It was confirmed that the Mg2Ca phase acts as a microanode in microgalvanic corrosion in both NaCl and Na2SO4 solutions, with the Mg matrix acting as the cathode and the Al2Ca phase acting as the microcathode to accelerate corrosion of the adjacent Mg matrix. It was also found that Cl− and SO42− have different sensibilities to microgalvanic corrosion.As a kind of potential biomedical material, Mg–Ca alloy has attracted much attention. However, the role of Ca-containing intermetallics in microgalvanic corrosion is still controversial. In 0.6 mol/L NaCl and Na They  process . The poo process ,13.2Ca precipitates were formed. This alloy demonstrated one of the highest combinations of tensile elongation and work hardening capacity among existing Mg alloys [Many alloying elements have been added to Mg alloys to increase corrosion resistance and strength for biomedical applications. Recently, rare earth elements ,15,16,17g alloys .17Al12 to the Al2Ca and Mg2Ca phases [17Al12 phase is nobler than the Mg matrix and acts as a cathode to accelerate dissolution of the Mg matrix [2Ca phase is unique as it possesses a more active corrosion potential than the Mg matrix of all Mg-based intermetallics. Yang [With the increase in Ca content in Mg–Al alloys, the precipitation phase changes from Mga phases . The eleg matrix . Ca is ag matrix  and Südhg matrix  claimed cs. Yang  added Cacs. Yang  added Al− is much more aggressive than SO42− to Mg alloys [2SO4 than in NaCl solution [2SO4 solution, where the second phase acts as anode and dissolves preferentially, is poles apart from that in NaCl solution, where the second phase acts as a cathode. It is worth investigating whether Ca-containing intermetallics have different microgalvanic behavior in NaCl and Na2SO4 solutions.Due to the potential application of magnesium–calcium alloys as biomaterials, their corrosion behavior is mostly studied in simulated body fluids ,28. Howeg alloys . The pre2SO4 solutions and the corrosion resistance of Mg–Ca and Mg–Al–Ca alloys in the two solutions.The present work aimed to study the role of Ca-containing intermetallics in microgalvanic corrosion in NaCl and Na2. Subsequently, the working surface was mechanically ground with SiC paper to P2000 grit, cleaned with deionized water and alcohol, and finally dried by flowing cold air.Mg–Ca and Mg–Al–Ca alloys were supplied by YinGuang Magnesium Industry Co., Ltd., Wenxi, China. The nominal composition is listed in The microstructure of the Mg–Ca and Mg–Al–Ca alloys was observed by optical microscopy (OM) and scanning electron microscope . The phase morphology of the alloys was detected by t backscattered electron (BSE).2SO4 solution for 2 min, 5 min, 30 min, 2 h, 5 h, and 24 h for corrosion morphology observation. All specimens were washed with deionized water after immersion, degreased with ethanol, and dried in cold air flow. At room temperature, the specimens were soaked in a chromic acid solution containing 180 g/L CrO3 for 5 min to remove the corrosion products. SEM–BSE and EDS were used to examine the corrosion surface after removing corrosion products as well as the cross-sectional morphology of the corroded samples.At 25 ± 0.5 °C, the sample size of 10 × 10 × 10 mm was exposed to 0.6 mol/L NaCl solution and 0.6 mol/L Na2SO4 solutions at 25 ± 0.5 °C. The funnel collected all the hydrogen bubbles over a specimen with the dimension of 30 mm × 30 mm × 10 mm in a beaker containing 2 L of testing solution. The hydrogen evolution was recorded every five minutes for a total of three hours. The weight loss test was carried out using a digital balance (JA3003) before and after soaking with the corrosion products removed by chromic acid.All the tests were performed in 0.6 mol/L NaCl and 0.6 mol/L Na2SO4 solutions using a CHI660D electrochemical workstation. At 25 ± 0.5 °C, a typical three-electrode setup was used with a Pt mesh as the counter electrode, an R232 saturated calomel electrode (SCE) as the reference electrode, and the samples as the working electrode.All the experiments were carried out in 0.6 mol/L NaCl and 0.6 mol/L NaEIS tests were performed at open-circuit potential over a frequency range from 100 kHz to 10 mHz, and the sinusoid potential perturbation had an amplitude of 5 mV. The EIS spectra were fitted using the ZSimpWin 3.20 software.−2. The scan rate was 0.5 mV s−1.After the EIS test, the cathodic polarization curves were carried out from 0 to −0.3 V vs. the OCP. All the electrochemical tests were replicated more than three times to ensure accuracy of the results. The anodic polarization curves were started at the OCP value and terminated at a final current density of 10 mA cm2Ca phase, and the types of second phase in Mg–Al–Ca alloys were determined by the mass ratio of Ca/Al [2Ca and Al2Ca phase) in Mg–Al–Ca alloys when the mass ratio of Ca/Al was higher than 0.8, while there was only an Al2Ca phase particle when the mass ratio of Ca/Al was lower than 0.8. Jiang [2Ca with the Ca/Al ratio of 0.50–0.67. The Ca/Al ratio of the Mg–Al–Ca alloy in this work was 0.70. Taking the results of EDS 2; thus, there should be the existence of Ca and Mg in this area in the mapping results. Perhaps the color in this area was weak but not void. This phenomenon requires further study.The mapping results of elements Mg, Ca, and O of the sample after immersion for 5 min e confirm2SO4 solution. The second phase still served as the anode and dissolved preferentially. However, it was found that the severity of the Mg–Ca alloy corrosion in Na2SO4 solution was much weaker than that in NaCl solution, meaning the corrosion resistance of the Mg–Ca alloy in Na2SO4 solution was much better than that in the NaCl solution. After immersion for 30 min and 2 h, there was no existence of second phase on the surface of the Mg–Ca alloy 2Ca.The mapping results of elements Mg, Al, and Ca of the sample after immersion for 24 h g, as sho2SO4 solution with corrosion products removed are shown in 2SO4 solution was much slower than that in NaCl solution. The slight corrosion of the surface morphology after immersion for 5 h is shown in 2Ca phase and Mg matrix like the corrosion behavior of the Mg–Al–Ca alloy in NaCl solution. Occasionally, there were one or two corrosion pits on the surface , and the weight loss rate of the Mg–Al–Ca alloy in NaCl solution (19.86 mm/y) was about 6 times as fast as that in Na2SO4 solution (3.33 mm/y). Both the Mg–Ca and Mg–Al–Ca alloys showed faster corrosion rates in NaCl solution than in Na2SO4 solution, which coincided with the corrosion morphologies. This phenomenon is attributed to Cl− being more corrosive than SO42− [The weight loss rates of the Mg–Ca and Mg–Al–Ca alloys immersed in 0.6 mol/L NaCl solution and 0.6 mol/L Naan SO42− . The WE42SO4 solution, the weight loss rate of the Mg–Ca alloy was about 5 times as fast as that of the Mg–Al–Ca alloy.On the other hand, the corrosion resistance of the alloy became better with the addition of Al. In NaCl solution, the weight loss rate of the Mg–Ca alloy was about 4 times faster than that of the Mg–Al–Ca alloy. In Na2SO4 solutions for 3 h are shown in 2SO4 solution. This result was consistent with the weight loss rate.The hydrogen evolution of the two alloys immersed in 0.6 mol/L NaCl and Na2SO4 solutions. The plots of the alloys in Na2SO4 solution consisted of a high-frequency and a medium-frequency capacitance loop, while the plots of the alloys in NaCl solution had a low-frequency inductance loop. The high-frequency capacitance loop can be associated with the electric double layer at the interface of the Mg substrate and electrolyte. The medium-frequency capacitance loop should be related to the surface film. The low-frequency loop can be attributed to the initiation of localized corrosion.s is the solution resistance, Rt is the charge transfer resistance, Qdl is the capacitance of the electric double layer, Rt and Qdl together indicate the first capacitance loop in high-frequency, and Rf and Qf are the surface film resistance and capacitance, respectively. In addition, L is the inductance of the electrochemical reactions at the film–substrate interface, and the RL is the resistance of the inductance [The equivalent circuit diagram is shown in ductance .2SO4 solution was much larger than that in NaCl solution. Meanwhile, the Nyquist plots of the two alloys in NaCl solution showed a distinctive low-frequency inductance loop. These results indicate that the corrosion resistance of two alloys in Na2SO4 solution is better than in NaCl solution, which coincides with the results of corrosion morphologies, weight loss, and hydrogen evolution.The capacitive diameter of the Mg–Ca and Mg–Al–Ca alloys in Na2SO4 solutions are shown in 2SO4 solution, Mg–Al–Ca alloys immersed in NaCl solution, and Mg–Al–Ca alloys immersed in Na2SO4 solutions were −1.6422, −1.5424, −1.5776, and −1.584, respectively.The cathodic and anodic polarization curves of the Mg–Ca and Mg–Al–Ca alloys immersed in 0.6 mol/L NaCl and Nacorr values of the two alloys in Na2SO4 solution were much smaller than those in NaCl solution. Meanwhile, the anodic polarization curve of the Mg–Al–Ca alloy in Na2SO4 solution showed passivation of the surface film, which terminated at the turning point of film breakdown. However, the other three curves were always active dissolution. This indicates that the surface film of Mg–Al–Ca alloy is more protective than that of the Mg–Ca alloy, and Cl− is more corrosive than SO42− to the surface film.It is generally believed that the cathode of the polarization curve represents a hydrogen evolution reaction, and the anode is related to the anodic dissolution of the magnesium alloy. The fitting results of the cathodic polarization curves in 2Ca) of the Mg–Ca alloy serves as the microanode in microgalvanic corrosion with the Mg matrix in NaCl and Na2SO4 solutions. However, the second phase (Al2Ca) of the Mg–Al–Ca alloy serves as the microcathode in microgalvanic corrosion in NaCl and Na2SO4 solutions.1. The second phase (Mg2SO4 solutions.2. The addition of Al can significantly improve the corrosion resistance of the Mg–Ca alloy in both NaCl and Na− ion is more sensitive to microgalvanic corrosion between different phases and more corrosive to the surface film than SO42− ion.3. Cl


