q–T. Thn by IRβ , C and D972 motif that helps the recognition and binding of the PTB domain of IRS to the IR (A and B and A and B). However, in Hepa1-6 cells, C646 treatment did not increase the tyrosine phosphorylation at Y972 and total tyrosine phosphorylation of IRβ in the absence of insulin (A and B and E and F), but C646 treatment drastically increased the binding of IRS1 and 2 to IRβ (C). We reasoned that C646 may bind directly to IRβ, or IRS1 and 2, therefore, facilitating the interaction of IRβ with IRS1 and 2. To test this notion, we conducted Surface Plasmon Resonance (SPR) to determine whether C646 can bind to IRS1 and 2 or IRβ. We found that C646 can bind directly to unphosphorylated IRβ, but not to IRS1 and 2 , and C646 binds to IRβ with relatively high affinity (KD = 4.34 μM). Due to the fact that C646 can bind directly to unphosphorylated IRβ, we tested whether C646 has any effect on the binding of unphosphorylated IRβ with IRS1 or 2 using SPR. Strikingly, unphosphorylated IRβ can bind to either IRS1 or IRS2 with extremely high affinity  in the presence of C646 at nM concentrations (E and F). These data are consistent with the findings that C646 treatment drastically increased the association of IRS1 and 2 with unphosphorylated IRβ in Hepa1-6 cells . Thus, in the absence of tyrosine phosphorylation of IRβ by insulin, it is the C646 that directly promotes IRβ bindings to IRS.The tyrosine phosphorylation of IRβ at Y972 generates the NPE(p)Yo the IR . We vali2 to IRβ C. We reatrations , E and Fin vitro phosphorylation assays, in which IRβ and IRS1 or IRS2 were incubated with different concentrations of C646, followed by addition of ATP to initiate the phosphorylation of IRS. We found that C646 significantly increased the tyrosine phosphorylation of both IRS1 and 2 by IRβ (A–D). Having seen these data, we asked whether C646 can affect the enzymatic activity of IRβ. To test this hypothesis, we incubated the insulin receptor with different concentrations of C646, then determined its kinase activity on the phosphorylation of tyrosine residue in peptide substrate by measuring the production of ADP from ATP using InsR kinase assay. C646 did increase the kinase activity of insulin receptor by 50% at 100 nM concentration (E). The above data show that C646 not only promotes the binding of IRβ to IRS, but also activates the enzymatic activity of IRβ, leading to drastically increased bindings of IRβ to IRS1 and 2 (C) and tyrosine phosphorylation of IRS1 and 2 (A and B).To determine whether C646 promoting the binding of IRβ to IRS has any effect on the tyrosine phosphorylation of IRS by IRβ, we conducted 2 by IRβ , A–D. Hantration E. The abS1 and 2 C and tyrS1 and 2 , A and BF and G), suggesting that C646 cannot affect the transphosphorylation of IRβ. These results are consistent with the findings that C646 treatment could not change the tyrosine phosphorylation at Y972 and total tyrosine phosphorylation of IRβ (A and B and E and F). Another potent CBP and P300’s acetyltransferase inhibitor, A-485, was unable to affect the tyrosine phosphorylation of IRS1 and 2 by IRβ (H–K). These findings may explain why C646 has a stronger effect than A-485 on the activation of the insulin signaling pathway (compared C and D).However, interestingly, C646 had no effect on the tyrosine phosphorylation of IRβ itself , F and G2 by IRβ , H–K. ThL and M). It is possible that the cleft-sitting C646 can cause conformational changes carried out by insulin-mediated autophosphorylation of tyrosine residues in IRβ, allowing IRβ binding to IRS with high affinity and also increasing the enzymatic activity through reduction of activation energy.Since the crystal structure of unphosphorylated and low activity form of the insulin receptor kinase domain (IRK) have been determined , we usedob/ob mice and treated the primary hepatocytes with control inactive compound C37 or inhibitor C646 after serum starvation. We found that, in the absence of insulin, C646 treatment drastically increased the tyrosine phosphorylation levels in positions that correspond to IRS1 and 2 