Primary care providers (PCP) - internists, family practitioners, nurse practitioners, physician assistants - play an integral role in the care of older adults, although many receive limited geriatrics education. We sought to examine what questions community-based PCPs had about geriatrics and clinical care of older adults. As part of large clinical continuing medical education (CME) conferences across 12 states , PCPs attended a live in-person 60-minute geriatrics-focused lecture and entered questions into a mobile application. Questions were then qualitatively analyzed using constant-comparison and tie-break methodology. At all sites, 103 questions were asked with 158 upticks (PCPs could check off that they had similar question) with a range of 3-18 questions per lecture. PCPs asked questions on the following common themes: 1.) Medication-related , 2.) Dementia  3.) Medicare Coding 4.) Falls 5.) Weight loss, and 6.) Insomnia. There were a number of questions referencing incorrect practices . In conclusion, community-based PCPs nationally experience gaps in geriatric knowledge and several utilize practices that could jeopardize older adult health. While attending CME-based lectures is one means of overcoming these gaps, some PCPs may not find time or realize geriatrics as an educational need. PCPs need to be better supported with opportunities to ask geriatric care-related questions in order to improve the care of older adults.


In anticipation of palatable food, rats can learn to restrict consumption of a less rewarding food type resulting in an increased consumption of the preferred food when it is made available. This construct is known as anticipatory negative contrast (ANC) and can help elucidate the processes that underlie binge-like behavior as well as self-control in rodent motivation models. In the current investigation we aimed to shed light on the ability of distinct predictors of a preferred food choice to generate contrast effects and the motivational processes that underlie this behavior. Using a novel set of rewarding solutions, we directly compared contextual and gustatory ANC predictors in both food restricted and free-fed Sprague-Dawley rats. Our results indicate that, despite being food restricted, rats are selective in their eating behavior and show strong contextually-driven ANC similar to free-fed animals. These differences mirrored changes in palatability for the less preferred solution across the different sessions as measured by lick microstructure analysis. In contrast to previous research, predictive cues in both food restricted and free-fed rats were sufficient for ANC to develop although flavor-driven ANC did not relate to a corresponding change in lick patterning. These differences in the lick microstructure between context- and flavor-driven ANC indicate that the motivational processes underlying ANC generated by the two predictor types are distinct. Moreover, an increase in premature port entries to the unavailable sipper – a second measure of ANC – in all groups reveals a direct influence of response competition on ANC development. The sights, sounds and smells of a familiar kitchen while dinner is being prepared can evoke a strong drive to eat but in refraining from doing so one can reserve their appetite for the main course. Such consummatory choice behavior is also at play in foraging animals where decisions based on prior knowledge of territorial food sources can result in animals passing up a nutritionally-insufficient option for one of greater value that is likely available  a progressive devaluation of the first food source when anticipating a preferred option; 2) competing behavioral responses, such as spatial competition with the unavailable port, reducing the amount of time dedicated to licking/eating; and 3) active inhibition of the urge to consume the less preferred option despite animals, in some experiments, being motivated by food restriction. While interpretations based on data from variations of the ANC paradigm mostly support ANC resulting from a reward devaluation of the first food source  and condensed milk (solution 2) sequence for further establishing a contrast effect with nutritionally-relevant food sources. Interestingly, different parameters of licking behavior have been related to distinct motivational processes  and housed in pairs. The room was kept at 21 °C and humidity of between 40% and 70% under a 12 hour light- dark cycle (lights on at 7:00). Rats had free access to standard lab chow  and water. Prior to the experiment, half of the rats were put on a mild food restriction, and given 12.5 g of lab chow per day. This food restriction continued on experimental days but rats were put on a free-access diet during the weekends. For food restricted animals, daily food ration was given immediately following the day's behavioural session. All rats were weighed daily Monday through Friday and a percentage of baseline weights calculated. All testing was conducted in accordance to the Animals (Scientific Procedures) Act 1986 (PPL# PFACC16E2).2.2Animals were trained and tested in two identical operant chambers , each located inside a sound- and light-attenuated aluminum outer chamber (1200 × 700 × 700 cm). The behavioral chambers were equipped with a house light located on the left wall and 2 retractable sippers located on the right wall, which when extended fully were located approximately 1 cm behind the chamber wall. Sippers were accessed via ports  in the wall through which rats needed to poke their noses. This arrangement ensured that only the tongue could contact the sipper and prevented the formation of fluid bridges meaning that individual licks were recorded with high fidelity. Contact lickometers (Med Associates) were used to detect licks and ports were fitted with infrared beam breaks for detecting port entries. The house light was turned on at the beginning of each daily session and turned off at the end of it. Equipment was controlled by a computer running Med-PC IV Software Suite (Med Associates). Webcams were used to monitor the animals’ behavior.Animals were pre-trained for 10 min per day to lick a palatable 10% sucrose solution in all experimental boxes (Context A and B) for 6 days prior to the experiment. For experiments using a contextual predictor, a standard Med Associates box with clear plexiglass walls and a barred floor was used for Context A and a modified version with a striped wall covering, a fine wire mesh floor and continuous white noise (75 dB level) played on a speaker was used for Context B. Both sippers were extended simultaneously for 5 min, retracted for 20 s and presented again for a further 5 min. Licks were recorded from both sippers at this time to check that there was no location bias within the contextual behavioral paradigm.The ANC conditioning protocol  consisteFor the flavor preference test performed before and after ANC conditioning, two sippers each containing one of two flavors (grape or cherry) + 0.2% sodium saccharin were extended one at a time for a maximum time of 30 s, or 10 s after first sipper contact. Overall, 10 "forced choice trials" where only 1 sipper was presented at a time was followed by 30 "free choice trials" where both sippers were presented together. In the flavor preference tests, we recorded licks at each spout and analyzed the total licks for each solution in each type of trial (free-choice vs. forced-choice). At the end of the experiments animals were humanely culled via a Schedule 1 method.2.3Behavioral data (lick and port entry timestamps) were extracted from data files and analyzed using custom Python scripts that measured numbers of licks for each solution. All raw data files and the analysis scripts are available at the following link: DOI: 10.5281/zenodo.4772860. From these measurements we obtained and analyzed the following variables: Total licks (at each spout); Premature port entries; Licks per cluster ; and Total clusters . From these, we determined total licks in each phase, total head entries in both unavailable ports, number of lick clusters and licks per cluster were recorded for each of the 32 days (16 sessions). Lick microstructure was analyzed by using interlick intervals to divide licks into clusters. Clusters were defined as runs of licks with no interlick intervals > 500 ms 33.13.1.1n = 10) as evidenced by reduced consumption during phase 1 specifically on days when a more preferred solution was expected. Statistically, total lick measurements revealed a significant effect by ANC conditioning day (session)  = 6.20, P = 0.03) and interaction between session and Malt-Malt and Malt-CM days (condition)  = 4.62, P = 0.0003) (P = 0.11). As expected in phase 2, FF rats  = 36.70, P = 0.0002; Interaction: F = 9.17, P < 0.0001; Bonferroni post-hoc differences - Sessions 2 through 8). Using a novel sequence of rewards in the current study, these results extend previous findings that FF rats can develop robust ANC [We used a modified ANC paradigm with a novel sequence of rewarding solutions to gauge the effectiveness of contextual or gustatory predictors for enhancing contrast effects in free fed (FF) and food restricted (FR) Sprague-Dawley rats . When us 0.0003) A1-left.  FF rats A1-right bust ANC , 22.Fig.n = 10) also showed strong ANC (P = 0.001) and by conditioning day  = 21.94, P = 0.001) and an interaction between the two was also revealed  = 6.03, P < 0.0001). Post-hoc Bonferroni tests showed significant differences between control and experimental conditions on sessions 3 through 8. These results are consistent with a recent within-subject ANC demonstrating that contextual cues are sufficient for ANC to develop in FR rats, albeit using a different sequence of rewards P = 0.0013; Sessions: F = 21.94, P = 0.001; Interaction: F = 6.03, P < 0.0001; Bonferroni post-hoc differences –Sessions 3 through 8). Overall, these results indicate that contextual cues can act as effective signals for decreasing animals’ intake of a less palatable Malt solution in anticipation of a more preferred CM option whose intake increases when it is made available. Moreover, FR animals developed ANC after fewer conditioning trials (session 3) than FF animals (session 6) suggesting that despite being hungry FR rats can act selectively in their feeding behavior.FR rats  = 6.54, P < 0.0001; Interaction: F = 10.05, P < 0.0001; Bonferroni post-hoc differences –Sessions 3,4,6–8)  = 4.16, P = 0.0008; Bonferroni post-hoc differences –Sessions; 3–8; No main effect by Session: F = 1.88, P = 0.087)  A1 (FR: 2= 0.087) B1 furtheP = 0.006; Sessions: F = 4.39, P = 0.0005; Interaction: F = 3.36, P = 0.0041; Bonferroni post-hoc differences –Sessions 6,8)  = 11.64, P = 0.008; Session: F = 3.84, P = 0.002; Bonferroni post-hoc differences –Session 8; No interaction: F  = 0.66, P = 0.70). No differences for port 1 entries were seen in any condition  = 1.20, P = 0.30; Session: F = 2.87, P = 0.05; Interaction: F = 1.32, P = 0.29)  = 2.27, P = 0.05; Interaction: F = 0.58, P = 0.77) (These premature entry results are in line with data collected during the IPI phase where port entries to unavailable sippers 1 and 2 were measured for 20 s - IPI. On = 0.29) A2-left ( = 0.77) B2-left. 3.1.3P = 0.009; Session:  = 2.43, P = 0.03; Interaction:  = 3.72, P = 0.002; Bonferroni post-hoc differences - Sessions 6,7)  = 2.577, P = 0.14 and Session: F  = 1.41, P = 0.21)  = 4.46, P = 0.0004; Bonferroni post-hoc differences –Sessions 1,5,7; No effect for Condition: F  = 0.93, P = 0.36)  = 2.34, P = 0.03; Bonferroni post-hoc differences –Session 3,4; No effect for Condition: F  = 5.06, P = 0.05)  - is a measure commonly used to quantify the palatability of the reward (liking) while total clusters has been shown to relate to a reward's incentive value (wanting) as well as signal its post-ingestive feedback properties , 8; revions 6,7) A1-left.  = 0.21) B1-left.  = 0.36) A1-right  = 0.05) B1-right.P = 0.15; Session: F = 1.13, P = 0.36; Interaction: F = 0.76, P = 0.62)   = 3.91, P = 0.08; Sessions: F = 1.38, P = 0.23; Interaction: F = 1.02, P = 0.42)  = 2.82, P = 0.01; Bonferroni post-hoc differences – Paired Sessions 1,6; No main effect by Session: F = 0.72, P = 0.65)  = 1.42, P = 0.21; Interaction: F = 1.79, P = 0.10)  = 2.45,  = 0.42) B2-left.  = 0.65) A2-right  = 0.10) B2-right.In summary, when using contextual predictors, ANC was seen to develop in both FF and FR rats and this may be associated with a change in palatability of the non-preferred maltodextrin solution on experimental vs. control sessions.3.23.2.1n = 10) and FR (n = 10) rats showed ANC when different flavoring was added to phase 1 Malt solutions used to predict either Malt or CM in phase 2. Specifically, in FF rats  = 3.99, P = 0.001; Bonferroni post-hoc differences –Sessions 4,5; No Interaction: F = 1.28, P = 0.27). Similarly in FR rats  = 4.26, P = 0.0007; Interaction: F = 3.53, P = 0.003) and was apparent on sessions 4, 6–8 (Bonferroni post-hoc differences). As expected, phase 2 consumption was greater for both groups  = 0.69, P = 0.68; No Interaction: F = 1.31, P = 0.26)  = 2.91, P = 0.01; Interaction: F = 7.15, P<0.0001; Bonferroni post-hoc differences – Sessions 1–4; No effect by Condition: F = 5.06, P = 0.05). In summary these results demonstrate that when Malt is followed by CM gustatory cues are sufficient predictors for ANC development.We next tested whether gustatory cues can be used as effective predictors for ANC to develop. In contrast to previous results , 32, FF  FF rats A1-left,  FR rats B1-left t3.2.2P = 0.0003; Session: F = 2.22, P = 0.04; Interaction: F(7 63) = 3.70, P = 0.002; Bonferroni post-hoc differences –Sessions 2–8)  = 6.50, P < 0.0001; Interaction: F = 4.77, P = 0.0002; Bonferroni post-hoc differences – Paired Sessions 2–8)  = 31.47, ons 2–8) A1  B1.Fig. 6P = 0.006; Session: F = 4.71, P = 0.0003; Interaction: F = 3.39, P = 0.004; Bonferroni post-hoc differences – Sessions 4,5,7)  = 2.50, P = 0.02; Bonferroni post-hoc differences – Sessions 7,8; No significant Interaction: F = 1.26, P = 0.28)  = 0.94, P = 0.48; Interaction: F = 1.56, P = 0.17)  = 5.47, P < 0.0001; Interaction: F = 1.09, P = 0.38)  = 13.09, s 4,5,7) A2-right  = 0.28) B2-right. = 0.17) A2-left ( = 0.38) B2-left. 3.2.3P = 0.10; Interaction, F = 0.77, P = 0.61; main effect by Session: F = 4.42, P = 0.0005)   = 2.21, P = 0.05; Session: F  = 4.0, P = 0.001; Bonferroni post-hoc differences – Session 8; No main effect by Condition: F = 1.54, P = 0.24) . In phase 2, both FF and FR animals showed a session-dependent change in licks per cluster for CM compared to Malt  = 2.88, P = 0.01; No main effect by Condition: F = 2.69, P = 0.14; or by Session: F = 1.68, P = 0.13)   = 3.63, P = 0.002; Bonferroni post-hoc differences – Paired Session 4–8; No main effect by Condition: F = 4.83, P = 0.06 or by Paired Session: F = 1.04 P = 0.41) . Notably, these results show that in either motivational state (FF or FR) licks per cluster in phase 2 shifted from high to low levels with conditioning trials  = 3.29, P = 0.36; Session: F = 0.41, P = 0.89; Interaction: F = 0.51, P = 0.82)   = 0.25, P = 0.62; Session: F = 0.22, P = 0.98; Interaction: F = 1.06, P = 0.40)  indicating that changes in total clusters do not contribute to changes in phase 1 total licks. There was however a significant increase in phase 2 total clusters in response to CM for both FF and FR animals throughout conditioning  = 11.73, P = 0.008; Sessions: F = 2.90, P = 0.01; Bonferroni post-hoc differences – Paired Sessions 4–7; No Interaction: F = 1.68, P = 0.13)   = 31.07, P = 0.0003; Session: F = 2.75, P = 0.01; Interaction: F = 4.06, P = 0.001; Bonferroni post-hoc differences – Sessions 4–8)  which is in contrast to the changes in lick microstructure for contextually-driven ANC. These differences in the lick microstructure between animals using contextual  = 0.90, ntextual  and gustntextual  suggest 3.2.4P < 0.0001; Condition: F=2.12, P = 0.18; Interaction: F = 1.83, P = 0.21) indicating that there was no initial preference before conditioning and that licking to both flavors, irrespective of which flavor predicted CM, increased after conditioning  = 0.34, P = 0.56; Interaction: F = 0.35, P = 0.57)  C3. These3.2.5P = 0.0002; Session and Predictor: F  = 2.07, P = 0.04; No effect of Diet: F = 0.3132, P = 0.58). Post-hoc Bonferroni tests, however, did not show a significant different on any session between any of the groups. The results here, however, need to be carefully considered as the experiments using flavor and contextual predictors were not performed simultaneously.In the current study, four unique contrast conditions were tested . To formally compare each condition, we next analyzed the normalized phase 1 total lick data  to determine the effectiveness of each condition compared to one another in promoting ANC . A 3-way4Using a novel sequence of nutritionally-relevant rewards, in the current study we tested the predictive ability of contextual and gustatory cues on ANC and the motivational processes that underlie this behavior. Our results indicate that both contextual and gustatory information are effective predictors for ANC to develop. The selective reduction of Malt consumption may be at least partially due to spatial competition between the two sipper ports as increases in premature port entries in phase 1 were significantly increased on experimental days. In contextually-driven anticipatory sessions, our lick microstructure analyses suggest that ANC may be determined by a change in the rewarding properties of the Malt solution that is in all other respects equivalent to the one presented on control days - the only difference being the context signaling the availability of CM in phase 2. In contrast, ANC driven by gustatory predictors was not mirrored by changes in the lick microstructure indicating that the contrasting effects seen here are perhaps driven by competing factors. Due to their biological relevance, gustatory cues can be robustly associated with food sources, potentially acting as strong secondary reinforcers that can compete with negative contrast effects , 32. SinPrevious research, however, has shown that within-subject ANC can develop without physical predictors but rather continuous alternating days alone can signal the different conditions (P = 0.02) suggesting an increase in the incentive value of the flavored Malt solutions – an interpretation which is consistent with the results of the flavor preference test. Thus, it is possible that the addition of a flavor to the maltodextrin solution in phase 1 enhances its palatability vs. a non-flavored maltodextrin solution. This change in Malt palatability may in effect be impacting the hedonic disparity between the two rewards by altering the incentive value of CM. Such changes are reminiscent of those seen in temporal discounting paradigms where a comparative analysis of two rewards separated by time can impact the incentive value of each [Stereotypical patterns of rodent licking behavior are thought to reflect different underlying motivational processes where the amount of licks in a cluster of continuous licks has been shown to relate to reward palatability (reward liking) while the amount of total clusters changes with incentive value (reward wanting) as well as signal its post-ingestive feedback properties . These results may be attributed to the novel food choice sequence  where the disparity in the hedonic value and nutritional content between the two was different than past investigations [The fact that contrasting effects are based on relative rather than the absolute value of the rewards make them sensitive to various factors. For instance, their value-based as well as temporal disparity can strongly impact contrast , 18, 37.igations , 18, 42.A number of different neural processes must be at play when animals learn to predict rewards of different hedonic value. The lick microstructure analysis performed in the current study suggests that gustatory and contextual predictors may be mediating ANC through different motivational or cognitive processes. Studies investigating the neurobiological mechanisms that underlie ANC have mostly focused on the changes in reward value that might contribute to ANC development. While dopamine activity encodes information predicting a future reward as well as the reward itself In summary, our results indicate that despite the potential benefit in taking an opportunistic feeding approach, food-restricted animals can use contextual predictors to act selectively in their feeding choice and that these changes may stem from learned changes in the hedonic properties of the readily available food source. Gustatory predictors can also be used to optimize intake of a preferred food option in both free-fed and food restricted rats but less effectively than contextual cues - a results that may be due to the high predictive strength of flavors linked to a food source that differentially impacts the underlying processes that drive ANC. Future investigation of the neural activity contributing to these motivational and cognitive changes will help elucidate the neurobiology that allows animals to optimize their foraging strategies.Jessica Hayes: Formal analysis, Investigation, Writing – review & editing. Celia Garau: Conceptualization, Formal analysis, Investigation, Writing – review & editing. Giulia Chiacchierini: Investigation, Writing – review & editing. Gonzalo P. Urcelay: Conceptualization, Writing – review & editing. James E. McCutcheon: Conceptualization, Formal analysis, Writing – review & editing, Funding acquisition. John Apergis-Schoute: Conceptualization, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Funding acquisition.The authors declare no competing financial interests.