The inserted Mn2+ forms coordination bonds with the oxygen of the host material and strengthens the interaction between the layers, preventing damage to the structure. Combined with the experimental data and DFT calculation, it is found that Mn2+ refines the structure stability, adjusts the electronic structure, and improves the conductivity of hydrated vanadium oxide. Also, Mn2+ changes the migration path of Zn2+, reduces the migration barrier, and improves the rate performance. Therefore, Mn2+‐inserted hydrated vanadium oxide electrode delivers a high specific capacity of 456 mAh g−1 at 0.2 A g–1, 173 mAh g–1 at 40 A g–1, and a capacity retention of 80% over 5000 cycles at 10 A g–1. Furthermore, based on the calculated zinc ion mobility coefficient and Zn(H2O)n2+ diffusion energy barrier, the possible migration behavior of Zn(H2O)n2+ in vanadium oxide electrode has also been speculated, which will provide a new reference for understanding the migration behavior of hydrated zinc‐ion.The interlayer modification and the intercalation pseudocapacitance have been combined in vanadium oxide electrode for aqueous zinc‐ion batteries. Intercalation pseudocapacitive hydrated vanadium oxide Mn 2+ is applied to regulate the interlayer atomic coordination structure, electronic structure, and affect Zn2+ migration behavior in the vanadium oxide (V10O24·nH2O). Zinc ion intercalation pseudocapacitance, ultra‐high rate performance, and cycling stability are achieved and the Zn(H2O)n2+ migration behavior is analyzed and speculated.The strategy of interlayer modification of Mn Therefore, it is essential to develop a new type of ion batteries to replace the lithium‐ion batteries. Multivalent ions such as Mg2+, Zn2+, Ca2+, and Al3+ can carry more charges per ion than that of the Li+, and they are believed to be promising working ions in rechargeable batteries. Among all multivalent ions mentioned above, zinc metals as anode electrode material for zinc‐ion batteries (ZIBs), have a low redox potential (−0.76 vs standard hydrogen electrode), high capacity (820 mAh g−1), easy production, high abundance, and intrinsic safety. So aqueous ZIBs have attracted tremendous attention. Recently, a series of cathode materials, such as manganese oxide, vanadium based composites, Prussian blue analogs, metal sulfides, and organic compounds, have been researched for ZIBs. Among them, vanadium oxide with tunnel, layered or open framework structure, possesses sufficient active site for Zn2+ and has been considered as ideal cathode materials for ZIBs. However, the poor conductivity and fragile open framework structure hinder the ion diffusion kinetics and lead to fast performance degradation during cycling. In order to solve the above problems, many strategies have been developed to optimize vanadium‐based cathode materials including pre‐inserted cations/molecules pillaring, structural modification, nanostructuring, and more. For instance, Li et al. synthesized Ni0.25V2O5·nH2O by hydrothermal method, which demonstrated a high reversible specific capacity of 402–147 mAh g–1 at 0.2‐5 A g–1. Geng et al. synthesized Mn0.15V2O5·nH2O by introducing Mn2+ that exhibited high rate performance of 150 mAh g–1 at 10 A g–1. Na0.33V2O5, NaCa0.6V6O16·3H2O, V10O24‐Aland more. have also been introduced by ion pillaring and deliver effectively improved stability of vanadium oxide cathode. However, in these battery‐type intercalation materials, kinetics limited by ion diffusion process and their energy storage performance is still not satisfactory. Also, a type of intercalation pseudocapacitive energy storage is easier to obtain high rate performance, with Faradaic charge transfer without crystallographic phase change. Over the past decade, the focus on intercalation pseudocapacitive materials is mainly in nanostructured, high defective, and metastable materials. But the structural stability of these intercalation pseudocapacitive materials is still unsatisfactory. Therefore, the interlayer tuning strategy and intercalation pseudocapacitive property have been applied to the hydrated vanadium oxide electrode materials, demonstrating an effective way to obtain high stability and rate performance.Lithium‐ion batteries have been successfully commercialized due to their high working voltage and high energy density.1 Howe21.4V10O24·nH2O (MnVOH) was synthesized by a mild spontaneous chemical reaction, and it exhibits a cobweb‐like crisscross microscopic morphology as shown in Figure10O24·nH2O (VOH) also exhibits a similar morphology with MnVOH  pattern in Figure 10O24·nH2O (PDF#25‐1006) and MnVOH is determined to be Mn1.4V10O24·nH2O. According to the Bragg's equation, the shift of the (002) peak from 6.1 to 6.3 degrees means that the interlayer distance is reduced from 14.3 Å to 13.5 Å, which is consistent with the HRTEM results.Mn1 Figure . The inse Figure , which iFigure which leads to more V4+ content in MnVOH as shown in Figure 2+ and promote the intercalation pseudocapacitive behavior. The Fourier Transform infrared spectroscopy and Raman spectra  is used to analyze the water loss in the heating process. Both samples were dehydrated in two stages as shown in Figure  MnVOH has a higher temperature of losing interlayer water than that of the VOH, which may be due to the electrostatic interaction between Mn2+ and interlayer water, reflecting the better thermal stability of interlayer water for MnVOH.X‐ray photoelectron spectroscopy (XPS) was further used to analyze chemical valences. The peaks of Mn 2p spectrum in 2+ to form coordination bonds and water to form hydrogen bonds. Our DFT calculation demonstrates that the inserted Mn2+ not only saturates [VO] surface dangling bonds to improve its stability, but also reduces interlayer distance  were obtained with typical coin‐type cell. Two pairs of redox peaks in both samples are displayed in Figure4+/V3+ appears around 0.6 V, and the peak pair at higher voltage around 1 V is related to the redox of V5+/V4+. It is worth noting that the broad peaks on CV curves and pseudo linear voltage responses on galvanostatic charge and discharge (GCD) curves  peak during charging and discharging demonstrates the reversibility of Zn2+ intercalation/de‐intercalation between the interlayer of MnVOH. The XPS spectrum of Zn 2p for MnVOH at 0.2 V/1.6 V also proves the reversible migration of zinc ions  measurements at a voltage range of 0.2–1.6 V . Compared with the capacities at 0.2 A g–1, the capacity retention of MnVOH electrode is 60% at 15 A g–1, which is higher than that of VOH electrode (42%). The MnVOH electrode delivers a similar specific capacity at small current density of 0.2 A g−1  as depicted in Figure 0.33V2O5, NaCa0.6V6O16·3H2O, Mn0.15V2O5·nH2O,δ‐Ni0.25V2O5·nH2O, and Cu3V2O7(OH)2·2H2O. And the stability of the MnVOH electrode (10 A g–1) is shown in Figure  Since both electrode materials of VOH and MnVOH have similar characteristics, such as large interlayer spacing and oxygen defects, the difference in rate performance may be due to the insertion of Mn2+. The average charge–discharge voltage during the cycles at 10 A g–1 are displayed in Figure 2+ migrates rapidly. In addition, the closer average charge–discharge voltage indicates that MnVOH has a smaller polarization and better reaction kinetics in the electrochemical processes, which is consistent with the high rate performance of MnVOH. In order to evaluate the electrochemical energy storage performance of the batteries, the rate and high current density charging–discharging were conducted. For the rate capability tests as shown in Figure 0.33V2O5,35 NaC−1. As the electrode polarization becomes more serious at the large‐scan speed, a voltage shift occurred in cathodic/anodic peaks as shown in FigureIn order to explain the electrochemical kinetics of the MnVOH cathode, the CV curve was tested at different scanning speeds of 0.1~1 mV sv refers to the scan rate (mV s−1), a and b are adjustable parameters. When b is close to 1, it reveals that this process is controlled by the capacitive process. When b is close to 0.5, it reveals that this process is controlled by the diffusion process. The b values of MnVOH are 0.71, 0.79, 0.94, and 0.86 respectively , the migration path of Zn2+ is changed as shown in Figure 2+ may cause Zn2+ migration path to change and it also occupies the adsorption sites of Zn2+ to a certain extent. Therefore, Mn2+ only improves the rate performance, but does not significantly increase the capacity. Further calculations show that the energy barrier corresponding to the migration path of Zn2+ in MnVOH is lower than that of VOH as placed in Figure 2+ diffusion coefficient (DZn) and the migration energy barrier can be explained by the following formula:p is a geometrical factor, *v is the vibrational frequency, k is the hopping distance, and EB is the migration energy barrier. Therefore, the low energy barrier can make the diffusion of Zn2+ in MnVOH easier.It is universally affirmed that the kinetic limiting step of insertion‐type materials is the solid‐state diffusion. Therefore, the influence of Mnficient DZn and thficient DZn and thFigure–1, at which the capacity of them are similar. The reaction resistance (RR) is calculated using the internal resistance (IR) drop. The RR of VOH fluctuates regularly with the maximum RR value of about 2.3 Ω g–1 which is placed in Figure –1. The better conductivity of MnVOH may be caused by the Mn2+ insertion, which is well confirmed by subsequent simulation results as depicted in Figure 2+ to VOH substrate, highest occupied molecular orbital (LUMO) orbitals of VOH are occupied and MnVOH displays good electronic conductivity.To further confirm the previous simulation results, the galvanostatic intermittence titration technique (GITT) was executed. The GITT plots Figure5a were Rct), which confirmed the low RR of MnVOH. The RCT may be related to the activation energy (Ea) for charge transfer during intercalation:At the same time, electrochemical impedance spectroscopy (EIS) as depicted in Figure A0 is a pre‐exponential constant, k is the ideal gas constant, and T is the temperature. The activation energy of charge transfer is related to the energy required for cationic solvent removal. Therefore, the low RCT of MnVOH represents a low activation energy, indicating that the energy required to desolvate Zn(H2O)62+ in MnVOH is lower, which promotes the insertion of Zn2+ into MnVOH. Therefore, the adjustment of the electronic structure of VOH by Mn2+ improves the electronic conductivity and further promotes the rate performance.Here, DZn), as shown in Figure DZn than that of VOH, which is consistent with its better rate performance. Structural simulation calculations are applied to explore the factors that may lead to changes of DZn. The simulation result indicates that hydration number can strongly affect the diffusion energy barrier EB of Zn(H2O)n2+, as shown in Figure 2+ and ligand orbitals of oxygen atoms of H2O and VOH, as shown in Figure n = 3 and octahedral coordination configurations with n = 5. Zn(H2O)n2+ has lower ligand field stabilization energy than other cases , as described in crystal field theory. According to the formula (3), it can be inferred that the bound water can promote the migration of Zn2+ by reducing the energy barrier. More detailed discussion of the relationship between Zn(H2O)n2+ and DZn will be demonstrated in another work since more calculations and experimental proof should be supply.The GITT also was used to calculate the zinc‐ion diffusion coefficients  and cycle life . The effects of Mn2+ insertion on the structure, Zn2+ migration behavior, and Zn2+ storage mechanism have been analyzed. First, Mn2+ forms a coordination bond with the oxygen of [VO] layers, which regulates the electronic structure of VOH and improves conductivity. Second, the strong coordination bonds reduce the interlayer distance and Mn2+ as “pillar” stabilizes the host structure. Third, the inserted Mn2+ affects the diffusion path of zinc ions and reduces the diffusion barrier. In addition, based on the relationship between the zinc ion diffusion coefficient and the diffusion energy barrier of Zn(H2O)n2+ with different bound water number, it is speculated that the bound water can promote the migration of Zn2+ by reducing the energy barrier. This work may provide a new understanding of the migration of zinc ions for ZIBs.In this work, the strategy of interlayer modification by inserting Mn4Experimental Section details are in the Supporting Information.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


