Staphylococcus aureus (MSSA) infections are an important cause of morbidity and mortality in neonatal intensive care unit (NICU) infants. MSSA colonization  is a risk factor for subsequent invasive infection . Active surveillance programs coupled with decolonization measures for MSSA-colonized infants have been associated with decreased invasive MSSA infection rates in the NICU setting.Invasive methicillin-susceptible In June 2020, an active surveillance program was implemented to detect MSSA nasal colonization for infants admitted to the St. Louis Children’s Hospital NICU, a 140-bed quaternary NICU. MSSA-colonized infants were decolonized with a 7 day course of intranasal mupirocin twice daily plus topical chlorhexidine baths on days 1, 4, and 7 . MSSA bloodstream infection (BSI) rates were followed quarterly pre- (September 2018-May 2020) and post- (June 2020-March 2022) implementation of MSSA surveillance and decolonization. Mean MSSA BSI rates pre- and post-implementation were compared via paired T-test. The number of infants treated with mupirocin was calculated pre- and post-implementation.Figure): 0.22 per 1000 patient-days pre-implementation, 0.16 per 1000 patient-days post-implementation (p=0.37). Since initiating MSSA decolonization in June 2020, the number of NICU infants exposed to mupirocin increased nearly fourfold: mean 10 infants treated per month pre-implementation, mean 38 infants treated per month post-implementation.After implementing MSSA surveillance and decolonization there was no significant change in the mean MSSA BSI rate .All Authors: No reported disclosures.


Strengthening muscles can reduce body fat, increase lean muscle mass, maintain independence while aging, manage chronic conditions, and improve balance, reducing the risk of falling. The most critical factor inducing effectiveness in strength training is neuromuscular connection by adopting attentional focus during training. However, this is troublesome for end users since numerous fitness tracking devices or applications do not provide the ability to track the effectiveness of users’ workout at the neuromuscular level. A practical approach for detecting attentional focus by assessing neuromuscular activity through biosignals has not been adequately evaluated. The challenging task to make the idea work in a real-world scenario is to minimize the cost and size of the clinical device and use a recognition system for muscle contraction to ensure a good user experience. We then introduce a multitasking and multiclassification network and an EMG shirt attached with noninvasive sensing electrodes that firmly fit to the body’s surface, measuring neuron muscle activity during exercise. Our study exposes subjects to standard free-weight exercises focusing on isolated and compound muscle on the upper limb. The results of the experiment show a 94.79% average precision at different maximum forces of attentional focus conditions. Furthermore, the proposed system can perform at different lifting weights of 67% and 85% of a person’s 1RM to recognize individual exercise effectiveness at the muscular level, proving that adopting attentional focus with low-intensity exercise can activate more upper-limb muscle contraction. Strength training has been the most fascinating exercise of the last decade. Strength training is used by default in workouts to build strength, muscle mass, and joint strength. However, everyone has different fitness goals. Long-term outcomes vary depending on the exercise routine; however, everyone wants to train efficiently every time. Depending on the program, exercise objectives have several definitions. Endurance, hypertrophy, maximum strength, and power are the four distinct goals outlined by the National Strength and Conditioning Association (NSCA) . For eacThe COVID-19 pandemic has resulted in social distancing measures and led to a “new normal” exercise practice. Instead of going to the gym, training at home became normal. In the field of fitness training, weightlifting equipment is used everywhere, but strength training does not take up space, and a dumbbell can be used to perform a variety of exercises. Emerging online platforms, including APPs, can also be used to guide and monitor practitioners. However, many people do not monitor their exercise performance on a variety of platforms because they are occasionally misaligned with their objectives, and there is no practical solution to give users feedback on training effects related to attentional focus at the muscle level ,8,9.The concept of attentional focus has important implications for fitness training. Sports science research refers to attentional focus as an individual focusing on muscles and movements while performing a given movement or activity . For exaIn , the autThe neuromuscular system comprises motor neurons, sensory neurons, and muscle fibers. Body movement, posture control, and breathing are also controlled by this system. In physical exercises that involve neuromuscular