proteins in the immunoblot (A). These data are consistent with the results that C646 treatment increased IRS tyrosine phosphorylation in Hepa1-6 cells (A and E). C646 treatment also significantly increased PI3K activity and the phosphorylation of AKT and GSK in these primary hepatocytes in the absence of insulin (B–D) and significantly suppressed glucose production (E). Moreover, in obese ob/ob mice, C646 treatment through intraperitoneal injection (30 nmol/g/day) for 10 days significantly improved insulin sensitivity and hyperglycemia without significant changes in body weight (F and G). The above results show that the augmentation of ΙRβ activity, binding to IRS, and subsequent IRS tyrosine phosphorylation by C646 leads to the activation of PI3K-AKT signaling and the suppression of glucose production in the liver and alleviation of hyperglycemia.We further tested C646’s effect on insulin signaling in hepatocytes from mice with obesity and diabetes. We prepared primary hepatocytes from obese munoblot A. These -6 cells , A and E insulin , B–D andoduction E. Moreovy weight , F and GH–K). Also, considering that the acetylation of IRS1 and 2 by P300 had no impact on the total tyrosine phosphorylation of these proteins, these data suggest that the interaction of p85 in PI3K with IRS proteins is not the main C646 targeting site. However, it is possible that the acetylation of IRS1 and 2 may have a negative effect on the phosphorylation of tyrosine residue in the YMXM motifs, thus affecting the binding of p85 to IRS.The phosphorylation of tyrosine residues in IRS1 and 2 by IRβ will generate pYMXM motifs and lead to the docking of p85 in PI3K to the membrane located IRS proteins and activation of downstream mediators in the PI3K-AKT signaling. We therefore determined whether IRS acetylation could affect the docking of p85 protein in PI3K to IRS1 and 2 proteins. First, we used IRβ to phosphorylate the tyrosine residues in IRS1 and 2, then P300 protein was added in the absence or presence of acetyl-CoA, followed by Far-Western blot to examine the docking of p85 protein to IRS1 and 2. We found that the acetylation of either IRS1 or IRS2 had no effects on the docking of p85 to these proteins , H–K. Alvia reduction of the ubiquitination and degradation of P300, and elevated P300 impairs insulin signaling by acetylating IRS1 and 2 YThe mouse IRS1 and 2 genes were gifts from Ronald Kahn  , and theMouse primary hepatocytes were cultured in William’s medium E supplemented with ITS (BD Biosciences) and dexamethasone. After 24 to 48 h of seeding, cells were washed with PBS twice, and the medium was changed to FBS-free DMEM supplemented with 20 μM of either C37 or C646. After 3 h of serum starvation, cells were washed twice with PBS, and 1 ml glucose production medium was supplemented with 20 μM of either C37 or C646. After 3 h incubation with glucose production medium, both the medium and cells were collected. The medium was used to determine glucose concentrations with EnzyChrom Glucose Assay Kit , 42, 43.3VO4, and 10 mM MgCl2 at 30 °C for 15 min to 2 h in the absence or presence of 0.2 mM ATP. To acetylate IRS1 and 2, 2 μg of IRS1 or 2 was added to the reaction containing 50 mM Tris-HCl, pH 8.0, 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 1 mM DTT, 1 mM PMSF, 10 mM sodium butyrate, 0.2 μg acetyl-CoA, and 1.5 μg P300 (Active Motif). Samples were incubated at 37 °C for 2 h.In phosphorylation assays, purified IRS1 and 2 proteins were incubated with 0.2 μg of IRβ  in reaction containing 25 mM Tris-HCl (pH7.5), 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Naob/ob mice were subjected to serum starvation for 2 h, then treated with 20 μM of C37 or C646 for 3 h. P110α antibody  was used to pull down PI3K, and its enzymatic activity was measured using the PI3K-Kinase Activity ELISA: Pico kit (Echelon Biosciences).Twenty-four hours after the seeding, primary hepatocytes from db/db mice, and ob/ob mice were purchased from the Jackson Laboratory. Floxed homozygous IR mice were fed on HFD  for 4 weeks, and AAV8-TGB-Cre (1 × 10ˆ11 GC/mouse) was