To assess the knowledge of certified athletic trainers (ATs) on the presenting signs and symptoms of type 1 diabetes (T1D).We conducted a 31‐question survey of secondary school ATs recruited from the National Athletic Training Association that established demographic information, knowledge of presenting signs and symptoms of T1D, and previous personal or professional exposure to individuals with T1D. We report descriptive statistics and univariate analyses evaluating the characteristics associated with T1D knowledge. We then report a multivariable model incorporating age, gender, years of experience and education level with T1D knowledge as the dependent variable.n = 123) and frequent urination  as common presenting signs and symptoms of T1D, while fewer participants identified weight gain  or joint pain  as incorrect presenting signs and symptoms of T1D. Participants with over ten years of experience or previous exposure to individuals with T1D had increased T1D knowledge. Participants with advanced education (Master's degree or Doctorate) had no statistically significant difference in T1D knowledge compared to those with a Bachelor's degree. The only factor that demonstrated a significant association with T1D knowledge on multivariable analysis was the female gender.128 participants (92f:34m) met inclusion criteria and were included in this study. The majority of participants correctly identified frequent thirst  to childhood contacts have successfully reduced the rate of diabetic ketoacidosis at diagnosis of T1D. No previous educational interventions have included athletic trainers (ATs) and no literature reported ATs knowledge of the presenting symptoms of T1D and characteristics associated with increased knowledge. We report experienced ATs (>10 years) or previous exposure to an individual with T1D was positively associated with T1D knowledge in univariate analysis, while the only factor that demonstrated an association in multivariable analysis was the female gender. Educational interventions on the common presenting symptoms of type 1 diabetes (T1D) to childhood contacts have successfully reduced the rate of diabetic ketoacidosis at diagnosis of T1D.No previous educational interventions have included athletic trainers (ATs). No literature reported ATs knowledge of the presenting symptoms of T1D and characteristics associated with increased knowledge.Experienced ATs (>10 years) or previous exposure to an individual with T1D was positively associated with T1D knowledge in univariate analysis. The only factor that demonstrated an association in multivariable analysis was the female gender. Our study highlights the importance of targeting newly trained ATs in future T1D education.1The overall incidence rate of type 1 diabetes (T1D) in United States youth has increased 1.9% each year from 2001 to 2015, with the greatest incidence rate being in children aged 10‐14 and 15‐19.Attempts to improve public awareness of the presenting signs and symptoms of T1D have been successful in decreasing rates of DKA during diagnosis of T1D.This study aimed to bridge that gap by assessing the knowledge of presenting signs and symptoms of T1D and identifying factors that influence this knowledge through a national survey of ATs in the United States.2A national survey was powered to include at least 125 participants to capture the participants estimated to obtain statistical significance for differences in AT knowledge of T1D presenting symptoms. The survey was distributed electronically to 2975 secondary school ATs at a cost of $0.15 per survey, thus collecting data beyond what was statistically necessary was beyond the scope of the budget. The survey was subsequently closed after reaching the desired sample size. The survey was distributed in accordance with the National Athletic Training Association internal process to obtain a broad representative sample of ATs throughout a three‐week span in October 2019. Inclusion criteria included ATs in the United States, able to read, write and communicate in English, have Internet access, and willing to consent and complete study requirements.3The cross‐sectional survey study was approved by the Institutional Review Board at Penn State College of Medicine. Before proceeding to the survey, participants read a summary of explanation of research and were asked if they agreed to participate in the study. They were also asked eligibility questions, which either allowed them to continue to the survey, or notified them that they were not eligible, and thanked them for their time.A 31‐question survey was constructed using Qualtrics survey generation software.3.1The T1D knowledge assessment (range: −18 to 18) was scored based on the ability to correctly identify true and false symptoms of T1D. The scoring incorporated the ATs confidence level, as shown in Figure First, differences in T1D knowledge were examined using two‐sample t tests for demographics , professional and personal encounters with individuals living with T1D, and years of experience as an AT. An analysis of variance test determined the temporal impact of years of experience on T1D knowledge. We then report a multivariable model incorporating age, gender, years of experience and education level with T1D knowledge as the dependent variable.4n = 92), aged 25‐34 years , white , worked in a suburban community , and had a Master's  or Doctorate  degree in athletic training. Most participants have encountered an athlete living with T1D in a professional setting  or had personal exposure to an individual living with T1D . The majority of participants did not feel adequately prepared for the management of an athlete living with T1D  and are interested in continuing education in presenting signs and symptoms of T1D .A total of 128 participants (response rate = 6.4%) completed the survey, including representation from 35 states. Demographic information is presented in Table n = 123), frequent urination , changes in behaviour and mood  and changes in appetite . Fewer participants identified weight gain  or joint pain  as incorrect signs and symptoms of T1D. Figure n = 60) or low (n = 68) T1D knowledge group based on their T1D knowledge score relative to the median score.The majority of participants identified common presenting signs and symptoms of T1D such as frequent thirst  and years of experience as an AT (P = .003) were positively associated with T1D knowledge, particularly among ATs with 11‐15 years (P = .02) or greater than 15 years (P = .001) experience. Both previous professional (P = .04) and personal (P = .02) exposure to an individual with T1D was positively associated with T1D knowledge. Participants with advanced education (Master's degree or Doctorate in athletic training) had no significant difference in T1D knowledge compared to those with a Bachelor's degree in both univariable and multivariable analysis. There were no statistically significant differences in T1D knowledge based on race or area of work . The only factor that demonstrated a significant association with T1D knowledge on multivariable analysis was the female gender (P = .03). While the majority of participants  never raised concern of T1D in an undiagnosed athlete, those with increased T1D knowledge were more likely to have reported their concern of undiagnosed T1D in an athlete (P = .03).An increased age .n = 123) and frequent urination  as common presenting signs and symptoms of T1D. There were fewer ATs able to identify weight gain  as an incorrect symptom of T1D. Knowledge of the four main presenting symptoms of T1D  is critical for early diagnosis of T1D because all children with new‐onset T1D present with one, and 98% present with two of the four main symptoms.Our first research objective is the initial determination of secondary school ATs knowledge of the presenting signs and symptoms of T1D. This study specifically focused on secondary school ATs because the incidence rate of T1D is highest among children ages 10‐14 and 15‐19.Our second research objective is identifying factors that influence ATs knowledge of T1D. We are the first to report that ATs with a Bachelor's degree had no significant difference in T1D knowledge than ATs with advanced education of a Master's degree or Doctorate in athletic training. We hypothesize this may result from limited requirements in post‐professional education for the identification and referral of T1D. Currently, undergraduate AT education consists of basic identification of T1D signs and symptoms for referral to the athlete's primary care provider or qualified members of the sports medicine team for disease confirmation. Following the diagnosis of T1D, ATs continue to monitor the athletes’ signs and symptoms and facilitate adherence to the athletes’ prescribed diabetic treatment plan. Management includes care of emergent and nonemergent hyperglycaemia and hypoglycaemia.,Beginning in 2022, The Commission on Accreditation of Athletic Training Education requires professional athletic training programs to be at the Master's degree level to maintain accreditation.Our data suggest ATs are learning from their on‐the‐job experience managing an athlete with T1D. Participants with over ten years of experience and previous exposure to individuals with T1D had increased T1D knowledge. These findings are consistent with a 2007 survey of 193 ATs, which identified AT age and years of experience as predictors of increased general diabetes knowledge.Dr Tamara Oser ‐Consultant, Cecilia Health (current). Consultant and grantee, Helmsley Charitable Trust (current). Consultant, Glucagon Multidisciplinary Working Group, Xeris Pharmaceuticals (2019). Primary Care Consultant, Afrezza Inhaled Insulin Working Group, MannKind Corporation (2019). No other authors report any conflicts of interest.KTB, TO, AZ, JP and MS performed and designed the research study and wrote and edited the final manuscript. KTB and JP performed the data analysis.