control, the body can produce forces as well as stabilize and reduce external forces dynamically through movement, which in essence keeps the body balanced. Many researchers have studied brainwaves and muscle tasks using EEG signals to map dynamic sources of cortical signals ,27. HoweWe develop a system with a wearable, eight-channel, noninvasive EMG fitness shirt to assist in sensing attentional focus during exercise.We develop a system comprising a multitask and multiclassification network to detect attentional focus on muscle contraction from EMG signals for tracking personal fitness at the muscular level.We implement and evaluate the system for attentional focus and muscle contraction at different lifting weights based on five standard exercises of isolated and compounded muscles.To date, we have acknowledged that attentional focus is an essential element of sports and exercise performance in hundreds of studies ,12,28. RWe begin the rest of the paper with a discussion on related works of attentional focus, followed by experiments and system design. Finally, we discuss the results and evaluate this new strength-training tracking method.We designed a fitness-training shirt integrated with EMG sensors to offer reliable signal recording, as shown in The system framework is illustrated in Signal collection was based on the use of electrodes on the training muscle as the source and reference electrodes. We investigated the signal when the user wore the shirt because the device’s position and orientation might not be identical if the gesture signal is sensitive to this position/orientation; therefore, the EMG might not be viable for practical applications. In this pre-experiment, five people were asked to wear the device and perform a bench press, producing natural diversity across the subjects. We recruited 12 healthy male college students who participated in personal weight training activities , height , weight , and BMI (body mass index) . To enable us to determine the force, participants underwent a strength assessment. We targeted five exercises recommended by the resistance training guide for healthy adults . The subInstruct the user to perform five exercises in the following order: Bench Press, Bicep Curl, Triceps Kickback, Front Raise, and Lying Pullovers;Ask the user to prepare for 5 min before start;Start session 1: 0% weight, 12 repetitions without attentional focus;Rest for 5 min;Start session 2: 0% weight, 12 repetitions with attentional focus;Rest for 5 min;Start session 3: 67% 1RM, 12 repetitions without attentional focus;Rest for 5 min;Start session 4: 67% 1RM, 12 repetitions with attentional focus;Rest for 5 min;Start session 5: 85% 1RM, 12 repetitions without attentional focus;Rest for 5 min;Start session 6: 85% 1RM, 12 repetitions with attentional focus;Rest for 5 min;Start a new exercise and repeat from Step 3;When the user completes the five exercises, the user is asked to rest.Procedure:ξ. When the frame energy exceeded ξ, it was recorded as the start frame, tf. When the frame energy is lower than ξ, it is recorded as the end frame ef. We removed signals with durations of less than 1.5 s or for which the maximum energy was lower than the threshold δ, and then obtained the corresponding starting point st and ending point et  of the original signal. We extended the signal forward and backward for 0.15 s (30 sampling points), respectively, to obtain a complete signal and finally obtain the 8-channel data of the EMG signal in a single movement for feature extraction.Our 8-channel EMG sensor was limited to 0–4096. When collecting the data, we normalized the data for each channel to a range of 0–1, with a minimum value of 0 and a maximum value of 4096. Then, we averaged the data from the eight channels for signal segmentation. First, we cut the average continuous EMG signal to obtain a single signal for each movement. Specifically, we performed high-pass filtering (cut-off frequency of 1 Hz) on the signal to remove the DC component. Next, we take 0.25 s as the frame length and 0.1 s as the frame step to calculate the frame energy. We set the signal threshold as The typical method to develop muscle strength is to contract the muscle to its maximum potential at any given load. In strength training, 1RM is the maximum number of repetitions that can be achieved with a given resistance or weight. Here, we asked subjects to exercise with dumbbells weighted to an estimated 67% and 85% of 1RM with and without attentional focus, respectively. The goal of estimating muscle contraction is to track the effectiveness of attentional focus during exercise. If muscle contraction with attentional focus is greater than that without attentional focus, it is interpreted that the targeted muscle is activated with respect to the minimum of the maximal low-intensity voluntary contraction ,15. The Seven features were selected from EMG signals ,29,30,31In this work, we propose a multitask and multiclassification network that considers