This functionalized graphene was confirmed with high atomic content of S (4.79 at % S) as well as the presence of C–S–C and N–H species on the L-cysteine functionalized graphene (FG-CYS). Raman spectroscopy evidently corroborated the modification of graphene to FG-CYS with an increased intensity ratio of D and G band, ID/IG ratio (0.3 to 0.7), full-width at half-maximum of G band, FWHM [G] (20.3 to 35.5) and FWHM [2D] (64.8 to 90.1). The use of ethanol as the reaction solvent instead of common organic solvents minimizes the chemical hazards exposure to humans and the environment. This direct attachment of multifunctional groups on the surface of pristine graphene is highly demanded for graphene ink formulations, coatings, adsorbents, sensors and supercapacitor applications.Functionalization of pristine graphene to achieve high water dispersibility remains as a key obstacle owing to the high hydrophobicity and absence of reactive functional groups on the graphene surface. Herein, a green and simple modification approach to prepare highly dispersible functionalized graphene via thermal thiol-ene click reaction was successfully demonstrated on pristine graphene. Specific chemical functionalities (–COO, –NH Graphene materials have sparked enormous attraction and interest from both the scientific and industrial community owing to the fascinating properties of graphene and its widespread applications across many technological fields, such as nanoelectronics, sensors, composites, coatings, batteries, supercapacitors and hydrogen storage . The ong2 carbons of pristine graphene, or by the formation of a covalent bond between organic molecules and the oxygen groups of graphene oxide, GO [2 carbon in the graphene framework appears to be a promising covalent functionalization approach that offers several benefits. It is highly efficient, simple to execute, insensitive to oxygen and water, has no side products and proceeds rapidly with high yield [Covalent attachment of organic functional groups to the graphene surface can be achieved either by chemical reaction of free radicals or dienophiles spxide, GO . The radgh yield . Severalgh yield ,10,11,12gh yield .2 honeycomb framework. In fact, limited reports were found on the direct functionalization of pristine graphene via thiol-ene click chemistry. Castelain et al. used thiol-ene reaction for a direct functionalization of the graphene surface with short chain polyethylene (PE) brushes which were successfully used to prepare graphene based high-density polyethylene nanocomposites [Surprisingly, up to now, the majority of the reports of functionalized graphene have primarily focused on GO and its reduced form, reduced graphene oxide (rGO), where the preparation methodologies usually involve the use of hazardous chemicals and toxic gases, with large quantities of chemical waste generated after the reactions . To perfmposites . Peng etmposites . In anotmposites . Althoug2 carbon of graphene via thermal thiol-ene click reaction, which is schematically presented in Herein, we present a simple, green and sustainable method to functionalize pristine graphene through thiol-ene click reaction to acquire water dispersible graphene materials with multiple functional groups that are important for many applications. The aims of this work are twofold: first, to improve an existing thiol-ene click reaction using more sustainable and green conditions for the functionalization of pristine graphene, and second, to demonstrate versatility of this method to generate graphene with multifunctional surface chemistry with different end groups including ester, amino, and thioether. The developed method is based on the covalent attachment of thiol molecules on the sp−1) was used throughout the work, unless otherwise stated.Graphene was obtained from a local company . L-cysteine , thionyl chloride , diethyl ether  2,2′-azobis-(2-methylpropionitrile) , ethanol  were used directly without prior purification. High purity milli-Q water (18.2 MΩ·cmEthyl esters of cysteine were synthesized according to a reported method . In briev/v) via sonication for 1 h. Subsequently, the sonicated mixture was purged with nitrogen gas for 30 min to create an inert environment. Then, L-cysteine ethyl ester and 2,2′-azobis-(2-methylpropionitrile) (AIBN) were added into the mixture with a further nitrogen gas purge for 30 min, followed by a 30 min sonication. After that, the reaction mixture was poured into a round bottom flask with an additional 30 min of nitrogen gas purge and heated in an oil bath at 65 °C under reflux conditions overnight. After the reaction, the as-synthesized product was washed thoroughly with ethanol and deionized water using centrifuge, dried in oven at 65 °C overnight, and stored for further characterization.Functionalized graphene with L-cysteine ethyl ester (FG-CYS), was prepared according to the adapted procedure reported by Yap et al. . In a ty−1 on a Nicolet 6700 Fourier Transform Infrared (FTIR) Spectrometer . Morphology of the materials was imaged using a scanning electron microscope  at an operating voltage of 10 kV and a transmission electron microscope at 120 kV . Chemical composition and chemical species were analyzed by X-ray Photoelectron Spectroscopy  equipped with a monochromatic Al Kα radiation source (hv = 1486.7 eV) at 225 W, 15 kV and 15 mA. XPS survey scans were performed at 0.5 eV step size over −10 to 1100 eV at 160 eV pass energy with peak fitting analysis executed using Casa XPSTM software. The core-level XPS spectra were calibrated at 284.8 eV. Raman spectrometer  with an excitation wavelength of 532 nm (mpc 3000 laser source) was applied from 500 to 3000 cm−1 with an integration time of 10 s for three accumulations to determine the vibrational features of pristine graphene, and thermogravimetric analysis (TGA) of functionalized graphene was performed at a heating rate of 10 °C/min in nitrogen atmosphere from 25 to 1000 °C on a METTLER TOLEDO TGA/DSC 2 instrument.FTIR spectra were collected at 500 to 4000 cmThe morphology of pristine graphene and the functionalized graphene was first examined using scanning (SEM) and transmission (TEM) electron microscopy, as shown in A simple and rapid water dispersion test was performed to examine the effectiveness of thiol-ene functionalization d–f. As e3/2 and S2p1/2 at around 164.1 eV and 165.2 eV, respectively, can be assigned to thioether sulfur bonded to carbon (C–S–C) species on the narrow XPS spectra of S2p of FG-CYS, and confirmed the successful attachment of the thiol groups from the thiol precursor via thiol-ene click reaction on the surface of the graphene [3/2 and S2p1/2 peaks with lower intensities deconvoluted at 164.8 eV and 166.1 eV, respectively, could be ascribed to C–S or S–S species on the surface of graphene [2, NH3+, N–H and C–N+, respectively [Chemical composition of the functionalized graphene was determined by XPS analysis  with thegraphene ,19. Meangraphene . Note thgraphene . High-reectively ,22. The ectively ,16,23. T−1, D band at around 1350 cm−1, and 2D band at 2700 cm−1 [−1) and a symmetrical 2D peak (~2700 cm−1) as clearly observed on the Raman spectra of Gr and FG-CYS samples. As depicted in −1 was detected on the Raman spectrum of FG-CYS, suggesting a certain degree of defect found on the surface of FG-CYS. This finding was in consistent with the average value of ID/IG ratio (a measurand of the level of disorder of graphene) of FG-CYS (0.7), which was higher than Gr (0.3), indicating more defects were experienced on the surface of FG-CYS than Gr due to thiol-ene click modification using thiol precursor, as shown in Detailed Raman analysis was performed to verify the modification created on the surface of pristine graphene and functionalized graphene, as illustrated in 700 cm−1 ,25. The −1 on the FTIR spectrum of the functionalized material, but not identified on the FTIR spectrum of pristine graphene [−1 (stretching of S–H group) in the functionalized graphene suggested that all the –SH groups in the thiol precursor had reacted with the sp2 carbon in pristine graphene through thiol-ene click reaction [−1 for the stretching of C=O in the ester group on FG-CYS. Peaks at about 1640 cm−1 (N–H bending), 1090 cm−1 (C–N stretching) and 1054 cm−1 (C–NH2 vibration) were also found on the FTIR spectrum of FG-CYS, implying successful grafting of the functional groups on the pristine graphene. Two common peaks at 2913 and 2846 cm−1, which can be attributed to the asymmetric and symmetric vibrations of C–H of –CH2 group, respectively, were also observed in the functionalized graphene [The obtained FTIR characterization results presented in graphene . This fireaction . Additiographene . Based oFurthermore, TGA was also conducted on pristine and functionalized graphene to qualitatively and quantitatively determine the presence of attached functional groups on the pristine graphene after the thermal thiol-ene click reaction. As presented in 2 carbon and the lack of reactive functional groups on the surface of pristine graphene, we successfully demonstrated an effective functionalization of pristine graphene through a green and scalable thiol-ene click modification to endow a highly dispersible graphene. A summary of our key findings with evidence supported by comprehensive techniques, as tabulated in Despite the key challenges of inertness of spTo date, functionalization of pristine graphene by covalent bonding has rarely been reported in relation to its derivative, GO, due to the lack of reactive functional groups on the pristine graphene surface . It is uNumerous attempts have been made in the last decade to introduce organic functional groups onto the surface of pristine graphene through thiol-ene click chemistry. The degree of functionalization can be quantitatively gauged using both bulk (TGA) and surface (XPS) characterization techniques, as summarized in Based on the bulk TGA characterization technique, L-cysteine was found to be more effectively grafted on FG-CYS (present study) than G-LCHa, as reflected by its lower functional group coverage of 113, compared to 137 carbon atoms per unit of L-cysteine molecule, as tabulated in Another example of direct functionalization on pristine graphene was manifested in G-CHI and GR-Cys, with 1 cysteamine molecule per 113 and 96 carbon atoms, respectively, based on the value of functional group coverage. Meanwhile, 1.13 at % N and 1.66 at % S were found on G-CHI, while 4.78 at % N and 4.52 at % S were detected on GR-Cys after the thiol-ene click modification ,16. In cDespite the growing body of work recently reported to functionalize graphene directly using thiol-ene click strategy, there are still numerous open research opportunities to address the graphene dispersion issue, to endow a better processability of