Oryza sativa), the PLASMA MEMBRANE INTRINSIC PROTEIN (PIP) family of aquaporin has 11 members, OsPIP1;1 to OsPIP1;3, and OsPIP2;1 to OsPIP2;8, which are hypothesized to facilitate the transport of H2O and other small compounds across cell membranes. To date, however, only OsPIP1;2, OsPIP2;1, and OsPIP2;4 have been demonstrated for substrate selectivity in their source plant (rice). In this study, OsPIP2;2 was characterized as the most efficient facilitator of H2O transport across cell membranes in comparison with the other 10 OsPIPs. In concomitant tests of all OsPIPs, four genes  were induced to express in leaves of rice plants following a physiological drought stress, while OsPIP2;2 was expressed to the highest level. After de novo expression in frog oocytes and yeast cells, the four OsPIP proteins were localized to the plasma membranes in trimer and tetramer and displayed the activity to increase the membrane permeability to H2O. In comparison, OsPIP2;2 was most supportive to H2O import to oocytes and yeast cells. After de novo expression in tobacco protoplasts, OsPIP2;2 exceeded OsPIP1;3, OsPIP2;1, and OsPIP2;4 to support H2O transport across the plasma membranes. OsPIP2;2‐mediated H2O transport was accompanied by drought‐tolerant responses, including increases in concentrations of proline and polyamines, both of which are physiological markers of drought tolerance. In rice protoplasts, H2O transport and drought‐tolerant responses, which included expression of marker genes of drought tolerance pathway, were considerably enhanced by OsPIP2;2 overexpression but strongly inhibited by the gene silencing. Furthermore, OsPIP2;2 played a role in maintenance of the cell membrane integrity and effectively protected rice cells from electrolyte leakage caused by the physiological drought stress. These results suggest that OsPIP2;2 is a predominant facilitator of H2O transport in relevance to drought tolerance in the plant.In rice ( In plans Brown, . These ps Brown, , 2015b. s Brown, .Oryza sativa) occupies a prominent position in global food security due to its vast production that is used to feed a huge population in the world. Rice also is representative of cereal crops with respect to the function of PIPs/AQPs in H2O transport tightly associated with water utility, osmotic response, and drought tolerance  and OsPIP1;2‐overexpressing rice plants  when it was infecting plants of the susceptible rice variety Nipponbare . PEG with molecular mass 4,000–8,000 effectively induces physiological drought in a variety of plant species, and PEG6000 has been mostly used  were reproduced and maintained in this lab. Rice and tobacco seeds were germinated in flat plastic trays filled with a substrate containing an industrial soil branded as PINDSTRUP. Five days later, the germinal seedlings were moved into 12‐L pots (two plants per pot) filled with the same soil. Seeds were incubated, and the plants were grown in environment‐controlled chambers under 28°C, 12‐h light at 250 ± 50 μmol quanta/m2/s and a relative humidity of 80%.Seeds of the rice variety Nipponbare were initially provided by Professor Hongsheng Zhang  and then reproduced and maintained in this lab. Seeds of tobacco 6000 was used as an inducer of physiological drought stress  and applied in parallel to the deionized water control to incubate plants by immersing the root system. In this study, 15‐day‐old rice seedlings growing in the PINDSTRUP soil were taken out carefully by pushing the soil away using a finger, cleaned gently by soft washing with tap water to remove the surrounding soil scraps, and then placed into plastic tubes containing deionized water. After a 12‐h acclimation, seedlings were moved into new tubes containing deionized water (control) or an aqueous solution of PEG6000 justified to 0%, 5%, 10%, 15%, 20%, 25%, and 30% w/v, respectively. Seedlings were monitored in the subsequent 10 h by automatic photography at 10‐min intervals. Chronological development of drought syndromes was analyzed.PEG2.2EF1α gene as a reference . All the qRT‐PCR experiments were performed with the QuantStudio®3 Real‐Time PCR Instrument (ThermoFisher Scientific) and using the constitutively expressed 2.32.3.1OsPIP1;3, OsPIP2;2, OsPIP2;2, and OsPIP2;4 were further investigated. Complementary DNAs (cDNAs) of OsPIPs were obtained by RT‐PCR amplification of total RNAs isolated from leaves of 15‐day‐old rice plants. For use in transformation of African clawed frog (Xenopus laevis) oocytes, every OsPIP cDNA was fused to the enhanced green‐fluorescent protein (eGFP) gene or a six‐his tandem sequence by conventional recombination methods  and then glued with T4 ligase (Thermo Fisher). The recombinant vector was linearized with restriction enzyme NotI (TAKARA) and purified in RNase‐free water. A constant amount of 1 μg linearized DNA was applied for an in vitro transcription using RiboMAX™ Large‐Scale RNA Production Systems‐T7 (Promega). The resulting cRNA was injected into oocytes. The transformed oocytes were incubated in sterile ND96 solution  amended with penicillin and streptomycin. After incubation for 36 h, oocytes were observed between 495 and 520 nm using 488‐nm argon‐ion laser excitation with a Zeiss LSM700 laser scanning confocal microscope. Fusion proteins were analyzed by immunoblotting of membrane proteins isolated from the transformed oocytes using a previously described protocol . The recombinant vector was transformed into competent cells of the wine‐brewing yeast (Saccharomyces cerevisiae) strain NMY51 in transformation solution . The positive transformants were cultured in SD‐Ura  media with 2% w/v galactose overnight at 30°C and harvested in phosphate buffer solution . Membrane proteins were isolated from the transformed yeast cultures using a previously described protocol  in leaves of tobacco was performed by the agroinoculation method  gene from a previously used yeast vector  and filtered with a 75‐μm nylon mesh. The filtrate collection was centrifuged at 100g and 4°C for 2 min. The supernatant was discharged. The precipitate was resuspended with the washing solution and centrifuged at 1000g and 4°C for 5 min. The supernatant was removed again. Tubes containing the protoplasts were placed on ice bath and suspended with the MMg solution  and adjusted to 5 × 106 protoplasts/ml. This protoplast suspension was supplied with 10 μl of the recombinant vector that carries an OsPIP insert, 110 μl of PEG‐Ca2+ , followed by incubation on ice bath for 30 min. At this time point, the transformation was terminated by adding 440 μl of the W5 solution . The suspension of transformed protoplasts was centrifuged at 1000g and 4°C for 5 min, the supernatant was removed, and the WI solution  was gently added. The resulting protoplast suspension was shifted into wells of a 96‐well plate, incubated for 12 h under room temperature and weak illumination, and used in further tests.This was performed by the chemical mediation method  vector pRBDV  was determined by measuring volume changes of the protoplasts as previously described  . Ten microliters of benzoyl PAs in methanol solution was injected into a 4.6 × 250‐mm reverse‐phase Kromasil C18 column  at 25°C. PAs were eluted from the column with 64% methanol at a flow rate of .7 ml/minute. PA peaks were detected with the plate reader at 254 nm.A previously described protocol  values.Electrolyte leakage from leaves of rice seedlings was measured using a conductivity meter. Briefly, 1‐cm leaf segments were immersed into deionized water by the aid of a vacuum pump, followed by incubation for 1 h under 25°C. The resulting solution containing electrolytes leaked from leaf segments were subjected to measurements with the Leitz DDS‐307 conductivity meter  under 220 V, 50 Hz, and 1.021 Kohlrausch coefficient. Similar measurements on deionized water were conducted in control. Extents of electrolyte leakage from WT, 2.6All experiments were repeated at least three times with similar results. Quantitative data were analyzed using the commercial IBM SPSS19.0 software package Shi, . Homogen33.1OsPIP genes in plants of the rice variety Nipponbare following PEG6000 treatment  caused no responses, but drought syndromes were induced when PEG6000 concentration was increased to 10% and higher  and those incubated in deionized water (−PEG6000) were used 3 days later in OsPIP expression assessments by qRT‐PCR. The qRT‐PCR analyses using total RNA isolated from the leaves indicated that all OsPIPs responded to the physiological drought