different exercises (task A), attentional focus (task B), and 1RM (task C) as three subtasks and classifies them uniformly to better guide people during exercise. The proposed network architecture is shown in A, B, and C represent the three subtasks, X is the input, Y is the real label, t is the task space {A, B, C}.Because each person’s physical function and muscle level were different, we trained a model for each subject. Each person’s data were divided into a 70% training set, 15% verification set, and 15% test set. In the training process, all these subtasks contribute to the overall loss function so that their performance can be considered. We define a loss function for the output vector of each subtask and then define the overall loss function as the (weighted) sum of these subtask loss functions. The loss function is defined as follows:tω that can measure the impact of the component loss function, which can limit the impact of the tasks. Therefore, if the output of only one task is important for the application, it can be prioritized by assigning it a greater weight. In our experiment, these three tasks were equally important. Therefore, its weight was 1.Because the subtasks in this study were supervised multiclassification tasks, the component loss function of each task was categorical cross-entropy. By defining loss as a linear combination of component losses, we obtain a set of super parameters The results of the experiment on practical scenarios are presented in the next section. Based on the evaluation, we divided the experiment into two categories: the robustness of the system and its impact on the user. A robust system analyzes how the system performs in practical scenarios on exercise recognition and attentional focus, whereas an impact system examines the relationship between a user’s attentional focus and the level of muscle contraction. To evaluate the robustness of the system for the recognition of attentional focus, we used precision  and specificity  as criteria for recognizing the correct attentional focus performance. In this context, precision refers to the total number of positives of the actual attentional focus covered while exercising (correct identification). The performance of recognized exercises without attention was measured by specificity. For exercise and 1RM recognition, accuracy was used to measure the proportion of both true positives and true negatives. Using RMS for muscle contraction level, the performance of subjects was determined based on their muscle contraction level in relation to their attentional focus in different 1RMs.We further investigated the results of exercise recognition in detail. If the position and orientation of the device are not identical and the posture signal is sensitive to this difference, the EMG may not be suitable for practical applications. The raw data are still reasonably consistent with the exercise in question. As shown in We first show the performance of the system through the recognition of attentional focus at different 1RMs. As shown in We used a support vector machine (SVM), a deep neural network (DNN) of a single task, and our multi-task and multi-classification network to compare the overall system performance. We found the best hyperparameter of all the models to evaluate the performance of the learning algorithms, and we evaluated the detection performance for each model. For our multitasking and multiclassification network, we used the Adam optimizer with a learning rate of 0.001. For the DNN of a single task, the three branches of the proposed network were trained independently. The 1RM recognition network lost its fusion layer, and other network structures and settings remained unchanged. For SVM, we used RBF as the kernel function and the one-versus-one method to design a multiclassification SVM. The results are shown in The results in In addition, we evaluated attentional focus under two conditions to determine the difference in muscle contraction. In this evaluation, we compared muscle contraction during each exercise. In this study, the detection of attentional focus was conducted in a practical scenario. Our system allowed us to monitor attentional focus during different 1RMs and exercises. The assistive methods currently used involve the verbal and physical gestures of the trainer or application. These methods emphasize form and maximum power when lifting heavy weights, perpetuating the myth of ‘no pain, no gain’ . In contWe believe that there is an interest in exploring our system in the real world for interactive sports trainers for all these reasons. The diversity of human physiology makes it difficult to conduct experiments on females and other age groups. The system is currently limited because it requires calibration per user and manually checks electrode placement. The system also requires users to test their lifting capacities in advance. Our study selected exercises based on previous studies on different muscle groups, both