injected via the jugular vein to generate liver-specific IR knockout mice. After viral injection, mice were treated with vehicle or C646 (20 nmol/g/day) for 2 weeks. To test C646’s effect on insulin sensitivity in obese ob/ob mice, 4-month-old ob/ob mice were randomly divided into two groups for which vehicle or C646 (30 nmol/g/day) was given through intraperitoneal injection for 10 days. Insulin tolerance test was performed after 6 h of fasting (0.8 unit/kg insulin). To test the effect of IRS1/2 mutants on their membrane translocation, 3-month-old homozygous double-floxed IRS1 and 2 mice were injected with adenovirus (1 × 1010 GC/mouse) through the jugular vein alone with AAV8-TGB-Cre (1 × 10ˆ11 GC/mouse). Two days after the viral injection, mice were placed on an HFD. After 19 days of HFD feeding, liver tissues were collected.All animal protocols were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University. Male floxed homozygous IR mice, Liver tissues were obtained from the left lateral lobe of the liver of three obese patients  and immediately frozen on dry ice and stored at −80 °C. Normal human liver tissues were obtained through the Liver Tissue Cell Distribution System, which was funded by National Institutes of Health contract no. HHSN276201200017C .We firstly employed different amounts  of insulin receptor to determine the enzymatic activity needed in the InsR Kinase Assay (Promega) and found a linear curve of ADP production (consumption of ATP by insulin receptor). Subsequently, 1 μg of the insulin receptor was used in a 25 ul reaction containing different concentrations of C646, 0.2 μg of substrate peptide, 50 μM of ATP, and 1 × provided reaction buffer. The reactions were incubated at room temperature for 60 min. ADP-Glo Kinase Assay (Promega) was used to determine the ADP levels generated by the insulin receptor. Twenty-five microliters of ADP-Glo reagent was added to deplete residual ATP , then 50 ul of Kinase Detection Reagent was added to determine the ATP levels converted from ADP followed by the measurement of luminescence in BioTek Synergy H1 plate reader.Cellular lysates were passed 15 times through a syringe needle to break cells on ice. ΙRβ, IRS1 and 2 were immunoprecipitated using ΙRβ antibody  and IRS1 and 2 antibodies (Millipore). The reaction was incubated at 4 °C for 16 h, followed by the addition of protein G beads (Active Motif) to pull down the target protein and its associated proteins. Cytosolic and nuclear extracts were prepared using the CelLytic NuCLEAR Extraction kit (Sigma). Membrane, cytosolic, and nuclear fractions were prepared from Hepa1-6 cells using Subcellular Protein Fraction Kit for Cultured Cells (Thermo Fisher) following manufacturer’s instruction.et al. , and 0.05% Tween-20) for 1 h at room temperature. Subsequently, the membrane was incubated in protein-binding buffer , 0.5 mM EDTA, 10% glycerol, 0.1% Tween-20, 2% skim milk powder, and 1 mM DTT, prepared freshly) plus 4 μg of p85 . After three washes with PBST buffer, the membrane was incubated with anti-p85  at 4 °C overnight.A far-Western blot was conducted using a modified method of Wu et al. . Purifiedb/db mice were fixed with 4% paraformaldehyde in PBS for overnight at room temperature, then incubated in 30% sucrose overnight and frozen in OCT. Tissues were sectioned. After antigen retrieval using a microwave, sections were permeabilized with 0.1 Triton X-100 (10 min) and blocked in 3% horse serum for 1 h at room temperature. Paraffin-embedded human liver samples were sectioned. Sections were incubated with P300-specific antibody  at 1: 20–200 dilution at 4 °C overnight.Frozen liver samples of heterozygous lean and homozygous via a Zeiss confocal microscope .Liver tissues were harvested, washed with cold PBS, and fixed immediately with 4% paraformaldehyde in PBS for 6 h at 4 °C, then incubated in 30% sucrose overnight, and frozen in OCT. The frozen sections were cut at 10 uM thickness. Sections were washed in PBS, blocked, and permeabilized in blocking buffer (5% goat serum with 0.3% TritonX-100 in PBS) for 1 h at room temperature. For primary hepatocytes, 24 h after the seeding of primary hepatocytes, cells were washed with PBS and cultured in FBS-free DMEM for 1 h, then C37 or C646 was added. 