stress, displaying significantly (P = 1.2 × 107) enhanced expression in contrast to the steady‐state expression levels in control  for additional OsPIPs. Evidently, OsPIP2;2 exceeds all the other OsPIP genes in response to the physiological drought stress and displays the highest level of induced expression under the stress condition, indicating that OsPIP2;2 is likely to take part in water relations of rice.To evaluate the effects of the physiological stress on ly P = 1. × 107 en3.2OsPIP1;3, OsPIP2;1, OsPIP2;2, and OsPIP2;4 have a role in mediating H2O transport in living cells of both the wine‐brewing yeast and African clawed frog oocytes. LSCM performed on oocytes 48 h after transformation indicated that all the fusion proteins  were localized to oocyte membranes, in contrast to the absence of any protein fluorescence in control  and OsPIP‐YFP, respectively. When transformed protoplasts were incubated in the ND98 medium that held an extracellularly low osmatic gradient, Pf increases over the control level were substantially induced by OsPIP2;1‐YFP, OsPIP2;2‐YFP, and OsPIP2;4‐YFP  are regarded as a physiological indicator of drought tolerance activation in plants  increased by OsPIP2;1‐YFP, OsPIP2;2‐YFP, and OsPIP2;4‐YFP in contrast YFP only. In these fusion proteins, OsPIP2;4‐YFP displayed the greatest activity in increasing proline and PAs, but OsPIP1;3‐YFP had little effect on concentrations of both compounds  caused considerable reductions in protoplast size, indicating protoplast contraction due to water outflux  decreases in diameters of the protoplasts no matter if they had been transformed with YFP only or with the OsPIP2;2‐YFP fusion gene  or display OsPIP2;2 silencing  or overexpression  mediated by the RBDV vector  increased by OsPIP2;2‐OE but decreased by OsPIP2;2‐Si compared to the canonical expression  induced in contrast to the steady‐state expression levels found in the PEG‐absent control plants. Moreover, COR413‐TM1 exceled OsABF1, OsPP48, and OsPP108 in expression extents in all plants. Noticeably, the OsPIP2;2‐OE plants highly exceeded but OsPIP2;2‐Si was considerably inferior to the WT in supporting PEG6000‐induced expression of all the genes . This is a small but substantial advance in exploring substrate selectivity and specificities of rice PIPs. The paucity in understandings of rice PIPs is incredible if we do not have the opportunity to scrutinize previous literatures. Previously to the present study, only three OsPIPs have characterized clearly to execute the substrate‐transporting function in their source plant  system and characterized OsPIP2;2 as a strong moderator for H2O import into cells of the PIP‐transformed yeast cultures  trafficking and by regulating the intracellular responses against drought stress, respectively (Figure 2O2 transport will better explain the molecular basis of the membrane integrity. We recently proposed that H2O‐transporting AQPs are also potential channels for H2O2 trafficking (Wang, Schoebel, et al., 2, H2O (Li et al., 2O2 transport across the PMs. H2O2 is generated in the apoplast upon induction by pathogen infection, transported by AtPIP1;4 into the cytoplasm, and therefore participates in immunity signal transduction (Tian et al., 2O2 transport, it may contribute to the membrane integrity by facilitating the stress‐induced apoplastic H2O2 into the cytoplasm and therefore reducing the oxidative pressure toward the cell membranes (Figure Based on these analyses, we propose a model that hypothesizes how OsPIP2;2 execute its functions toward He Figure . In the y Figure . In the y Figure , which hy Figure . Furthers Figure . VerificOsPIP2;2‐silencing, and OsPIP2;2‐overexpressing plants and protoplasts in response to the physiological drought stress demonstrate the critical role of OsPIP2;2 in H2O transport and drought tolerance. This finding should stimulate further studies to characterize the functional relationship of OsPIP2;2 in H2O transport and drought‐tolerant responses. It is necessary to study the functional relationship between OsPIP2;1‐mediated drought tolerance and ABA signaling. It is also necessary to study if OsPIP2;10 has a role in H2O2 transport in relevance to plant defenses against biotic and abiotic stresses.In conclusion, the physiological, molecular, and cytological performances of the WT, J.B., X.W., L.Z., and H.D. designed the experiments. J.B., X.W., X.Y., X.C., Y.H., Z.W., and Y.M. performed the experiments and analyzed the data. X.W., L.Z., and H.D. wrote the article. L.Z. and H.D. conceived the study. All authors approved the final manuscript.FIGURE S1 Alignments of 11 OsPIP coding sequences.FIGURE S2 Northern blotting of the OsPIP RNAs isolated from leaves of PEG6000‐stressed rice plants.Click here for additional data file.


A fundamental goal in the drive to understand and find better treatments for dementia is the identification of the factors that render the aging brain vulnerable to neurodegenerative disease. Recent evidence indicates the integrity of the blood–brain barrier (BBB) to be an important component of functional failure underlying age‐related cognitive decline. Practical and sensitive measurement is necessary, therefore, to support diagnostic and therapeutic strategies targeted at maintaining BBB integrity in aging patients. Here, we investigated changes in BBB permeability to endogenous blood water in the aging brain.A multiple‐echo‐time arterial spin‐labeling MRI technique, implemented on a 9.4T Bruker imaging system, was applied to 7‐ and 27‐month‐old mice to measure changes in water permeability across the BBB with aging.We observed that BBB water permeability was 32% faster in aged mice. This occurred along with a 2.1‐fold increase in mRNA expression of aquaporin‐4 water channels and a 7.1‐fold decrease in mRNA expression of α‐syntrophin protein, which anchors aquaporin‐4 to the BBB.Age‐related changes to water permeability across the BBB can be captured using noninvasive noncontrast MRI techniques. Click here for author‐reader discussions Our previous work demonstrates the sensitivity of a multiple‐echo‐time (multi‐TE) ASL technique to the presence of AQP4 water channels, an important route for water transport across the BBB.22.1ad libitum.All experiments were performed in accordance with the European Commission Directive 86/609/EEC  and the United Kingdom Home Office Animals (Scientific Procedures) Act (1986). All mice were acclimatized in an animal house, prior to data acquisition, with a 12‐hour light/12‐hour dark cycle with food and water provided Eight male C57Bl/6JRj mice at 27 ± 1 months old  and 10 male C57Bl/6JRj mice at 7 ± 1 months old  were used in this study. All mice were induced with 2% isoflurane anesthetic in a mixture with room air at 1.0 L/min, which was manually adjusted between 1.75%‐1.5% to maintain the respiration rate at approximately 100 bpm, which was measured using a pressure pad and monitored throughout the scan. A rectal probe  was used to monitor the core body temperature, which was maintained at 37.0 ± 0.5°C via an adjustable water bath supplied to a mouse heating pad .aδ) were also captured in the same group of adult and aged mice with a separate acquisition using imaging parameters: TI = 200 ms, 300 ms, 400 ms, 500 ms; TE = 10 ms; TR = 10 000 ms; data matrix = 64 × 64; slice thickness = 2 mm; partial Fourier = 32 + 4 lines of k‐space; and repetitions = 10.Images were acquired on a horizontal‐bore 9.4T Bruker preclinical system  using a 440‐mT/m gradient set with an outer and inner diameter of 205 mm and 116 mm, respectively (BioSpec B‐GA 12S2), a 86‐mm volume transit RF coil, and a four‐channel receiver‐array coil designed for the mouse brain (Bruker). The ASL image acquisition was based on a flow‐alternating inversion recovery sequence with a single‐shot spin‐echo echo planar imaging readout. A single‐slice flow‐alternating inversion recovery protocol was implemented (slice thickness = 2 mm), with a slice selective inversion pulse thickness of 8 mm and a global nonselective pulse (no slice‐select gradient). Imaging parameters were inflow time (TI) = 800 ms and 1500 ms; echo times (TEs) = 8 ms, 10 ms, 12 ms, 15 ms, 18 ms, 23 ms, 30 ms, 40 ms, 50 ms, 65 ms; pulse repetition time (TR) = 5000 ms; data matrix = 64 × 64; field of view = 25 × 25 mm; slice thickness = 2 mm; partial Fourier = 32 + 4 lines of k‐space; and repetitions = 10. The ASL data at TI = 800 ms and TE = 10 ms were used to estimate cortical cerebral blood flow (CBF). Measurements of the arterial transit