graphene materials  with desired properties. Beyond the conventional chemical modification of graphene such as the thermal heating process, other methods including microwave irradiation, plasma etching, electrochemical modification or a combination of these methods could be explored as promising alternative routes to resolve the low reactivity of graphene. Based on our current work, functionalization parameters such as the ratio of graphene to thiol precursor, reaction time and temperature, could be tuned to accommodate a more effective grafting of functional groups on the graphene surface. The future direction of graphene functionalization may be structured towards room temperature modification of graphene, which can be feasibly achieved via the photoinitiated thiol-ene click approach. In addition, more research efforts should be dedicated to the optimization of functionalization methods to achieve not only the better dispersion of graphene, but also the scalability of processing of new graphene materials to be more economical.In summary, the synthesis of functionalized pristine graphene by a green thermal thiol-ene click approach using L-cysteine ethyl ester was successfully demonstrated. The use of ethanol in replacing highly toxic organic solvents in this click reaction provides a generic, green, and scalable approach to graft graphene with desired functional groups, which can be achieved by selecting different types of thiol precursors bound with functional groups including hydroxyl, ester and amine groups. Successful modification of pristine graphene with the thiol precursor was confirmed by Raman, FTIR, TGA and XPS analyses. This green modification process provides a significant contribution towards the scalable production of functionalized graphene due to a simple and scalable chemical route which is beneficial for many applications, such as biomedical, sensing, graphene polymer composites, inks, supercapacitors, etc.


TARDBP, forms aggregates in the motor cortex. This aggregate formation may be triggered by an increase in the TDP-43 level with aging. However, the amount of TDP-43 is autoregulated by alternative splicing of the TARDBP 3′UTR, and how this autoregulation is affected by aging remains to be elucidated. We found that DNA demethylation in the autoregulatory region in the TARDBP 3′UTR reduced alternative splicing and increased TARDBP mRNA expression. Furthermore, in the human motor cortex, we found that this region was demethylated with aging, resulting in increased expression of TARDBP mRNA. The acceleration of DNA demethylation in the motor cortex was associated with the age of ALS onset. In summary, the dysregulation of TDP-43 autoregulation by age-related DNA demethylation in the motor cortex may explain the contribution of aging and motor system selectivity in ALS.In amyotrophic lateral sclerosis (ALS), TAR DNA-binding protein 43 (TDP-43), which is encoded by  In order to assess the effects of aging on the autoregulation of TAR DNA-binding protein 43 (TDP-43) and the potential effects of this on the role of TDP-43 in Amyotrophic Lateral Sclerosis (ALS), Koike et al examined post-mortem motor cortex tissue from ALS patients. They found that DNA demethylation in the autoregulatory region of the TARDBP 3′UTR, which encodes TDP-43, increased with age and was associated with the onset age of ALS and thus could be indicative of a role for dysregulation of TDP-43 autoregulation in ALS pathology. It has been speculated that an increase in the amount of causative proteins contributes to aggregate formation3. Thus, one pathological hypothesis of system selectivity in neurodegenerative diseases is that the amount of disease-causing protein is increased in the impaired system. In fact, alpha-synuclein levels have been shown to be increased in neuronal systems affected by alpha-synucleinopathy4. However, the association between the amount of the causative protein and aging is unclear.In neurodegenerative diseases with aging as a primary risk factor5. In more than 95% of sporadic ALS cases, the nuclear protein TDP-43 forms aggregates in the cytoplasm and is depleted from the nucleus in diseased cells6,8. Increased intracellular concentrations of TAR DNA-binding protein 43 (TDP-43) have been speculated to be a factor in this aggregate formation10. However, to date, the mechanism linking TDP-43, motor neuron selectivity, and aging has not been elucidated.Sporadic amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that selectively impairs primary motor neurons in the motor cortex and alpha-motor neurons in the spinal cord, and similar to other sporadic neurodegenerative diseases, aging is a major risk factorTARDBP pre-mRNA 3′UTR, and the level of TDP-43 is strictly autoregulated via its alternative splicing13. An increased level of nuclear TDP-43 promotes its splicing to produce nonsense-mediated mRNA decay-sensitive TARDBP mRNA and reduce the level of TDP-4314. However, when the level of TDP-43 in the nucleus is reduced, this splicing is repressed, and the TARDBP mRNA levels are increased. Therefore, TARDBP mRNA expression is continually increased in ALS-affected cells with reduced nuclear TDP-43 levels14. Following the formation of cytoplasmic TDP-43 aggregates, which are presumed to reduce the TDP-43 level in the nucleus by either entrapping newly generated TDP-43 or inhibiting the nuclear transport of TDP-43, the progression of the disease accelerates16. Some mutations in the TARDBP gene can alter its alternative splicing and affect the autoregulatory mechanism18. However, in sporadic ALS, the factor triggering the disruption of this autoregulatory mechanism leading to increased TDP-43 expression and TDP-43 aggregate formation in the motor cortex is unclear.Nuclear TDP-43 binds the 19. Indeed, an accelerated epigenetic age based on DNA methylation is associated with the age of onset in ALS patients with C9orf72 expansion repeats20 and in sporadic ALS patients21. DNA methylation not only regulates gene expression but also affects alternative splicing26. Based on these observations, we hypothesized that the DNA methylation status of the TARDBP-3′UTR changes with age in the motor cortex, which in turn alters the splicing of the TARDBP-3′UTR in the direction of increased TDP-43 expression.Aging alters the methylation status of DNA. The DNA methylation status defines the epigenetic age independent of the chronological age. In age-related diseases, this epigenetic age contributes more than the chronological age to mortality and morbidityTARDBP 3′UTR. We found that the demethylation of this region inhibited the alternative splicing and increased the expression of TARDBP mRNA. Furthermore, in the human motor cortex, this region was demethylated with aging, and the degree of demethylation was correlated with the level of TARDBP mRNA expression. An accelerated DNA methylation age of the TARDBP 3′UTR in the ALS motor cortex was associated with a younger age of onset, suggesting its involvement in ALS pathogenesis.To explore this hypothesis, we used the dCas9 system to selectively demethylate the CpG region of the alternative splicing-related site in the TARDBP pre-mRNA and induces the alternative splicing of intron 6 (exonic intron) and intron 7 (3′UTR intron), leading to nonsense-mediated mRNA decay14. We found that 15 CpG sites were clustered around the 5′ splicing site of alternative intron 7. An analysis of publicly available data revealed that these CpG sites are moderately to highly methylated in the human prefrontal cortex   Fig. , c. In t36. In addition, the methylation-linkage diagram of the two youngest (middle-aged) and two oldest (elderly) individuals showed generally lower pairwise correlations in the elderly individuals  was applied to demethylate the target DNA region  was cut at the AflII site, and a guide RNA fragment was inserted using Gibson assembly master mix (New England Biolabs). Four guide RNAs were selected using Benchling (https://benchling.com/). To construct the DNMT3A-TARDBP-target vector, pdCas9-DNMT3A-EGFP (Addgene plasmid 71666) was cut at a BbsI site, and a guide RNA fragment was inserted in the same manner. The sequences of the guide RNAs are described in Supplementary Table To construct the TET1-21. During the analysis, one ALS patient was excluded due to the detection of a mutation in TBK1. To analyze the relationship between the DNA methylation levels of CpGs 10–15 in the motor cortex and age, we included the motor cortexes of three ALS patients and three controls and analyzed data from 10 ALS cases and 11 controls . Using 25 mg of human postmortem tissue, genomic DNA was purified using a DNeasy Blood and Tissue kit (QIAGEN). According to the manufacturer’s protocols, the purified genomic DNA was bisulfite treated with an EpiTect Fast DNA Bisulfite kit (QIAGEN). In both HEK293T cells and human postmortem tissues, bisulfite amplicon sequencing was performed using primer sets A and B . The sequenced reads were mapped to the human reference genome hg19 using the methylation analysis tool Bismark v0.18.1 (Babraham Bioinformatics)38. The CpG density at each CpG site was calculated by a kernel-density estimation (bandwidth = 10.73). Principal component analysis was performed using the average methylation rates, average pairwise correlations, and epipolymorphism scores of 3′UTR CpGs 1–9 and 3′UTR CpGs 10–15. The ‘pcaMethods’ R package54 was used to apply the nipals algorithm with unit-variance scaling.The epipolymorphism scores were calculated and methylation-linkage diagrams were generated based on a previous report5 HEK293T cells, the total RNA was extracted using a Nucleo Spin RNA XS kit (MACHEREY-NAGEL). First-strand cDNA was synthesized from the total RNA using Prime Script Reverse Transcriptase (TAKARA Bio). RT-PCR was performed using LA Taq (TAKARA Bio). The primer pairs employed for RT-PCR are listed in Supplementary Table From 2.5 × 10One patient with an unknown postmortem interval (control 9) was excluded from the RNA analysis , and centrifuged at 100,000 × g for 30 min at 4 °C. The insoluble pellet was dissolved in 100 μl of urea buffer  supplemented with a protease-inhibitor cocktail, sonicated , and centrifuged at 100,000 × g at 22 °C for 30 min. The supernatant was used as the urea-soluble fraction. The protein concentration of the RIPA-soluble fraction was measured using a BCA protein assay kit (Thermo Fisher Scientific). The protein concentration of the urea-soluble fraction was assumed to have the same ratio as the RIPA-soluble fraction. The supernatant was resuspended in Laemmli Sample Buffer (BioRad) and treated at 96 °C for 5 min. The proteins were separated by SDS-PAGE using a 10% polyacrylamide gel  and transferred to a PVDF membrane (Millipore). After transfer, total protein on the membrane was visualized using No-Stain Protein Labeling Reagent (Thermo Fisher Scientific). The blot was immersed in the primary antibody overnight at 4 °C. The antibodies used for immunoblotting included a rabbit anti-TDP-43  polyclonal antibody  and a mouse anti-GAPDH monoclonal antibody (MBL). HRP-conjugated secondary antibodies (Dako) were used. The bands were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and quantitatively analyzed using Amersham Imager 680 .The frozen motor cortex remaining after the DNA methylation analysis was homogenized in 10 volumes of ice-cold RIPA buffer  supplemented with a protease-inhibitor cocktail (Sigma). After sonication on ice , 400 μl of lysates were centrifuged at 100,000 × p < 0.05.For each experiment, the corresponding statistical test, which was two-tailed, is indicated in each figure legend. The number of samples per group is indicated in each figure legend. Statistical significance was considered to exist at Further information on research design is available in the Peer Review FileSupplementary informationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