4 h later, medium was removed. Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 1% Triton X-100, and blocked with 3% horse serum in PBS. Antibodies against IRS1 (1:50), IRS2 (1:50), and Na/K ATPase (1:100) were used . Fluorescein-conjugated AffiniPure donkey anti-mouse IgG (1:150) and Cy3-conjugated AffiniPure donkey anti-rabbit IgG (1:250)  were diluted in PBS with 3% horse serum and incubated at room temperature for 2 h. Fluorescent images were acquired (These experiments were conducted in the Biosensor Core at the University of Maryland School of Medicine). For the determination of C646 bindings to IRβ, IRS1 and 2 proteins, these proteins were immobilized on the surface of a CM5 chip at flow cell 2, 3, and 4, respectively. Binding reactions were done in HBS-P buffer from Biacore (Cytiva Inc), containing 10 mM Hepes, 150 mM NaCl, and 0.05% (v/v) surfactant p20, pH 7.4. This buffer was filtered (0.2 μM) and degassed before use. In order to minimize mass transport effects, the binding analyses were performed at flow rate of 30 μl per minute at 25 °C. The analyte (C646) was dissolved in HBS-P buffer with 0.05% P20 and 2% DMSO and injected into flow cell 1, 2, 3, 4, and the association of analyte and ligands  was recorded respectively by Biacore T200 . Next, the surface was washed with buffer for 600 s to dissociate the analyte–ligand complexes. The signal from the Blank channel (flow cell-1) was served as a control to subtract from the channel containing IRβ, IRS1 and 2 proteins. The binding was removed by injecting 30 μl of glycine (pH 1.5) followed by injection of 30 μl of HBS-P (pH 7.4). For the determination of IRβ binding to IRS1 and 2 proteins, IRβ was immobilized on the surface of a CM5 chip, and the IRS1 or IRS2 protein was injected with different concentrations of C646. Sensorgrams of the interaction were generated by the instrument and analyzed using the software BIAeval 4.0 (Biacore Inc). The reference surface data were subtracted from the reaction surface data to eliminate refractive-index changes of the solution, injection noise, and nonspecific binding to the blank surface. A blank injection with buffer alone was subtracted from the resulting reaction surface data. Data were globally fitted to the Langmuir model for a 1:1 binding.t test and ANOVA test. Significance was accepted at the level of p < 0.05. Sample size (number of mice) was determined on the basis of our previous studies. At least three samples per group were chosen for statistical meaningful interpretation of results. Statistical significance (p < 0.05) was evaluated in GraphPad Prism using one-way ANOVA for multigroup comparisons or using Student’s t test for two-group comparison.Statistical significance was calculated with a Student’s heling@jhmi.edu) upon request.All data are contained in this manuscript, and supporting information is available from the author: Ling He (This article contains The authors declare that they have no conflict of interest with the contents of this article.


Giardia lamblia is a pathogenic unicellular eukaryotic parasite that causes giardiasis. Its genome encodes the canonical histones H2A, H2B, H3, and H4, which share low amino acid sequence identity with their human orthologues. We determined the structure of the G. lamblia nucleosome core particle (NCP) at 3.6 Å resolution by cryo-electron microscopy. G. lamblia histones form a characteristic NCP, in which the visible 125 base-pair region of the DNA is wrapped in a left-handed supercoil. The acidic patch on the G. lamblia octamer is deeper, due to an insertion extending the H2B α1 helix and L1 loop, and thus cannot bind the LANA acidic patch binding peptide. The DNA and histone regions near the DNA entry-exit sites could not be assigned, suggesting that these regions are asymmetrically flexible in the G. lamblia NCP. Characterization by thermal unfolding in solution revealed that both the H2A–H2B and DNA association with the G. lamblia H3–H4 were weaker than those for human H3–H4. These results demonstrate the uniformity of the histone octamer as the organizing platform for eukaryotic chromatin, but also illustrate the unrecognized capability for large scale sequence variations that enable the adaptability of histone octamer surfaces and confer internal stability.  