isolated and compound exercises. For example, pullovers and bench presses are designed to train chest muscles. Females may not be suitable for our prototype shirt, and heavy exercise may result in injury in certain age groups. Considering that women and other age groups differ physiologically from men of the age group studied here, it is necessary to exclude them from training. However, we envision that the proposed system can be used in various daily activities. Future work will include field studies with a broader focus on fatigue-inducing conditions. Collecting data from a larger pool can extend our findings.To evaluate the user experience of our system, we asked all subjects to complete modified versions of the intrinsic motivation inventory (IMI) , a widelIn the IMI, responses are graded on a seven-point Likert scale divided into seven subscales. Three subscales are relevant to our application: interest/enjoyment, perceived competence, and value/usefulness. All questions were randomly assigned to categories and averaged for statistical analysis. User encounters. In general, all participants were enthusiastic about the system: “(P6) Compared to other similar products on the market, the biggest difference of the system is that it can be worn on the body and accurately detect the muscle activity of a specific part.” and “(P1) It also helps me focus on my muscle while doing exercise.” Almost everyone mentioned that the other products they used could not provide helpful suggestions while strengthening the muscles: “(P10) My smartwatch only detects the heartbeat, which cannot guide or detect specific muscle activation.”Improvements and suggestions. Participants provided improvements and suggestions for the system. Although the equipment was very portable, “(P3) it felt obvious pressure when worn during reuse” and “(P4) it made me feel uncomfortable after being used for a long time.” Furthermore, one of the participants “(P3) felt a little afraid of touching the equipment during the experiment.”Interactions. The participants enjoyed the effective interaction that helped them recognize muscle contraction and track the progress of the exercise: “(P7) It definitely helped me feel and focus on my muscle when I workout.”; “(P6) it can intuitively and easily reflect whether my muscle force is correct and whether I can borrow it.” Many participants agreed that it was beneficial to let them focus on the exercise and provide helpful feedback. (P2) mentioned that “there were not only some incomprehensible data.” After the experiment, half of the participants showed great interest in the system and spent more time exercising. In addition, they all want to recommend the system to their family and friends who regularly participate in fitness.In this paper, we present a system that utilizes a multitasking and multiclassification network and a wearable EMG sensing device to monitor attentional focus with a robust signal quality. The purpose of our study was to develop a noninvasive, eight-channel EMG fitness shirt for the detection of attentional focus during exercise. Using a multitask and multiclassification network, we developed a system for tracking personal fitness at the muscle level by detecting attentional focus on muscle contractions from EMG signals. As a result of the implementation and evaluation of the system, we were able to obtain information regarding attentional focus and muscle contraction for a variety of lifted weights based on five standard exercises for isolated and compound muscles. Our study suggests that the system can classify the user’s attentional focus by analyzing the user’s training stage via the EMG signals obtained with the proposed device, with an average precision of approximately 94.79% among subjects at various maximum forces. In addition, the proposed system can recognize 67% and 85% of the 1RM to investigate the effectiveness of muscle activation in each session among the subjects. In addition, the findings show that low-intensity exercise at 67% of the 1RM can increase upper-limb muscle contraction more than at 85% of the 1RM.


Graphene has stimulated great enthusiasm in a variety of fields, while its chemically inert surface still remains challenging for functionalization towards various applications. Herein, we report an approach to fabricate non-covalently functionalized graphene by employing π–π stacking interactions, which has potentialities for enhanced ammonia detection. 