In full-thickness (FT) wounds, autologous skin cell suspension (ASCS) is used in combination with widely meshed split-thickness skin grafts (mSTSGs) to obtain definitive closure, reducing donor skin requirements compared to traditional autografting (AG) techniques. In the treatment of these wounds, dermal matrices (DMs) are also often utilized to address various challenges including need for temporization, mitigation of contour defects, covering avascular structures, and modulation of scar formation. Varying DMs exist and are composed of different biologic and synthetic materials designed to address these clinical needs. The purpose of this study was to compare outcomes obtained in FT wounds using 3 different DMs coupled with ASCS+mSTSG in immediate or delayed AG procedures.A FT excisional porcine wound model was used. DMs evaluated included a single-layer dermal matrix composed of bovine dermal collagen and elastin (Col/E), a bilayer construct composed of a bovine collagen-glycosaminoglycan with a silicone epidermal layer (Col/GAG), and a synthetic bilayer polyurethane DM (Poly/U).DMs were applied, managed, and grafted following manufacturer’s recommendations. AG included ASCS+mSTSG . Wounds were evaluated for inflammation, infection, DM take, AG take, re-epithelialization, and contracture over 49 days post-excision. Additionally, biopsies were evaluated to further inform tissue generation and healing outcomes.Results related to healing outcomes are reported in the table below. Inflammation at wound margins was noted during acute phase of healing for all DMs and visual cues for infection were noted in 1 wound for Col/E, 3 wounds for Col/GAG causing partial loss of DM, and 1 wound for Poly/U. All signs of infection were resolved by day 14.ASCS+mSTSG can be used successfully over DMs composed of various materials in both immediate and delayed AG procedures. No difference was observed on percent AG take between the DMs, however the data suggest that time to definitive closure is impacted based on utilized DM and AG strategy, with potential implications on contracture.


With the increasing importance of the internet to our everyday lives, questions are rightly being asked about how its' use affects our wellbeing. It is important to be able to effectively measure the effects of the online context, as it allows us to assess the impact of specific online contexts on wellbeing that may not apply to offline wellbeing. This paper describes a scoping review of English language, peer-reviewed articles published in MEDLINE, EMBASE, and PsychInfo between 1st January 2015 and 31st December 2019 to identify what measures are used to assess subjective wellbeing and in particular to identify any measures used in the online context. Two hundred forty studies were identified; 160 studies were removed by abstract screening, and 17 studies were removed by full-text screening, leaving 63 included studies. Fifty-six subjective wellbeing scales were identified with 18 excluded and 38 included for further analysis. Only one study was identified researching online wellbeing, and no specific online wellbeing scale was found. Therefore, common features of the existing scales, such as the number and type of questions, are compared to offer recommendations for building an online wellbeing scale. Such a scale is recommended to be between 3 and 20 questions, using mainly 5-point Likert or Likert-like scales to measure at least positive and negative affect, and ideally life satisfaction, and to use mainly subjective evaluation. Further research is needed to establish how these findings for the offline world effectively translate into an online measure of wellbeing. A single, standardized definition of wellbeing does not currently exist in the literature. Previous attempts to define wellbeing could be argued to be a “description,” rather than a “definition,” as they focus on the dimensions of wellbeing rather than wellbeing itself  is a cognitive-judgmental process  that reviews evidence on the science of happiness and wellbeing and ranks countries by how “happy” they are   reports that 99% of 16–44 year olds in the UK were recent internet users at the time of survey, and only 7.5% of adults in the UK have never used the internet in 2019  and well?being].ti.Titles only were searched because it was felt that it was more likely that this would focus the search on studies that were specifically looking at scales. Words describing the type of wellbeing  were not included as some studies refer to SWB as simply “wellbeing,” leading to potentially missed studies. The term “happiness” was also excluded, as it is often a vague term with multiple meanings  , studies were not appraised for quality, and one researcher performed the analysis. These are discussed in greater detail in The following sections report common features of all the included scales, including how scales are structured, what components of SWB they measure, and how they measure it. Percentages on some tables may not add up to 100% because some scales can be counted under multiple categories. See Additional Material for (1) full list of included and excluded scales and (2) validation and evaluation status of included scales.The mean number of questions was 13 (median 10), with a minimum of 1 and a maximum of 37. Most of the 38 scales (57.9%) had up to 10 questions, and only 18.4% had more than 20 questions. The vast majority of scales used Likert or Likert-like measures (92.1%), with that being the only type of question used for most (86.8%); one also used interviews, and one used Likert, Yes/No, and multiple-choice questions. Only one measure used only multiple-choice questions and one only Yes/No questions, while one had variable question types depending on researchers choices, using the Experience Sampling Method (ESM) ; 55.3% used only these measures in their scale  , this will push the boundaries of what “the online world” is. Moreover, with the arrival of 5G mobile technology, it is expected to further increase consumer demand for online content and services Ofcom, . This siIf questions are not adapted or reworded to fit the online context, it is not guaranteed that online wellbeing is measured. Rewording questions [for example, from “I have been feeling interested in other people”   are underrepresented is unclear, but it may be speculated that Likert-type scale questions offer the ideal mix of characteristics. Yes/No questions suffer from a limited range of responses and does not determine how much respondents agree or disagree with a statement. Multiple choice offers more freedom, depending on the context of the question. Descriptive questions make mass administration of the scale difficult, as a lack of standardization means that each response will need to be analyzed; this is challenging for quantitative analysis, although it provides rich qualitative data in small samples.In terms of the number of points on a Likert scale, measures ranged from 3 to 11. A higher number of options for participants to most accurately represent how they feel may be particularly useful for tracking changes in online wellbeing over time, capturing smaller increases or decreases in mood: an increase from three to four on a 5-point scale is a greater increase than three to four on a 10-point scale, so respondents may be more likely to stick to the lower number. On the other hand, longer scale items may provide too much choice or take longer for respondents to complete, leading to less consistency between answers measuring the same thing: if four or five are seen as very close together, the choice between them may be random and differences missed. “The Paradox of Choice” also argues that excessive choice increases difficulty in choosing an option and decreases satisfaction with the choice , and in general, people may be more receptive to “rises in wellbeing” than reporting increases in poor wellbeing. This would suggest that an OWS might be better scored positively; however, in some contexts, this might not make sense. For example, if a questionnaire is asking how often something has occurred, for example, feeling happy or sad, it might be more intuitive for higher scores to equal more often, whether or not the statement was positive. For scales that measure several components of SWB (see next section), having more often high scores for positive items and low for negative items would be confusing. This leads to discussion of the possibility of reverse scoring. The majority of scales did not use reverse scoring. Equal numbers of scales used reverse scoring for only positive or only negative affect items. If positive scoring is used, negative affect questions can be reverse scored; otherwise, a high negative affect score would be taken to mean good wellbeing. There is a theoretical risk that including a high number of reverse-scored questions could increase the complexity; having a mix of positive and negative statements may also cause mistakes from misreading. This has been demonstrated in  (Larson and Csikszentmihalyi, The vast majority of scales measured wellbeing by directly asking for a subjective evaluation from respondents, with some of these using additional alternative measures. Very few scales only rely on alternative measures; an example is the laughter frequency scale (Murakami et al., However, the notion that objective indicators are not useful and do not correlate to SWB is not true (Oswald and Wu, how much social interaction do you receive?,” a potentially better question to ask is, “do you receive enough social interaction to satisfy you?.” The advantage of the latter is that it accommodates for people who prefer not to interact much with others, and