time (2.2aδ) reflects the arrival time of the labeled bolus of blood water to the imaging region of interest and in this work aδ is estimated using a separate multiple short inflow time (multi‐TI) ASL acquisition. At short TIs the ASL signal, ΔM, has, approximately, a linear dependence TI, according to the pulsed arterial spin‐labeling biophysical modelA is a constant factor, dependant on CBF (A = 2α· MOA· CBF/λ), T1a is the longitudinal relaxation of the arterial blood , R1a is the longitudinal relaxation rate of the arterial blood, τ is the temporal length of the labeled bolus (previously measured at 1.7 seconds in the mouse brain), and λ is the partition coefficient .MIV and ΔMEV are intravascular and extravascular ASL signal‐weighting factors, respectively, TE is the echo time, T2IV is the T2 value of the intravascular compartment, and T2EV is the T2 value of the extravascular tissue.The multi‐TE ASL method is used to separate IV‐EV compartments to model BBB permeability to water. This is achieved by fitting the ASL signal decay to a two‐compartment biexponential model at intermediate TIs  indicates the time for magnetically labeled vascular water to transfer across the BBB into brain tissue after entering the imaging slice:The water exchange time  and α‐syntrophin protein (SNTA1) to probe possible age‐related changes to AQP4 polarization. mRNA expression measurements were taken from the cortical brain region of seven C57Bl/6JRj mice at 27 ± 2 months old and seven C57Bl/6JRj at 7 ± 2 months old (the same mice cohort used for MRI BBB water permeability measurements). All mice were euthanized by overdose with an intraperitoneal injection of sodium pentobarbital (10 mL/kg), the brain was removed, hemisected, and the cortex dissected and snap frozen on dry ice.The noninvasive BBB water permeability measurements were accompanied by assessment of mRNA expression profiles of aquaporin‐4 water channels  were used. Each reaction contained 10‐µL Mastermix, 8‐µL RNase‐free water, 1‐µL gene expression assay, and 1‐µL cDNA, and were performed in triplicate. Each of the mRNA expression levels were determined using the 2−∆∆Ct methodTotal mRNA from each brain region was extracted using the RNeasy Plus Microkit . Total mRNA was converted to cDNA using the QuantiTect Reverse Transcription kit (QIAGEN), which was quantified using the Eppendorf Mastercycler with Realplex software  and the TaqMan Gene Expression Mastermix and Taqman Gene Expression assays . Taqman Gene Expression assays for 2.5All imaging data were processed using MATLAB 2018a . Automatic scaling of image intensity was applied during the online reconstruction on the Bruker scanner; this was corrected offline to ensure the correct relative signal intensities across different TE and TI values.MIV and ∆MEV variables . All data are reported as the mean and the associated error (± standard deviation [SD]) across each group. A two‐way ANOVA with Bonferroni’s multiple comparisons test was performed on the intravascular fraction to evaluate the interaction between the two inflow times and the effect of age between the adult and aged mice. A two‐tailed unpaired Student 3P = .016; Figure The mean cortical water exchange time was 32% lower in the aged mice (345 ± 71 ms) relative to the adult mice . The intravascular fraction was measured to be 0.76 ± 0.12 for adult mice compared with 0.61 ± 0.08 for the aged mice at TI = 800 ms, and 0.28 ± 0.05 for adult mice compared with 0.22 ± 0.09 in the aged mice at TI = 1500 ms  in the cortical region was measured to be 13.5 ± 1.2 ms and 11.3 ± 4.1 ms for the aged mice compared with 20.6 ± 1.4 ms and 14.3 ± 4.0 ms for the adult mice, at TI = 800 ms and TI = 1500 ms, respectively . The extravascular tissue T2EV  was measured at 37.1 ± 1.6 ms in the adult mice compared with 35.6 ± 1.5 ms in the aged mice at TI = 800 ms, and 34.5 ± 1.3 ms in the adult mice compared with 32.8 ± 1.6 ms in the aged mice at TI = 1500 ms (P = .003).P = .77; Supporting Information Figure P = .93; Supporting Information Figure There were no significant age‐related differences in the arterial transit time in the cortical region in aged mice (125 ± 34 ms) and adult mice  compared with the adult mice , which indicates a downregulation of the α‐syntrophin protein in the aging mouse brain, which is in part responsible for anchoring AQP4 to the BBB  in the aged mouse brain cortex relative to the adult mice, indicating decreased BBB resistance to water penetration with age. To our knowledge, this is the first demonstration of measurable changes to water permeability across the BBB in the aging brain. An increase in water exchange across the BBB has previously been reported in a rat model of AD, which led the authors to suggest that changes in BBB permeability to water may elucidate early breakdown in the BBB, as the gold‐standard DCE technique was unable to differentiate between the animal groups.Aqp4 knockout mice.Aqp4 mRNA expression to be significantly higher (2.1 ± 0.9‐fold increase) in the aged mice. We also observed a marked downregulation (0.14 ± 0.09‐fold change: ~7‐fold decrease) of mRNA expression of SNTA1 in the aged mouse brain, an anchoring protein of AQP4 to the BBB that gives an index of the extent of AQP4 polarization. We hypothesize that an increase in AQP4 expression and decrease in AQP4 polarization would have opposing effects on the rate of water exchange time, which makes it difficult to predict the overall influence of AQP4 water‐channel proteins on water permeability across the BBB. Therefore, further studies are required to elucidate the precise changes to the structure of the BBB that account for the increased permeability to water with aging, recorded here.Brain AQP4 proteins are intramembrane bidirectional channels, central to water exchange across the BBB. We have previously demonstrated sensitivity of multi‐TE ASL to the presence of AQP4, recording a 31% longer cortical water exchange time in Several studies report an increased expression of AQP4 to be associated with the aging process, with evidence of further enhancement in AD cases.1, which may change with aging. The arterial transit time was only measured in the cortical brain region, however it replicates the results from a previous study measuring the arterial transit times in different regions of the aging mouse brain.There were no measurable differences in cortical CBF detected with aging in the present study. A relatively subtle (13%) reduction of cerebral perfusion has been previously reported in aged mice (23‐month‐old compared with 6‐month‐old mice).The multi‐TE ASL technique has potential as a clinical tool to measure BBB water permeability with aging in the human brain. In the present study the technique has been optimized for the cortical region of the mouse brain because of its relatively large size and proximity to the surface coil. A higher resolution readout would be necessary to target smaller brain regions, such as the hippocampus, which are associated with AD. To our knowledge, there have not been any techniques that have measured BBB permeability to water in the aging brain. Previous studies have measured an increased BBB permeability in the hippocampus and the caudate nucleus in the aging brain using contrast agent with DCE‐MRI techniques.In summary, we have demonstrated evidence for compromised BBB integrity in the aging brain via noninvasive detection of increased BBB permeability to water. This finding indicates that an increase in BBB permeability to water occurs with age, which provides encouragement that ASL‐based BBB water permeability assessment techniques can noninvasively capture age‐related changes to the BBB that may play a key mechanistic role in the pathogenesis of neurodegenerative conditions such as AD.FIGURE S1 ASL signal decay across the range of echo times (TE) at inflow times of 800 ms and 1500 ms, for individual adult mice (n = 9) with the mean value and the associated error (± standard deviation) indicated on each plotFIGURE S2 ASL signal decay across the range of echo times (TE) at inflow times of 800 ms and 1500 ms, for individual aged mice (n = 8) with the mean value and the associated error (± standard deviation) indicated on each plotFIGURE S3 A, Cortical arterial transit time measurements. B, Cortical cerebral blood flow (CBF) measurement in adult and aged mice. Each plot indicates the individual animal measurements along with mean value and the associated error (± standard deviation)Click here for additional data file.