5,5′-Di(4-biphenylyl)-2,2′-bithiophene (BP2T) molecules are used in our work for the non-covalent functionalization through strong π–π interactions of aromatic structures with graphene, and systematic investigations by employing various spectroscopic and microscopic characterization methods confirm the successful non-covalent attachment of the BP2T on the top of graphene. From our gas sensing experiments, the BP2T functionalized graphene is promising for ammonia sensing with a 3-fold higher sensitivity comparing to that of the pristine graphene, which is mainly attributed to the enhanced binding energy between the ammonia and BP2T molecules derived by employing the Langmuir isotherm model. This work provides essential evidence of the π–π stacking interactions between graphene and aromatic molecules, and the reported approach also has the potential to be widely employed in a variety of graphene functionalizations for chemical detection. Non-covalent functionalization of graphene has been achieved by employing π–π stacking interactions, and it is promising for ammonia detection with greatly enhanced sensitivity. Despite the great potentialities, the inert surface of the graphene results in numerous challenges for selective detection of various gases.9–11 To address this concern, various functionalization approaches have been exploited to conjugate selective ligands on its surface, including both covalent and non-covalent modifications.12Graphene with its single-atom-thick two-dimensional (2D) conjugated structure has been extensively explored as an ideal material for chemical detection owing to its exceptional properties, 13 Aromatic molecules have been demonstrated useful for non-covalent functionalization by π–π stacking interactions.14,15 Owing to the aromatic structure, the molecules can strongly absorb onto the graphene surface, while at the same time, the functional groups of the aromatic molecules can be exposed and employed to recognize the target molecules directly.16 Therefore, the aromatic molecule based non-covalent functionalization of graphene is promising to be widely employed to achieve the surface modification of graphene towards chemical detections.Non-covalent functionalization approach based on π–π stacking interactions shows unique advantages since it does not necessitate chemical modifications and thus can maintain the unique physical and structural properties of graphene.3) is a typical hazardous and irritant gas which can induces toxicity to the environment and sensitive ecosystems, therefore, a range of techniques have been employed to achieve the sensitive detection of NH3, including nanowires,17,18 metal oxide,19 reduced graphene oxide,20 laser-coupled spectroscopy,21etc. Despite the great potentialities as an ideal candidate for solid state gas sensors, graphene based ammonia sensors are still struggling as the sensor response is relatively much lower  as compared to other gas species sensed with pristine graphene. Even if the fact that many methods such as doping,22 defect insertion23,24 and chemical functionalization,25 have been applied, it is still a challenge to improve the sensitivity of graphene-based gas sensors to NH3.Ammonia -2,2′-bithiophene (BP2T) molecules are used for the non-covalent functionalization via strong π–π interactions between the aromatic structures of BP2T and graphene. Systematic analysis by utilizing different spectroscopic and microscopic characterizations was performed and the result confirms the successful non-covalent functionalization of the BP2T molecules on graphene surface. Moreover, our gas sensing experiments show that the BP2T molecule functionalized graphene demonstrates a 3-fold higher sensitivity for NH3 comparing to that of the pristine graphene, which opens up possibilities for graphene functionalization towards various chemical detections.In this work, we reported a systematical investigation of the graphene surface functionalized by employing π–π non-covalent stacking mechanism and also demonstrated its potential application in NH−1) and G peak (at 1580 cm−1) (I2D/IG) of more than 3, while the negligible D peak (at 1350 cm−1) indicates the good quality of the graphene with a very low level of defects. With the increase of the non-covalent functionalization time, both I2D and IG are decreased. More specifically, in the samples of immersing time of 1 h and 2 h, graphene still maintains a monolayer feature of graphene where the intensity ratio of I2D/IG is more than 3. Nevertheless, when the immersing time is longer than 2 h, the intensity ratio of I2D/IG tends to be 1, implying the tendency of the structure evolution towards forming a bilayer graphene.26–29 As for the D peak, it can be seen that all of the samples after non-covalent functionalization maintains a low intensity D peak, indicating a low defect level of the graphene, which is quite different comparing to covalent graphene functionalizations.5,30,31To better understand the structure evolution of the surface after modifications, graphene with various non-covalent BP2T functionalization time ranging from 1 h to 5 h were prepared and investigated. 