the question asks respondents to compare what they currently have relative to their ideal.Alternative measures should also be phrased in a way that accommodates for different individual ideals. For example, instead of asking “The search for wellbeing scales was intended to be comprehensive but focused, but some literature will be missed, and therefore, there are SWB scales that are not included. To consider more wellbeing scales is outside of this review's aims but is certainly an area for further exploration. Far more wellbeing scales exist, and reading is available such as Boyle et al. . AnotherStudies were not appraised for quality, in favor of identifying as many scales as possible and not looking into the research carried out with them. Some scales reviewed may be low quality or biased, which could in turn bias the results and conclusion of this paper. However, the vast majority of the scales had some form of validation reported, and only three of the scales were produced solely for the purpose of a single study. Additionally, many of the recommendations were made based on several scales, including many that are strongly validated in previous research. This study was also not designed to determine the reasoning behind design choices in SWB scales, focusing on observing trends in current scales instead. Scoping reviews are intended to be a narrative overview of the literature (Arksey and O'Malley, Another limitation is that without a standardized definition of SBW, it can be problematic to recommend existing SWB measurements to the development of a new OWB assessment tool, especially when assuming that the psychometric qualities transferred from SBW to OWB remain the same. This is a limitation that should be kept in mind when applying the recommendations derived from this scoping review.Finally, one researcher performed the analysis using agreed definitions for terms related to SWB, which could lead to mistakes or biased judgments. However, topic experts were able to agree on stringently defined terms before analysis took place, so it is hoped that another researcher using the same definitions would arrive at similar results. Regardless, multiple researchers analyzing data independently are still preferred.This paper describes a broad scoping review of existing measures of SWB, highlighting the lack of online wellbeing measurement research, and compiling features of existing scales to offer recommendations for an OWS. Online wellbeing should be distinguished from generalized SWB, with an OWS focusing on areas of wellbeing that are contextually important to the online world. To summarize, an OWS is recommended to be between 3 and 20 questions and consider developing a brief measure if longer; use mainly 5-point Likert or Likert-like scales, with predominantly positive scoring, using reverse scoring if negative affect is to be measured alongside other SWB components; measure at least positive and negative affect and ideally life satisfaction; ask for evaluations within 1–2 weeks and a month at the maximum; and use mainly subjective evaluations with alternative measures being used as supporting questions if needed.The search and proposed recommendations for building an OWS are based on how current SWB scales are used in research. By focusing on field-tested SWB scales, the paper examined what is used in practice instead of what works in theory. It needs to be stressed that there is much research to be done to test these recommendations, especially in assessing the accepted components of SWB and how the online world might be unique. Most critically, a clear, accepted definition of the “online world” and what it means in relation to online wellbeing needs to be developed for research into online wellbeing to fruitfully continue. We welcome future work that incorporates the recommendations from this paper to develop and validate a new measure for OWS alongside existing SWB measures to test the assumptions surrounding what effects a persons' online wellbeing.ZO carried out the data collection and analysis and drafted the first version of the paper. LD edited and formatted all drafts of the paper. EP acted as main supervisor for the study, with LD, MR, and YL providing advice at various stages. EP, MR, and YL also commented on versions of the paper and approved the final manuscript. 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.


Elder abuse and neglect (EAN) comprises multiple dimensions, is experienced by about 10% of older adults in the U.S in diverse communities, and is severely detrimental to older adults’ (OA) health and wellbeing. However, documentation of EAN among refugee OA is greatly lacking as are services for these communities. Refugee OA are overall underserved members of marginalized communities. This paper reports on a community-engaged study to collect information and raise awareness of EAN among OA in 2 North Carolina refugee communities - Nepali-speaking Bhutanese and Congolese. Research partners included University researchers and community refugee-serving organizations. Surveys and focus group interviews were conducted. 17 Nepali-speaking Bhutanese and 13 Congolese filled out survey questions, including the Elder Abuse Suspicion Index. They participated in focus group discussions (FGDs), separately for men and women of each community. Survey results indicated EAN more among Congolese than Nepali-speaking Bhutanese. FGD results showed both communities prefer to depend on family members, and experience difficulties with language, transportation, and economic insecurity. No EAN was reported in the FGDs. In line with principles of community-engaged approaches, a capacity-building event to increase awareness of EAN was held, attended by 25 persons from the two communities. This study adds documentation on an under-researched area and marginalized communities. Action recommendations include disseminating culturally appropriate EAN information, strengthening English language and job skills and transportation options, encouraging cooperation across state, nonprofit, educational, and service organizations to address needs of older refugee adults.


Since its discovery in 2004, graphene has attracted worldwide attention due to its excellent physical and chemical properties. Herein, this review summarizes the latest developments in graphene frameworks, heteroatom-modified graphene, and graphene composite frameworks in sulfur cathodes. Moreover, the challenges and future development of graphene-based sulfur cathodes are also discussed.The global energy crisis and environmental problems are becoming increasingly serious. It is now urgent to vigorously develop an efficient energy storage system. Lithium-sulfur batteries (LSBs) are considered to be one of the most promising candidates for next-generation energy storage systems due to their high energy density. Sulfur is abundant on Earth, low-cost, and environmentally friendly, which is consistent with the characteristics of new clean energy. Although LSBs possess numerous advantages, they still suffer from numerous problems such as the dissolution and diffusion of sulfur intermediate products during the discharge process, the expansion of the electrode volume, and so on, which severely limit their further development. Graphene is a two-dimensional crystal material with a single atomic layer thickness and honeycomb bonding structure formed by sp Facing the depletion of fossil fuels and gradual serious environmental pollution problems, people have slowly realized the necessity of clean energy development. Sustainable energy such as solar and wind energy has been extensively developed . However−1 and a theoretical energy density of 2600 Wh·kg−1 . B. B35]. Bne plane ,37. IntrIn recent years, a variety of graphene/sulfur configuration materials have been developed as sulfur cathodes. In order to maximize the utilization of active materials, researchers have studied the graphene/sulfur configuration from a variety of perspectives. The basic configuration is divided into the following types: in-plane, three-dimensional sandwich, three-dimensional core-shell, and so on. This section summarizes the various configurations of sulfur cathodes based on unmodified graphene.There are several forms for in-plane: graphene sheets, graphene paper, graphene nanoribbons, etc. ,42,43,44−1 at 0.1 C was also due to the effective physical limitation of the sandwich structure. Fang et al. prepared a full graphene sandwich structure composed of high-porosity graphene (HPG), highly conductive graphene (HCG), and partial graphene oxide (POG)  a [48]. S0 cycles d. There 0 cycles b. This sThe core-shell structure has been proven to be able to solve the above-mentioned problems of LiPS leakage and poor volume buffer capacity. Graphene is used as an outer packaging to firmly bind the sulfur particles in the shell e,h. The + with polysulfides. The oxygen-containing groups and a large amount of space in the rGO aerogel inhibit the migration of LiPSs from the cathode . . 2 is alr method , and fin3S2) has also received attention in supercapacitors. Ni3S2 is not only low-cost but is also highly conductive. Guo et al. synthesized a Ni3S2/-rGO hybrid material , , 141], g−2 had a better specific capacity when doped with 2 wt% CNT .The amount and form of loading sulfur are also some of the evaluation criteria for electrode performance. For GO/CNT/S prepared by the freeze-drying method, Hwa first added sulfur and its sulfides during the electrode preparation process and finally removed the impurities , while G3 in the electrolyte. Lee et al. prepared a graphene-loaded N-doped carbon framework (NCF-G) [−1. Sun et al. decorated graphene with N and S co-doped carbon nanowalls (NSCNWs) to effectively trap polysulfides [The abundant functional groups on graphene are also one of the breakthrough points of G/CNT composites. Combined with the heteroatom-modified graphene structure mentioned in previous sections, single-atom doping or diatomic co-doping are both effective ways to improve the adsorption performance of G/CNT. For example, Su et al. converted Prussian blue (dehydrated sodium ferrocyanide) into a N-doped graphene-carbon nanotube hybrid material through one-step pyrolysis . The act (NCF-G) . The inisulfides . The NSC+. The most important thing is that it can fix polysulfides well. Zhou et al. designed N-doped double-shelled hollow carbon spheres (G–NDHCS–S) coated with graphene to capture sulfur [−1 at a current rate of 0.2 C. The graphene/carbon nanosphere composite material synthesized by Jia et al. possessed a specific surface area of 3200 m2·g−1 [−1 after 100 cycles. Therefore, the potential performance of the spherical structure electrode in the LSBs has a very broad development prospect.In addition to the CNT structure, electrodes