In the nucleus of eukaryotic cells, genomic DNA is packaged into chromatin as a crucial structure for the maintenance of genome organization. Chromatin stability and conformational dynamics play an essential role in regulating biological events, such as gene transcription, DNA replication and DNA repair . The nucThe NCP is composed of 145–147 base-pairs of DNA wrapped around a histone octamer, containing two molecules each of the four histone proteins, H2A, H2B, H3 and H4 . These hGiardia lamblia is a unicellular eukaryotic microorganism classified as a diplomonad that parasitically infects the small intestines of humans and other vertebrates to cause giardiasis, one of the most common waterborne diarrheal diseases throughout the world . The structural and biophysical implications of such large histone sequence variations in the Giardia nucleosome have not been investigated. In the present study, we determined the cryo-electron microscopy (cryo-EM) structure of the G. lamblia NCP at 3.6 Å resolution, and characterized the stabilities of the nucleosome and histone octamer in solution. Our studies revealed a number of structural features that can vary while still retaining the characteristic fold and DNA wrapping of the nucleosome.The histones of parasitic protozoa are generally evolutionally diversified, with lower identity to metazoan histones than other eukaryotes ,30. EvenG. lamblia histones H2A, H2B, H3, H4, and H2AG.lambliaCTD were cloned into the pET-15b vector, which encodes the hexahistidine (His6)-tag and thrombin protease recognition sequences. The G. lamblia histones H2A, H2B, H3 and H2AG.lambliaCTD were produced in Escherichia coli BL21 (DE3) cells, and the G. lamblia histone H4 was produced in Escherichia coli Rosetta-gami B (DE3) cells, by induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside. The cells were cultured overnight at 37°C. The G. lamblia histones H2A, H2B, H3, and H2AG.lambliaCTD were purified by the same method used for the purification of human histones, as described previously  agarose column chromatography (QIAGEN) with a linear gradient of 5 mM to 1 M imidazole in 50 mM Tris–HCl buffer (pH 8.0), containing 0.5 M NaCl, 6 M urea, and 5% glycerol. The eluted samples were dialyzed against  50 mM Tris–HCl buffer (pH 7.5), containing  2 mM 2-mercaptoethanol. The N-terminal His6 tag was removed from the histone H4 portion by thrombin protease treatment. Afterwards, the histone H4 without the His6 tag was subjected to MonoS column chromatography (Cytiva). The column was washed with  20 mM sodium acetate buffer (pH 5.2), containing 0.2 M NaCl, 5 mM 2-mercaptoethanol, 1 mM EDTA and  6 M urea, and the histone H4 was eluted by a linear gradient of 0.2–1.2 M NaCl in 20 mM sodium acetate buffer (pH 5.2), containing 5 mM 2-mercaptoethanol, 1 mM EDTA and 6 M urea. All purified histones were dialyzed against water containing 2 mM 2-mercaptoethanol, and then freeze-dried.The DNA fragments encoding the eviously . The G. G. lamblia or human histones H2A, H2B, H3 and H4, or H2AG.lambliaCTD, human histones H2B, H3 and H4 were mixed at a 1:1:1:1 molar ratio in 50 mM Tris–HCl buffer (pH 7.5), containing 7 M guanidine hydrochloride and 20 mM 2-mercaptoethanol. For the histone H3–H4 tetramer and H2A–H2B dimer reconstitution, the G. lamblia or human histones H3 and H4, or H2A and H2B, were mixed at a 1:1 molar ratio, respectively, in 50 mM Tris–HCl buffer (pH 7.5), containing 7 M guanidine hydrochloride and 20 mM 2-mercaptoethanol. The histone octamer, tetramer, and dimer were assembled by dialysis against 10 mM Tris–HCl buffer (pH 7.5), containing 2 M NaCl, 1 mM EDTA and 5 mM 2-mercaptoethanol, and then purified by size exclusion chromatography on a Hiload 