2 substrate, it is realistic to measure the film thickness of graphene T (T = Tgraphene + TBP2T layer). According to the work of Cumpson and co-workers,32,33 the graphene thickness T on top of SiO2 substrate can be expressed as:λ is the mean free path of the photoelectrons, θ is the emission angle, Ic and Io are the measured peak intensities of the C 1s peak of the film and O 1s peak of the substrate, respectively, and Sc and So are the XPS elemental sensitivity factors of carbon and oxygen. The obtained BP2T molecular thickness TBP2T layer as a function of immersing time is shown in In order to investigate the formation of BP2T molecule layer on graphene surface through non-covalent π–π interactions, X-ray photoelectron spectroscopy (XPS) measurements were performed as shown in 3 gas sensing measurements were performed in a gas sensing probe station under ambient pressure, and the current–time (I–t) characteristics of the gas sensors are recorded. To ensure reliable sensing results, the gas sensing chamber is purged by using dry N2 for more than 1 h prior to introducing the target gas in all measurements. The gas flow of 100 standard cubic centimeter per minute (sccm) is used in the experiment, which is controlled by a mass flow controller. Here, the conductance response S of the gas sensor can be defined as the normalized conductance change response as:G2N denotes the initial conductance of the graphene gas sensor and G3NH is the final conductance of the sensor under NH3 gas exposure. Owing to the electron donor character of the ammonia gas, the measured conductance response S is normally smaller than the initial conductance G2N.8,343 at room temperature, and S is observed upon the NH3 exposure. As for the response time, it is found from the curves that all of the devices demonstrate a similar response time of ∼250 s. Meanwhile, it is worthy to be mentioned that the recovery process of all graphene gas sensors is also very fast, around 200 s, and does not require any external treatment such as high temperature annealing or UV light illumination, showing unique sensor properties compared to conventional NH3 sensors that normally require higher temperature annealing for the recovery to the initial state.To facilitate the electrical analysis and gas sensing experiments, a pristine monolayer graphene is fabricated into a gas sensor and graphene is electrically contacted by Au (60 nm)/Cr (5 nm) as illustrated in 35,36 and thus thiophene groups have a great potential for the detection of NH3 molecules.37 To further understand the gas sensing behavior of the non-covalently functionalized graphene, the Langmuir isotherm model is applied to estimate the binding energy between the NH3 molecules and functionalized graphene. In the Langmuir isotherm, the gas sensing response S is proportional to the gas coverage, therefore the sensitivity of the gas sensor can be written as:38S(∞) is the sensor response at equilibrium state, S0 is the saturated response, Ka is the adsorption constant, K is the equilibrium constant, υ is the attempting frequency of the gas molecules, P is partial pressure and t is response time. Under the room temperature of 300 K, the adsorption energy Ea of the gas can be expressed as:kB is the Boltzmann constant, T (300 K) is the temperature, υ (1012 s−1) is the attempt frequency, K is the equilibrium. Hereby, S0 (0.08) and K (0.32 Pa−1) can be derived by linear fitting of response S of as a function of partial pressure P according to the following formula:10Theoretical studies have predicted that graphene functionalization can result in an enhanced binding energy between graphene and target molecules, leading to a considerably increased sensitivity in gas sensing,Ka can be precisely derived. Based on the equations above, it is possible to extract the binding energies of ammonia to graphene samples with different treatment time, by fitting the obtained electrical gas response curves for various samples, and the obtained binding energy as a function of immersion time is shown in 3, while sample treated with 3 h and 4 h have a lower binding energy comparing to pristine graphene and correspond well with the decreased sensitivity observed in the electrical gas sensing experiments. As for the sample after 5 h treatment, no data is derived because due to the complete loss of sensitivity.3 molecules, DFT calculations have been performed in this work /Si wafer by employing a polymer-assisted method.40 The graphene was patterned and electrically contacted  by using standard electron beam lithography (EBL) in a Nanobeam nB Series EBL system, oxygen plasma etching in an Advanced Vacuum Vision 320 Reactive Ion Etching (RIE), and physical vapor deposition (PVD) in a Lesker PVD 75 system. To prepare the molecule solution for the non-covalent functionalization, 10 mg BP2T molecules (Sigma-Aldrich) were dissolved in 10 ml of toluene. The non-covalently BP2T functionalized graphene sensors for NH3 detection were prepared by immersing the monolayer CVD graphene devices in the BP2T/toluene solutions for 1 h, 2 h, 3 h, 4 h and 5 h, respectively, then being washed with the toluene and followed by a blow drying.Large-scale monolayer graphene (Graphenea) was synthesized by using chemical vapor deposited (CVD) approach on a copper foil3 at room temperature.Scanning electron microscopy (SEM) and light optical microscopy (LOM) images