with spherical structures have also been studied accordingly. The nanosphere structure can hold a large amount of sulfur and increase the contact area with the electrolyte to promote the transport of Lie sulfur . Due to 0 m2·g−1 . Its strAdditionally, current collectors based on other carbon materials and graphene have shown multiple advantages in LSBs. First, they show excellent flexibility. This is particularly prominent in CNT/graphene electrodes and provides an important foundation for the research of flexible batteries. Second, they mostly act as porous containers to hold polysulfides during the oxidation–reduction reaction process and improve the cycle stability of the battery.In summary, this article took graphene as a theme and summarized the potential applications of the pure graphene framework, heteroatom-modified graphene framework, and graphene composite framework in LSB cathodes in detail, aiming at obtaining LSBs with high energy density and long cycle capability and promoting their long-term development and application. The idea of graphene as a sulfur cathode carrier originates from its high conductivity, large specific surface area, and strong adsorption capacity for LiPSs on a surface defect. This viewpoint has also been confirmed by the composite structure of sulfur and the simple graphene framework. Sulfur cathodes have been designed with various structures such as sandwich, core-shell, and hybrid structures. Due to the flexibility and controllable assembly capabilities of graphene, the electrochemical performance of LSBs has improved rapidly. In the field of heteroatom-modified graphene cathodes, researchers gradually discovered that pyridinic-N had stronger chemisorption energy than other forms. It is worth noting that in addition to chemical bonding, transition metal doping also exhibits additional electrocatalytic capabilities. In order to solve the problem of limited content of single atom doping, researchers have gradually used diatomic doped graphene to achieve the common capture of LiPSs by a variety of chemical bonds. Metal compound additives have two main functions in the S electrode: one is to fix polysulfides, and the other is to catalyze redox reactions to promote polysulfide conversion. A small number of metal compounds can also serve as active materials to store lithium, increasing the lithium storage capacity. The advantage of the composite of other carbon materials and graphene is to achieve a diversified conductive network structure, thereby improving the conductivity of the skeleton, increasing the sulfur content and expanding the pore volume.However, although many efforts have been made to achieve better electrochemical performance, there are still many new challenges in LiSBs. For instance, although the stronger bonding force of pyridinic-N has been confirmed through repeated experiments, accurate control of the type and content of nitrogen doping cannot be achieved. The cost of boron doping is high, the doping process is complex, and the accuracy of characterization is difficult. The theoretical system for the interaction of transition metals and graphene bonds is not yet clear. There are also many challenges in the field of heteroatom doping. For metal additives, although catalytic conversion and effective chemical bonding inhibit the diffusion of polysulfides, the conductivity of such additives is not ideal, which inevitably reduces the battery capacity and cycle capacity. In this respect, the conversion of the catalyst into quantum dots may produce unexpected performance results.For graphene substrate materials, the key to improve the electrochemical performance of LSBs is the dissolution of polysulfides. The inhibiting of polysulfide diffusion has mainly focused on the physical properties of macroscopic physical encapsulation and microscopic van der Waals forces. In terms of chemical properties, it has the function of chemical bonding and catalysis, which accelerates the reaction process. The combination of physical action and chemical action is expected to be the most effective way of inhibition. This is a long way toward the industrial production of lithium-sulfur batteries, and it is necessary to coordinate development in many aspects such as the preparation process, applicable environment, high energy density, and economic benefits to promote the commercial application of LSBs.


Yarrowia lipolytica cells. Our treatment process can be integrated with the growth procedure and achieved the release of 50% of total cellular lipids from Y. lipolytica cells. Based on this result, we propose that nanoparticles coated with axially oriented graphene could pave efficient, environmentally friendly, and cost-effective way to release intracellular lipids from yeast cell factories.Engineering of microbial cells to produce high value chemicals is rapidly advancing. Yeast, bacteria and microalgae are being used to produce high value chemicals by utilizing widely available carbon sources. However, current extraction processes of many high value products from these cells are time- and labor-consuming and require toxic chemicals. This makes the extraction processes detrimental to the environment and not economically feasible. Hence, there is a demand for the development of simple, effective, and environmentally friendly method for the extraction of high value chemicals from these cell factories. Herein, we hypothesized that atomically thin edges of graphene having ability to interact with hydrophobic materials, could be used to extract high value lipids from cell factories. To achieve this, array of axially oriented graphene was deposited on iron nanoparticles. These coated nanoparticles were used to facilitate the release of intracellular lipids from  Recently, environmental concerns regarding the use of fossil fuels has increased, due to their contribution to the greenhouse effect, acid rain, ozone depletion and climate change2. Hence, the interest towards the production and utilization of biofuel is growing. Different species of yeast, bacteria and microalgae are being genetically engineered to enhance the production of various biofuels and oleochemicals5. Lipids produced by these microorganisms can be used as an alternative for fossil fuels, both as a sustainable source of energy or chemicals otherwise derived from oil. This production is sustainable, because cheap and widely available carbon sources, even including a variety of industrial waste and biowaste, can be used to grow such microorganisms producing biofuels and oleochemicals7.Microbial cell factories provide a sustainable source of biofuels and olechemicals4. Yarrowia lipolytica is one of the most promising yeast cell factories for production of oleochemicals and biofuels, because of its extremely high yield of lipids4, high levels of lipid accumulation8 and due to its metabolic diversity, which allows for production of a broad spectrum of unusual lipids9. However, Y. lipolytica and other cell factories do not actively secrete the produced lipids10. Instead, the lipids remain trapped inside the cells. Hence, different approaches for release of intracellular lipids from Y. lipolytica and other cell factories have been attempted, including microwave, ultrasound, thermal and mechanical treatments11, bio-based solvents12 and detergents13. However, most of these established methods are not selective for a specific product, they require high anounts of energy and require additional steps for lipid recovery14. Thus, these efforts failed in making biofuel and oleochemical production in yeasts and other cell factories economically feasible. The extraction of lipids from microbial cell factories remains a major bottleneck.Yeast cell factories are particularly promising in this respect, due to their excellent ability to synthesize and accumulation of lipids16. The contact of graphene edges with microbial cell surfaces leads to penetration of the cell membrane and displacement of phospholipids onto the graphene surface19. In extreme cases, the interaction can lead to irreparable membrane damage and leakage of intracellular content20. The key parameter for effective membrane penetration, identified by our group and others, is the contact angle between graphene and the cell surface, which must be perpendicular to each other25.Nanosheets of graphene have been previously shown to interact with microbial membranes via computational simulations and experimentallyY. lipolytica that has been genetically engineered for high-level production of lipids26. The interaction between graphene and membrane lipids is known to be mainly based on short-range van der Waals attractions17. These attractions promote the movement of lipid molecules from the biological membrane onto the graphene surface. For displacement of lipids from the membrane onto the graphene surface, the optimal contact angle between the graphene sheet and the biological membrane surface is 90º18.In this study, we hypothesized that the exposed sharp edges of graphene spikes, deposited axially on magnetic nanoparticles, could interact with the membranes of yeast cells and thereby facilitate the release of valuable lipids  produced by these cell factories. To test this hypothesis, we decided to work with a strain of Y. lipolytica. The treatment can be performed directly in the growth culture, without any interruption, and it can therefore be compatible with continuous release of intracellular lipids in fermenters. Our approach is based on metallic (iron oxide) nanoparticles coated with axially oriented graphene29, which show a considerable potential to release intracellularly accumulated lipids from yeast cell factories.Herein, we demonstrate a novel strategy to mechanically compromise the cell envelope in order to release intracellular lipids from 24. Briefly, iron oxide nanoparticles dispersed in water were drop-casted onto a SiO2 surface and heated at 80 °C for 20 min to remove water. After that, samples were kept at 350 °C for 10 min in the presence of H2 and Ar, and the temperature was increased to 450 °C for 10 min. The SiO2 surface with deposited nanoparticles was then loaded into a cold wall CVD system . There the samples were heated rapidly (~ 300 °C/min) to the growth temperature of 775 °C and annealed in H2 and Ar atmosphere. A 75 W DC glow discharge plasma was ignited, and the graphene growth was initiated by introducing 15 sccm C2H2, 15 sccm H2 and 1000 sccm Ar. The density and orientation of coatings was confirmed by SEM . The samples for SEM were sputter coated with gold (5 nm) before imaging. Raman spectra were measured by a WITec alpha300 R confocal Raman spectrometer equipped with a 100 × objective and spectrometer spectral range from 420 to 830 nm. Each spectrum was recorded in the range 500–3000 