16/60 Superdex 200 prep grade column (Cytiva).For the histone octamer reconstitution, the G. lamblia or human histones, the DNA fragments were mixed with a histone octamer or the H3–H4 tetramer in 10 mM Tris–HCl buffer (pH 7.5), containing 2 M KCl, 1 mM EDTA and 1 mM dithiothreitol. For the reconstitution of the NCPs containing the G. lamblia H3–H4 and the human H2A–H2B or the G. lamblia H2A–H2B and the human H3–H4, the DNA fragment containing a palindromic human α-satellite sequence was mixed with the G. lamblia H3–H4 tetramer and the human H2A–H2B dimer, or the G. lamblia H2A–H2B dimer and the human H3–H4 tetramer, in 10 mM Tris–HCl buffer (pH 7.5), containing 2 M KCl, 1 mM EDTA and 1 mM dithiothreitol. The NCPs and the H3–H4–DNA complexes were reconstituted by the salt-dialysis method, with the KCl concentration gradually decreased to 250 mM  sequence or a 146 base-pair palindromic human α-satellite sequence were prepared . For theo 250 mM ,32. The o 250 mM ,32. The G. lamblia NCP containing the 601L sequence was stabilized by the GraFix method, using paraformaldehyde  were successively collected from the top of the gradient. The absorbance of the fractions at 260 nm was monitored, and the peak fractions were collected. Using a PD-10 column (Cytiva), the buffer was exchanged to 20 mM HEPES–KOH (pH 7.5) buffer, containing 0.1 mM Tris–HCl (pH 8.0), 50 mM potassium acetate, and 0.2 μM zinc acetate. The resulting sample was concentrated with an Amicon Ultra centrifugal filter. The sample concentration was adjusted to 0.8 mg/ml for DNA. For the cryo-EM specimen preparation, a 2.5 μl sample was applied onto a Quantifoil R1.2/1.3 200-mesh Cu grid, which was treated with a PIB-10 glow discharge device . The grids were blotted for 8 s under 100% humidity at 16°C in a Vitrobot Mark IV (Thermo Fisher Scientific), and then immediately plunged into liquid ethane. The cryo-EM data collection conditions were screened with the Talos Arctica electron microscope (Thermo Fisher Scientific) installed at KEK.The aldehyde . A gradiG. lamblia NCP were imaged at 300 kV on a Krios G3i microscope (Thermo Fisher Scientific), equipped with an energy-filtered K3 detector calibrated at a pixel size of 1.05 Å with a slit width of 25 eV, using the SerialEM auto acquisition software  with the 0.143 criterion .The crystal structures of the nucleosomal human histones (PDB ID: 6R93) and 601L DNA (PDB ID: 3UT9) were placed in the cryo-EM map of the  Chimera . The amial Omega , and theith COOT  followedith COOT . The G. 2 and 1.9 mM dithiothreitol. The reactions were stopped by adding an aliquot (10 μl) to 5 μl of a deproteinization solution, consisting of 20 mM Tris–HCl (pH 8.0), 20 mM EDTA, 0.1% SDS and 0.5 mg/ml proteinase K (Roche). The reaction products were analyzed by non-denaturing polyacrylamide gel electrophoresis, followed by ethidium bromide staining. Three experiments were independently performed, and the reproducibility was confirmed.The reconstituted NCPs containing the palindromic human α-satellite or 601L sequence (1.2 μg for DNA) were incubated with 0.6 or 0.4 units of MNase , respectively, at 37°C, in a reaction solution containing 40 mM Tris–HCl (pH 8.0), 25 mM NaCl, 2.5 mM CaClE. coli BL21(DE3) cells, and purified by Glutathione Sepharose 4B (Cytiva) column chromatography. The G. lamblia and human canonical NCPs containing the 601L DNA fragments were mixed with the purified GST-LANA or GST in a solution containing 14 mM Tris–HCl (pH 7.5), 4 mM HEPES–NaOH (pH 7.5), 0.45 mM EDTA, 150 mM NaCl, 0.8 mM dithiothreitol, and 4% glycerol. The LANA–NCP complexes were analyzed by 6% non-denaturing PAGE in 0.5× TBE  buffer, followed by ethidium bromide staining.The DNA fragment encoding residues 1–23 of the viral latency-associated nuclear antigen (LANA) was ligated just downstream of the PreScission recognition sequence in the pGEX6P1 plasmid. The LANA peptide was produced as a fusion protein with GST (GST-LANA). The GST-LANA and GST proteins were produced in ScaI restriction enzyme (Takara), which cleaves the linker DNA regions of the nucleosome arrays, as