were recorded in a Zeiss 1550 SEM and an Olympus AX70 research microscope, respectively. Raman spectroscopy was carried out in a Renishaw inVia Raman spectroscopy with a 532 nm excitation laser. X-ray photoelectron spectroscopy (XPS) was performed in a PHI Quantum 2000 spectroscopy using monochrome Al Kα radiation (1486.7 eV) with a 45° angle of electron emission. The electrical characterizations of the graphene sensor devices were carried out in an Agilent B1500 semiconductor parameter analyzer with tungsten probes, and the noise level of the current measurement was lower than 50 aA. The gas sensing experiment was performed in a home-built gas sensing probe station equipped with a gas sensing chamber and a Keithley 6430 sub-femto amp source meter inside a Faraday cage, and the whole assembly is inside fumehood. The chamber is also coupled to several mass flow controllers (Brooks 5878), which are used to regulate the speed of different gases flowing into the chamber. The electrical response of the graphene sensors to the gas can be obtained by recording the current variations as a function of gas exposure time through the source meter under the exposure of 10 ppm NH41 In the initial setup the BP2T molecule had perfectly aligned hexagons over graphene sheet with unit cell taken in  direction along molecule and  or 7.3830 Ang across. NH3 molecule was placed coordinated to sulphur atom, with preferable position to be hydrogen bonded via N–H⋯S and C–H⋯N bonds. All atoms were allowed to fully relax. Core electrons are modeled using Troullier–Martins42 soft norm-conserving pseudopotentials, with the mesh cut off was 200 Ry and Brillouin zone integration for the supercell was sampled by 1 k-point. The valence electrons are expanded in a basis set of local orbitals using a double-ζ plus polarization orbital (DZP) set. The GGA was used for the exchange–correlation functional.43To understand the binding mechanism for molecular sensing, calculations were carried out from first principles with a method based on DFT as implemented in the Siesta package.Hu Li: conceptualization, formal analysis, funding acquisition, investigation, writing – original draft. Tianbo Duan: formal analysis, methodology, writing – review & editing. Omer Sher: formal analysis. Yuanyuan Han: formal analysis, methodology. Raffaello Papadakis: investigation, writing – review & editing. Anton Grigoriev: formal analysis, investigation, writing – review & editing. Rajeev Ahuja: supervision, writing – review & editing. Klaus Leifer: conceptualization, funding acquisition, methodology, writing – review & editing.There are no conflicts to declare.RA-011-D1RA06879B-s001


Small interfering RNA (siRNA) therapies require effective delivery vehicles capable of carrying the siRNA cargo into target cells. To achieve tumor-targeting, a drug delivery system would have to incorporate ligands that specifically bind to receptors expressed on cancer cells to function as portals via receptor-mediated endocytosis. Cell-targeting and internalizing aptamers are the most suitable ligands for functionalization of drug-loaded nanocarriers. Here, we designed a novel aptamer-based platform for the active delivery of siRNA targeting programmed cell death-ligand 1 (PD-L1) to triple-negative breast cancer (TNBC) cells. The generated nanovectors consist of PLGA-based polymeric nanoparticles, which were loaded with PD-L1 siRNA and conjugated on their surface with a new RNA aptamer, specific for TNBC and resistant to nucleases. In vitro results demonstrated that these aptamer-conjugated nanoparticles promote siRNA uptake specifically into TNBC MDA-MB-231 and BT-549 target cells, along with its endosomal release, without recognizing non-TNBC BT-474 breast cancer cells. Their efficiency resulted in an almost complete suppression of PD-L1 expression as early as 90 min of cell treatment. This research provides a rational strategy for optimizing siRNA delivery systems for TNBC treatments. Triple-negative breast cancer (TNBC), which constitutes 10% to 20% of all breast cancers, is one of the most aggressive subtypes of breast cancer with the worst outcome ,4. So faProgrammed death-1 (PD-1) is a prominent immune checkpoint receptor, which is expressed on T cells and interacts with its ligand PD-L1 on cancer cells to induce inhibitory responses, which could promote immune evasion and tumor progression . PD-L1 iPoly(lactic-co-glycolic)-block-poly ethylene glycol (PLGA-b-PEG) has gained great attention in recent decades as a major component for nanovectors, because of its capability to create biodegradable polymeric nanoparticles (PNPs), which are congenial to targeted drug delivery approaches: indeed, thanks to its amphiphilic property, PLGA-b-PEG is able to self-assemble under mild conditions into a micellar-like structure, protecting the drug, siRNA, or other small molecules in its core while exposing the PEG shell to the external environment, thus ensuring the dispersibility of the whole nanosystem in