cm−1 with 10 min accumulation time and approximately 4 cm−1 resolution. The graphene-coated nanoparticles were detached from the SiO2 substrate carefully by using a surgical scalpel and used in all subsequent experiments.Axial graphene coating was deposited on iron oxide  magnetic nanoparticles using PE-CVD, as described previouslyλ = 1.5418 Å) in the range 5° < 2θ < 70°. Bragg’s Law, λ = 2d sin(Ɵ), was used to calculate the distance d between adjacent graphene layers, where λ is the wavelength of the incident X-Ray beam and Ɵ is the diffraction angle. For FT-IR spectroscopy measurements, graphene powder was dispersed in lipids for 30 min, rinsed with water and dried. The absorption of lipid by graphene was examined by FT-IR. Infrared spectra of the samples were recorded using an attenuated total reflection (ATR) Alpha FT-IR spectrometer from Bruker, with a diamond crystal as refractive element, in the range 400 to 4000 cm−1 at a resolution of 2 cm−1.Lipid absorption by different graphene coatings  was evaluated by contact angle measurements of lipid droplets on the coated surfaces. Commercially available lipids extracted from bacterial cells  were used in these assays. The purchased lipid solution was diluted in 5% of dimethyl sulfoxide (DMSO). Droplets of DMSO (negative control) and lipids were placed onto graphene-coated surfaces and contact angle was measured for 1 h with an interval of 4 s. To evaluate the lipid absorption ability of graphene using X-Ray diffraction (XRD) and Fourier-transform infrared (FT-IR) spectroscopy, graphene nanoflakes in powder form (purchased from Graphitene) were used. The powder was ground before XRD measurements and mixed with lipids (or Mili-Q water), until visibly saturated. The X-Ray diffractograms were obtained using a Siemens Bruker D5000 powder diffractometer operating at 40 kV and 40 mA with nickel-filtered Cu Kα radiation  were divided into three different fractions for further processing: a control fraction containing only Y. lipolytica culture, a fraction containing Y. lipolytica culture plus iron oxide nanoparticles and a fraction containing Y. lipolytica culture plus graphene-coated iron oxide nanoparticles. All samples were further incubated for 2 h with continuous agitation (1000 rpm) and then examined for release of intracellular lipids and morphological alterations. To examine the release of lipids, a fraction of treated cells was stained with 1 μL of BODIPY 493/503 (Thermo Fisher Scientific) solution (1 mg/mL in ethanol), which binds specifically to lipids and provides a green fluorescent signal. The stained yeast cultures were observed using a fluorescence microscope . The acquired images were further processed to calculate the percentage of intracellular lipids (green stain appearing within cells) and released lipid droplets (stain appearing outside cells) by using Image J. The percentages of intracellular and released droplets were quantified by measuring the image area occupied by the droplets. The cumulative data represent quantification of intra- and extracellular lipid obtained by measuring the area of intracellular and extracellular lipid droplets in microscopy images with 200 cells, for both the control experiments (non-treated cells and cells treated with bare nanoparticles), and cells treated with graphene-coated nanoparticles. The morphological alterations of yeast cells were examined with SEM. A fraction of treated cells was fixed in 3% glutaraldehyde overnight. The fixed cells were dehydrated with graded series of ethanol . The dehydrated cells were transferred to a clean cover glass and dried for 24 h at room temperature. The dried samples were sputter-coated with gold and examined by SEM . Further, the effect of graphene coated nanoparticles on growth pattern of Y. lipolytica was evaluated. Graphene-coated nanoparticles was collected from two substrate plates (each having an area of 1 cm2) and dispersed in 1 ml of medium. This represents the standard working concentration of coated nanoparticles in all our experiments. Other concentrations were obtained by diluting the working concentration in culture medium. In order to evaluate the impact of nanoparticles on the growth profile, Y. lipolytica was grown overnight and inoculated in 150 µl of nitrogen limiting media with various concentrations of graphene coated nanoparticles to a starting OD600 of 0.05 in 96-well plates. The cells were cultivated at 30 °C and 200 rpm for 96 h. The growth was measured every 30 min by the Growth Profiler 960 .24. Considering this phenomenon, initially we chose to compare the lipid absorption capacity of two types of differently oriented graphene coatings: a graphene monolayer parallel to the coated surface  and Fourier-transform infrared (FT-IR) spectroscopy Fig. . Figure −1 (C–H stretching (asymmetry)), 2854 cm−1 (C–H stretching (symmetry)), 1746 cm-1 (C=O stretching), 1465 cm−1 (C–H bending (scissoring)), 1165 cm−1 (C–O stretching and C–H bending), and 722 cm−1 (C–H bending)30. Taking together the findings from contact angle measurements  being higher than the G peak (~ 1590 cm−1) is indicative of axial orientation and high density of flake boundaries. The low intensity of the 2D peak (~ 2650 cm−1) (intensity decreases with increasing number of graphene layers) indicated that the samples are a mixture of monolayer and multilayer graphene. Combining high magnification SEM imaging and Raman data, we concluded that the iron oxide nanoparticles were successfully axially coated with PE-CVD graphene, resulting in a complete and homogenous coating, with a large proportion of exposed sharp edges, containing on average < 10 layers of graphene.Iron oxide nanoparticles (average diameter of 25 nm) were axially coated with graphene, using the method of plasma-enhanced chemical vapor deposition (PE-CVD) Fig. a. As a r32. According to this mechanism, the graphene nanosheet cuts through the lipid membrane, with the edge of the nanosheet sinking into the lipid bilayer. Thereafter, the lipid extraction mode commences, where the nanosheet extracts the phospholipid molecules from the lipid bilayer onto its surface. This massive draining of lipids from the membrane onto the graphene sheet results in membrane deformation, and ultimately in loss of cell membrane integrity34. Following this extraction of phospholipids, strong hydrophobic interactions between the graphene and the lipids have been speculated to play an important role through nanoscale ‘dewetting’. This strong hydrophobic interaction can be compared to the hydrophobic collapse of many biomolecular self-assemblies, such as cell membrane formation and protein folding36. Hence, it has been suggested that nanoscale dewetting can provide the driving forces required for complete cellular collapse, leading to release of all intracellular content37. However, all available mechanistic simulations and experimental data showing this mechanism is based on the interaction of graphene with bacterial cell membranes. To the best of our knowledge, there are no reports showing the interaction of graphene nanosheets with yeast cells.Next, we asked whether these coated nanoparticles can effectively compromise the integrity of yeast cells to release intracellular lipids. Computational simulations of graphene interaction with cellular membranes describe the insertion of edges of free graphene nanosheets into the membrane, driven by strong van der Waals attractions and hydrophobic interactions with membrane lipidsY. lipolytica culture grown for 5 days were treated with bare nanoparticles and graphene coated nanoparticles for 2 h with continuous agitation (1000 rpm). In order to assess if the release of lipid would be compatible with continuous growth in a fermentation process, no other treatment was applied. After 2 h of treatment with bare nanoparticles and graphene coated nanoparticles, yeast cells were examined by brightfield and fluorescent microscopy. The latter was combined with BODIPY staining (specific to lipid drops) that allows for visualization of lipids (including triglycerides), stained bright green  iron oxide nanoparticles were examined, and their lipid content was fully comparable to that of untreated cells, with only about 4.3% of total lipid present in the extracellular space. Thus, we concluded that the graphene coating is the key factor for release of lipids from cells. To more closely examine the morphology of Y. lipolytica cells exposed to graphene-coated nanoparticles, we further examined them by SEM (Fig. A direct interaction between graphene-coated nanoparticles and smaller lipid droplets can be seen in Fig. SEM Fig. . The morSEM Fig. b was indSEM Fig. a. This wY. lipolytica cells were clustered, apparently held together by a lipid matrix presumably formed by lipids released from the damaged cells (Fig. Y. lipolytica was examined. As shown in Fig. 600 = 3. However, the impact of nanoparticles on the final OD was minimal. This finding is very promising, since it indicates that lipid release from yeast cells can be achieved continuously without inhibiting growth, and therefore without compromising the metabolic yield of lipids. This technology could provide a significant improvement over existing methods to extract biofuels from microbial cell factories, thereby reducing energy consumption, extraction time and the use of hazardous chemicals. This encouraging finding is of course preliminary, and further optimization in scaled-up production setup should be performed in follow-up studies, to achieve an optimal balance between lipid production and lipid release.By contrast, cells treated with graphene-coated nanoparticles exhibited various morphological defects Fig. c, indicalls Fig. d. FinallY. lipolytica (Fig. 24. Hence, we propose that our graphene-coated magnetic nanoparticles could be developed into a promising tool to break yeast cells to release intracellular lipid. The magnetic properties of the coated nanoparticles can be used to collect graphene a side while collecting the released lipid and could be compatible with an uninterrupted fermentation process. We propose that this simple technology could be used to resolve the major bottleneck for opening of yeast cells and release of intracellular lipid from yeast cell factories, thus paving the way for commercial and sustainable production of oleochemicals and biofuels. However, future studies should focus on finding the optimal size of graphene coating and concentration of coated nanoparticles which maximize the release of intracellular lipid while minimizing damage to yeast cells.In conclusion, our results demonstrate that nanoparticles that are coated with sharp graphene spikes can be used to effectively break the cells and release intracellular lipids from ica Fig. . Graphenica Fig. , and by ica Fig. , 2. In o