Sci., 2020, 11, 3986–3995, DOI: 10.1039/D0SC00738B.Correction for ‘Transforming colloidal Cs After the publication of our manuscript, an inquiry from a reader pointed out an earlier publication that was not cited in the context of prior art relevant to our study. We thank the reader for their interest in our work. Prompted by the inquiry, we thought it would be appropriate to acknowledge a few earlier and relevant publications that escaped our attention.et al.1 have reported room temperature excitation–emission maps (photoluminescence maps) of powders containing a mixture of Cs4PbBr6 and CsPbBr3 compounds 3 show a qualitatively similar dip in the intensity of ∼504 nm emission from CsPbBr3 when excited at ∼314 nm, the absorption wavelength of Cs4PbBr6. In contrast to the above-mentioned observations, Cs4PbBr6–CsPbBr3 heterostructured nanocrystals were not emissive in UV at room temperature but showed a weak ∼376 nm emission from Cs4PbBr6 only when cooled down to ∼35 K .3 The three studies share similar photoluminescence measurements and chemical formulas of the studied compounds. However, the synthetic origins and structures of the samples, together with discussions of the observed phenomena, are different in the three studies.The room temperature photoluminescence maps of Cset al.4 have reported effective colloidal stabilization of CsPbBr3 nanocrystals over a wide range of concentrations, from 400 mg ml−1 to 4 × 10−6 mg ml−1 of inorganic content in toluene  compound (PMAO) used in our work to transform Cs4PbBr6 nanocrystals into CsPbBr3 nanocrystals yielded colloids of PMAO-capped CsPbBr3 nanocrystals which survive several rounds of washing and are stable in the concentration range of ∼26 mg ml−1 to ∼1 × 10−4 mg ml−1 . It is notable that both lecithin and PMAO increase the colloidal stability of CsPbBr3 nanocrystals despite an apparently different surface binding chemistry and a different way of being introduced into the nanocrystal preparation.Krieg The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


The NF-ĸB transcription factor is a critical regulator of immune homeostasis and inflammatory responses and is a critical factor in the pathogenesis of inflammatory disease. The pathways to NF-ĸB activation are paradigms for signal-induced ubiquitination and proteasomal degradation, control of transcription factor function by subcellular localisation, and the control of gene transcription and physiological processes by signal transduction mechanisms. Despite the importance of NF-ĸB in disease, the NF-ĸB pathway remains unexploited for the treatment of inflammatory disease. Our understanding of NF-ĸB comes mostly from studies of transgenic mice and cell lines where components of the pathway have been deleted or over expressed. Recent advances in quantitative proteomics offer new opportunities to understand the NF-ĸB pathway using the absolute abundance of individual pathway components. We have analysed available quantitative proteomic datasets to establish the structure of the NF-ĸB pathway in human immune cells under both steady state and activated conditions. This reveals a conserved NF-κB pathway structure across different immune cell lineages and identifies important differences to the current model of the NF-ĸB pathway. These include the findings that the IKK complex in most cells is likely to consist predominantly of IKKβ homodimers, that the relative abundancies of IκB proteins show strong cell type variation, and that the components of the non-canonical NF-ĸB pathway are significantly increased in activated immune cells. These findings challenge aspects of our current view of the NF-κB pathway and identify outstanding questions important for defining the role of key components in regulating inflammation and immunity. •Quantitative proteomic datasets offer new insights into the NF-κB pathway.•The structure of the NF-κB pathway is highly conserved in human immune cells.•The IKK complex is likely composed mainly of IKKβ homodimers.•The relative abundancies of IκBα, −β and -ε show strong cell type variation.•Components of the non-canonical NF-ĸB pathway are greatly increased by activation. The top layer (plasma) was discarded and the layer beneath containing PBMCs was collected. PBMCs were washed three times in PBS and centrifuged at 300g for 10 min between the washes. CD14+ monocytes were isolated from PBMCs with EasySep Human CD14 Positive Selection kit according to the manufacturer's instructions . The isolated monocytes were subsequently resuspended at 1 × 106 cells/ml in complete Alpha Minimum Essential Medium  supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% l-glutamine. Cells were stimulated with 100 ng/ml LPS  for 16 h prior to analysis by western blotting.Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood of healthy donors by density gradient. Blood was diluted 1:1 in sterile phosphate-buffered saline , carefully applied to 3 ml of Ficoll-Paque Plus  and layered by centrifuging at 4002.3Whole cell lysates were prepared from cells previously washed twice in ice-cold PBS and suspended in RIPA buffer containing 50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% deoxycholate, 150 mM NaCl, 1 mM EDTA, supplemented with 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4, 2 μg/ml aprotinin, 1 μg/ml pepstatin and 1 μg/ml leupeptin. Lysates were resolved using Tris-Glycine SDS PAGE, transferred to nitrocellulose membranes and immunoblotted with specific antibodies. Anti-p105/p50 (HPA027305) and anti p100/p52 (HPA008422) were obtained from Atlas Antibodies. Anti-cRel (sc-71) and anti-RelB (sc-226) were obtained from Santa Cruz Biotechnology. Anti-p65 (A301-824A) was obtained from Bethyl Laboratories. Anti-α-tubulin antibody (T6074) was purchased from Sigma. Western blots were imaged using a digital chemiluminescence scanner (LiCor).3To determine the structure of the NF-κB pathway in human immune cells we analysed the quantitative proteomic dataset recently generated by Reickman and colleagues 3.11:IKKβ1:NEMO2 has been recently proposed based on crystallographic and quantitative analyses of the interaction between N-terminal NEMO and C-terminal IKK fragments + T cells, naïve B cells, monocytes and myeloid dendritic cells (mDCs) 2:NEMO2 configuration would require the presence of significant pools of IKKs unbound by NEMO. This analysis also reveals that the majority of IKK complexes are composed of IKKβ homodimers, assuming all IKKs are dimerised. In addition, there is significant variation in IKKα/β:NEMO ratios across cell types which suggests that the composition of the IKK complex may vary in different cells (+ T cells (IKKα/β:NEMO 2.87 ± 0.32) would allow an IKK complex composed entirely of IKKβ and NEMO alongside free IKKα. In contrast, neutrophils appear to have by far the lowest ratio of IKKα/β:NEMO (0.54 ± 0.26) suggesting that there are significant amounts of NEMO not bound to IKKs in these cells. Much of the previous work on the composition of the IKK complex has come from studies involving cell lines such as HeLa The IKK complex is a tripartite complex composed of the homologous kinases IKKα and IKKβ, and the scaffold protein NEMO s (mDCs) C. Calculs (mDCs) D. Assumint cells E. For ex3.2+ T cells, IĸBβ is the most highly expressed IĸB protein  while BCL-3 protein is detected in T regulatory cells, basophils, myeloid and plasmacytoid dendritic cells and NK cells E. IĸBζ wonocytes F. This i3.36 in neutrophils to 3.4 × 105 in naïve B cells with an average of 5.9 × 105 per cell across all cell types . This revealed that across all cell types the abundances of the IKK complex, RelA and IĸBβ are not substantially altered following activation B. Howevedy state B. As exp B cells B. We con B cells C and D. 3.5The activation induced degradation and re-synthesis of IĸB proteins is a critical regulatory feature of the NF-ĸB pathway As previously noted, the atypical IĸB proteins BCL-3, IĸBζ and IĸBNS are absent or expressed at very low levels in cells at the steady state To provide a further comparison of the overall structure of the NF-ĸB pathway in resting and activated cells we next generated a similarity matrix based on average protein copy number per cell of all factors across all cell types. This analysis revealed that the core NF-ĸB components in the steady state are the IKK complex, IĸBα, IĸBβ, IĸBε, RelA, NFKB1 and cRel , essenti4Quantitative proteomic data offer a unique opportunity to assess the current model of the NF-ĸB pathway. In this study we have used open access data from primary human immune cells in the steady and activated states to measure the abundance and stoichiometry of the core components of the pathway. Overall the analysis shows a strong conservation of pathway structure across different cell types and lineages. The analysis also reveals that the structure of the NF-ĸB pathway is dynamic in response to activating stimuli and that increased RelB and NFKB2 protein levels is a general feature of activated immune cells. The majority of the core pathway components are present in highly similar abundancies in different cell types, revealing that the NF-ĸB pathway in different cell types is broadly the same in terms of structure and abundance. An exception to this is the low abundance of NF-κB pathway components in T regulatory cells, an observation which correlates strongly with the requirement for these cells to supress NF-κB activity to maintain function 1:IKKβ1:NEMO2. However, assuming that there is no free pool of these proteins outside the IKK complex, the analysis here indicates a stoichiometry of (IKKα/β)2:NEMO1. Moreover, in contrast to cell lines such as HeLa, the relative excess of IKKβ over IKKα in primary cells indicates that the majority of IKK complexes are likely composed of IKKβ homodimers. Indeed, in some cell types such as CD4+ T cells, the excess of IKKβ over IKKα suggests the possibility that the IKK complex is composed exclusively of IKKβ and NEMO, since NEMO does not directly bind IKKα While the broad similarity of the NF-κB structure in primary immune cells will allow for the extrapolation of observations in one cell type to another, the obvious dissimilarity to cells commonly used to study NF-ĸB suggests caution in extrapolating findings from cell lines to primary cells. This is clearly illustrated when considering the stoichiometry of the IKK complex which is widely accepted to be composed of equal amounts of IKKα and IKKβ, with a number of different stoichiometries proposed including IKKαThe non-canonical pathway of NF-ĸB activation has been predominantly linked to a relatively small number of TNFR superfamily members including CD40, RANK, Lymphotoxin β receptor and BAFF receptor Nfkb1 deletion in mice leads to a relatively moderate immune phenotype when compared to deletion of Nfkbia+ and CD8+ T cells + and CD8+ cells RelA is the most abundant component of the NF-κB pathway while in human CD4+ and CD8+ T cells NFKB1 is the most abundant component. It is noteworthy that Nfkb1+\- mice do not have an overt immune phenotype NFKB1 haploinsufficiency is the most common monogenic cause of combined variable immunodeficiency in Europeans IĸBα is the prototypic member of the IĸB family of proteins and its deletion in mice results in death shortly after birth The atypical IĸB proteins BCL-3, IĸBζ, IĸBNS and IĸBη are located predominantly in the nucleus where they act to modulate the activity of specific NF-ĸB dimers An overview of the patterns of abundance between the components of the NF-ĸB pathway in all cell types identifies the core pathway components in both the steady and activated states. Unsurprisingly, the IKK complex, the canonical subunits RelA, NFKB1 and cRel, and the IĸB members IĸBα, IĸBβ and IĸBε form the core in the steady state. In the activated state the core components of the pathway are different and includes all the NF-ĸB subunits including NFKB2 and RelB and the atypical IĸB proteins BCL-3, IĸBζ and IĸBNS, reflecting changes in the abundance of RelB, NFKB2 and the atypical IĸB proteins. This analysis raises important questions when considering how the NF-ĸB pathway may be exploited for therapeutic benefit. To date, the key strategy for preventing NF-ĸB dependent inflammation has been to inhibit the IKK complex using IKKβ kinase inhibitors, which unfortunately leads to severe toxicity that precludes their clinical application In summary, quantitative proteomic analysis offers unique insights into the structure of the NF-ĸB pathway in primary cells which differs in important ways from those of cell lines commonly used in its study. It raises important questions on the nature of the IKK complex and the role of individual IĸB proteins that require further experimental investigation in order to answer. Moreover, it shows that the structure of the NF-ĸB pathway is dynamic following cellular activation suggesting new approaches to harnessing this pathway for therapeutic benefit.The authors have no conflict of interest.