previously described (ScaI in a 10 μl reaction solution (10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 0.5 mM MgCl2 and 0.1 mg/ml BSA) at 22°C for 12 h, and the resulting mononucleosomes were analyzed by electrophoresis on a non-denaturing 5% polyacylamide gel in 1× TBE buffer , followed by ethidium bromide staining. The gel image was acquired with an Amersham imager (Cytiva). The sedimentation velocity analysis of the nucleosome arrays (35 ng/μl of DNA) was performed using a Beckman Coulter ProteomeLab XL-I centrifuge with an 8-hole An-50Ti Analytical Rotor, at 22 000 rpm for 3 h in 10 mM Tris–HCl (pH 7.5). The collected data were analyzed with the SEDFIT software (3/g.The DNA fragment containing twelve tandem repeats of a 177 base-pair 601 sequence was prepared as described previously . The DNAescribed . Brieflysoftware . The sedF(T)normalized = [F(T) – F(26)]/[F(95) – F(26)]. F(T) indicates the fluorescence intensity at a particular temperature. The averages of three independent experiments were plotted with standard deviation values. The thermal stabilities of the histone octamers were evaluated as described previously  and human (PDB ID: 6R93) NCPs are 0.49–0.85 Å. A 125 base-pair DNA region (out of 145 bp) was wrapped around the histone octamer in a left-handed supercoil, with a mean RMSD of 2.01 Å from the canonical NCP with the same DNA sequence  assay to test whether these DNA regions are differentially accessible in solution, since MNase preferentially accesses and digests the DNA regions detached from the histone surfaces in the NCP. The DNA ends of the G. lamblia NCP were substantially more susceptible to MNase than those of the control human NCP  phenotype, and the mutagenesis of H2A Leu115 or Leu116 in metazoan histones affects nucleosome stability in vitro , where LmaH3 weakens the histone-DNA association in the NCP . Th. ThG. las Figure  and B, ae Figure . InteresP Figure . In contP Figure , suggest the NCP . To testG. lamblia histone octamer, we reconstituted the histone octamers with the G. lamblia and human histones in the absence of DNA (G. lamblia and human histone octamers efficiently formed under these conditions, and could be purified by gel filtration chromatography (G. lamblia histone octamer rapidly increased in the temperature range of 28–49°C, which is considerably lower than that of the human histone octamer, in the range of 39–74°C (Figure G. lamblia NCP and indicate that the combined sequence divergence of the G. lamblia histone octamer makes it less stable and potentially more dynamically accessible than the orthologous human histone octamer.To evaluate the histone–histone interactions in the e of DNA . H3 and tography . The theC Figure . These rG. lamblia NCP, which revealed that it retains the similar overall structural organization as metazoan NCPs despite the very high degree of histone sequence divergence. However, we also observed that histone sequence variation gives rise to unique structural and biochemical features in the G. lamblia NCP. The DNA and histone regions near the DNA entry-exit sites of the G. lamblia NCP are remarkably flexible, and the morphology of the G. lamblia NCP acidic patch is profoundly different. In addition, the G. lamblia NCP is quite unstable as compared to the human canonical NCP, probably due to the weakened binding of the H3–H4 complex to both H2A–H2B and DNA. These structural and physical characteristics of the G. lamblia NCP illustrate the potential for the diversity of chromatin architecture and function, because G. lamblia may be less constrained by the complex interdependencies of chromatin interacting factors. The NCP structure may be useful in future drug design and discovery to treat infections by this important parasite.We have determined the cryo-EM structure of the G. lamblia NCP have been deposited in the Electron Microscopy Data Bank under the accession code EMD-30591, and in the Protein Data Bank under the accession code PDB ID 7D69.The cryo-EM structure and the atomic model of the gkab644_Supplemental_FileClick here for additional data file.