However, there has been a lasting debate about the fact that the octahedra are found to exclusively elongate. In contrast, for Na9Bi5Os3O24, the octahedron around Os6+(5d2) is heavily compressed, lifting the degeneracy of the 2gt set of 5d orbitals such that in the sense of a JT compression a diamagnetic ground state results. This effect is not forced by structural constraints, the structure offers sufficient space for osmium to shift the apical oxygen atoms to a standard distance. The relevance of these findings is far reaching, since they provide new insights in the hierarchy of perturbations defining ground states of open shell electronic systems.The Jahn–Teller (JT) theorem constitutes one of the most fundamental concepts in chemistry. In transition‐element chemistry, the 3d 6 octahedron resulting in an unprecedented large splitting of the t2g levels and a diamagnetic ground state has been observed.A Jahn–Teller‐compressed OsO Dealing with multitudes of particles constitutes an inescapable and major challenge in chemistry, even within the framework of the Born‐Oppenheimer approximation. While for molecular species the numbers of atoms involved are finite, they are virtually infinite for extended solids. In coping with the task of structuring and classifying chemical knowledge for purposes of for example, communication, teaching, understanding or planning of chemical research the enormous richness of empirical and computational results need to be cast into comprehensible concepts.9Bi5Os3O24 in which the additional degrees of freedom have enabled a complex composition and a singular crystal structure to form, where the constituents Na, Bi, Os, and O come close to their characteristic structure and bonding properties. As a surprising result, although containing Os6+ with a 5d2 electron configuration, the compound is diamagnetic and exhibits a substantially compressed octahedron around osmium, which causes an exceptionally strong crystal field splitting of the 2gt levels thus lifting the degeneracy of unequally occupied orbitals according to a Jahn–Teller (JT) effect. This finding is very special in another, more general respect. In known solids the transition metal (TM) ions with partially‐filled 2gt levels always obey the first Hund's rule, that is, electrons are arranged such that the maximum S results. We are not aware of any exception of this rule for 2gt shells in extended solids. In one incident, La4Ru2O10, it was initially suggested that Ru4+ has an S=0 ground state.6+(5d2) really has a singlet ground state with S=0, violating the first Hund's rule. This particular configuration may form because of a synergetic action of weaker structural matrix effects of the many‐component material and a strong local JT effect, seen for the first time to act on the 2gt levels of an octahedral complex and to induce a compression.Here we report on an illustrative example of a novel four‐component oxide Na9Bi5Os3O24 was obtained by reacting the binary constituent oxides at high oxygen pressure and hydrothermal conditions. Black reflective hexagonal blocks or thick plates were harvested after washing the product with water and rinsing with ethanol  basically confirming the composition obtained from EDAX. The compound starts decomposing in a stepwise manner at 600 K  but the decomposition is not complete till 1273 K.Nal Figure . SeveralThe powder X‐ray diffraction pattern was refined using the final atomic parameters from the single crystal structure determination as a starting model. No apparent impurity phase was observed in the powder pattern. Figure The determination of the crystal structure by X‐ray diffraction did not proceed straight forwardly and required to employ both powder and single crystal techniques in an alternating fashion, for details see experimental section and Tables S4–S7 in SI.9Bi5Os3O24 exhibits a singular and peculiar crystal structure that cannot be related to any of the prominent aristotypes of typical oxide structure families. Here, for providing a comprehensive description, we resort to the tool of analyzing extended structures in terms of rod packings. via common edges O1‐O2 and O3‐O3, respectively. The rods are linked to form a three‐dimensional framework, basically via the apical oxygen atoms of the Bi5+ and Os6+ octahedra. Now, the key role played by the distorted trigonal prisms around trivalent Bi2 and Na3 in controlling the bond length to the apical oxygen atoms is becoming evident: The particular sequence of Bi2 and Na3 is generating a breathing mode of the oxygen atoms reflected by small triangles between Bi2 and Na3 atoms and wider ones between each two subsequent Bi2 and Na3, respectively. The oxygen atoms constituting the smaller triangles are giving space to Bi5+ to adopt a virtually regular octahedron, while the wider triangles enable Os6+ to develop the strikingly short bonds to the apical oxygen atoms, that is, to form a substantially compressed octahedron. Interestingly, this contraction is not forced by any matrix effect of the crystal structure. From Figure From the structure and chemical formula it was deduced that osmium is hexavalent and bismuth is mixed‐valent with three out of five (Bi1) being pentavalent and the remaining trivalent Bi2). Na. Na9Bi5O3+ and Na+ (Bi2 and Na3) are hosted in virtually the same kind of trigonal prisms, see Figure As one of the remarkable characteristics of the crystal structure found, cations as different as BiPeff=0.2 μB mol−1 corresponding to 1.3 % of a spin 1/2impurity (from a Curie fit of the susceptibility in the entire temperature range). Electrical resistivity data . This separation virtually corresponds by magnitude to the octahedral 2gt‐ge splitting (10Dq) in conventional 3d transition metal oxides. Considering Hund's energy for 5d ions of ≈0.5 eV,xy orbital is filled by two electrons of Os6+, resulting in a singlet ground state, S=0. Thus, it is clearly the strong crystal‐field (ratio of short and long Os−O bonds is ≈0.89), which leads to the non‐magnetic state in the investigated material. It is worthwhile mentioning that, based on various experimental evidences, there have been claims of a low‐spin state realized in 2gt configurations.9Bi5Os3O24 is thus the only oxide, where such a situation affecting a set of 2gt levels is actually realized.In order to uncover the physical origin of the low‐spin state stabilization in Nad derived states, we performed crystal structure optimizations with an artificially reduced number of electrons in the system of six per formula unit (compensated by a corresponding surrounding charge) or by substituting W6+ (5d0) for Os6+ . The results are qualitatively similar and rather illustrative. In the first case, of reduced number of electrons, the distance from the plane formed by equatorial oxygen atoms to apical ligands increases (1.87 and 1.97 Å), while the in‐plane M−O bond lengths decrease (1.87 Å(2×) and 1.89 Å(2×)), for positional parameters see Table S13 in SI. When giving the 5d2 electrons back to the system (or reinstating Os for W), the structure returns to the original experimental configuration upon computational relaxation. These observations clearly demonstrate that the compression of the OsO6 octahedron is basically caused by electronic effects, that is, due to a JT effect lifting the 2gt orbital degeneracy.For checking the role of the degree of filling of the 59Bi5Os3O24. It is imperative to have two electrons on a JT active orbital and lattice connectivity allowing for a strong distortion. In a normal situation for a single d electron or hole the decrease in one‐electron energy due to JT effect is −gδ ; it competes with the elastic energy ≈Bδ2/2, and this would result in JT distortions δ=g/B and a JT energy gain of EJT=−g2/2 B.2 configuration we of course lose Hund's coupling energy, but it can be readily seen that in this case the JT distortion is much stronger, δ=2 g/B, and the energy gain is 4 times larger, EJT=−2 g2/B .The following consideration can be proposed to explain how the anti‐Hund low‐spin state, which normally corresponds to an exited state, can be stabilized in Na9Bi5Os3O24 with other functional oxides and coordination compounds containing Os6+. In principle, open shell 5d systems can arrive at a nonmagnetic state due to strong spin‐orbit interaction even for a cubic crystal field. This has been encountered for 5d4 ions like Ir5+. which seemingly constitute another analogy. Here, short Os−O bonds and diamagnetic behavior are understood in terms of local bonding, in particular enforced by (p‐d)π bonding.It appears advisable to compare the situation in Na9Bi5Os3O24 which features a unique crystal structure and an unparalleled electronic ground state: a strongly compressed octahedron of the oxygen atoms coordinating osmium(VI) is giving rise to a nonmagnetic configuration by hosting the 5d2 electrons paired in the xyd derived state. The splitting of the 2gt levels amounts to 1.4 eV, substantially surpassing the Hund's coupling energy for 5d ions of ≈0.5 eV. This pronounced JT distortion is not enforced by structural matrix effects, the structure would offer sufficient space to enable a virtually regular octahedron without causing strain. Apparently, the energy landscape of configurations for 5d2 systems, like Os6+, in approximately octahedral coordination by oxygen is rather flat and, depending on the balance of local electronic factors of influence vs. structural constraints, diverse ground states may develop. Against this background it appears rewarding to inspect these kind of competing impacts, structural frustration vs. strive for an as low as possible local electronic state, on the basis of the structure type presented, which offers plenty of options for modifying the relevant conditions by aliovalent or isovalent substitution of any of the cations present. Such computational studies, at best followed by experimental validation, would have the potential to provide valuable insights in the hierarchy of perturbations defining the ground states of open shell transition element compounds.We report on new NaThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.