water or the physiological environment ,20. MoreThese nanosystems have been long used for the trapping of many anti-cancer drugs  specifically into TNBC cells. The sTN145 2’Fluoro-pyrimidines (2’F-Py) RNA aptamer, which we generated from TNBC cell-SELEX ,29, was In this study, we developed novel PNPs decorated with the sTN145 aptamer and loaded with siPD-L1, succeeding in highly efficient siRNA entrapment. We show that, according to the TNBC cell type specificity of the sTN145 aptamer, the aptamer-decorated PNPs efficiently deliver the siRNA cargo into TNBC MDA-MB-231 and BT-549 cells, but not into TPBC cells. Importantly, the siRNAs delivered by the sTN145-aptamer-decorated nanoparticles resulted in efficient PD-L1 gene silencing and suppression of PD-L1-induced epithelial–mesenchymal transition (EMT) factor Snail in TNBC cells.Taken together, these results present a viable platform for the active delivery of siRNA to cancer cells and strongly support further investigation to evaluate its efficacy for boosting anti-tumor immunotherapy in TNBC.2-terminated 2’F-Py-containing RNA sTN145 and scrambled (SCR) aptamer used as a control were synthesized by LGC Biosearch Technologies . The sequences of the aptamers were previously reported [NHreported .Human PD-L1 siRNA sequences entrapped in nanoparticles  were: sense sequence 5’-GUAGCAAUAUGACAAUUGATT-3’ and anti-sense sequence 5’-UCAAUUGUCAUAUUGCUACCA-3’. siPD-L1 and its 5’ Fluorescein (6-FAM)-labeled version (FAM-siPD-L1) were purchased from Qiagen .w/w 6:1). Then, the two-phase mixture was emulsified, in an ice bath, with a tip probe sonicator  for 45 s. Subsequently, 144 mL of 1.25% sodium cholate solution in water was slowly added to the obtained emulsion: the resulting two-phase mixture was further emulsified for 3 min, in an ice bath, at the amplitude shown above. The FAM-siPD-L1@PNPs were likewise prepared.The preparation of the PLGA-PEG nanoparticles loaded with siPD-L1 followed a previously reported procedure ,22. BrieAfter the final water-in-oil-in-water (w1/o/w2) emulsion thus created was ready, the chloroform was completely evaporated under reduced pressure, and the resulting particles were washed and concentrated using centrifugal filter devices  to a final volume of 5 mL and, finally, filtered using nylon syringe filters .2O to reach a volume of 8 mL, then a solution of N-hydroxysulfosuccinimide 2.3 mM (4.3 mL) and a solution of 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) 0.28 M (1.8 mL) were added to the vial. The -COOH activation was carried out at room temperature (RT) for 10 min, then 102 pmol of the sTN145 aptamer or of the scrambled sequence, dissolved in 1 mL of water and suitably activated with a cycle of 5′ at 85 °C, 3′ at 0 °C, and 10′ at 37 °C, was added and left to react for 24 h. After that, the particles obtained were washed and purified by repeating the same passages described above (centrifugation with filtering devices and filtration on syringe filters). The final volume was adjusted to 5 mL with sterile water.For the conjugation onto the outer shell of the aptamer sequence, siPD-L1@PNPs or FAM-siPD-L1@PNPs were diluted in HDynamic light scattering (DLS) analysis and ζ-potential values were acquired with a Zetasizer Nano-S  instrument, working with a 532 nm laser beam at 25 °C, using standard cuvettes or DTS1070 Clear Disposable zeta cells, and the results are expressed as the average of three measurements.The siPD-L1 encapsulation efficiency was determined by measuring the amount of extractable siRNA in the PNPs. To this aim, 0.5 mL of siPD-L1@PNPs (2 mg of dry matter) was added to 0.2 mL of chloroform and 0.25 mL of TE buffer, then rotated end-over-end for 90 min at RT to facilitate the extraction of siRNA from the organic phase in the aqueous phase. The aqueous and organic phases were separated by centrifugation for 15 min at 13,200 rpm at 4 °C. The upper aqueous phase containing the RNA was transferred to a new tube and incubated for 5 min at 37 °C to remove the residual chloroform, concentrated using centrifugal filter devices , and 8 µL of each sample was loaded onto the denaturing PAGE (15% polyacrylamide with 7 M urea). The gel was stained with ethidium bromide and UV exposed to visualize the RNA bands. Band intensity was quantified using ImageJ (v1.46r), and the amount of siPD-L1 extracted was extrapolated from a linear standard curve obtained with different amounts of siPD-L1.Another determination, albeit indirect, of the amount of FAM-siPD-L1 was assessed by quantitative fluorescent analysis using an Edinburgh FLSP920 spectrofluorimeter equipped with a 450 W Xenon arc lamp. The measurement was performed using a subtractive strategy, checking the fluorescence emission of the PNPs’ washing waters after each fabrication/conjugation step. The spectrofluorimetric determination was performed using an excitation wavelength of 485 nm and an emission wavelength of 520 nm and FAM-siPD-L1 standard solutions at different concentrations (1 to 20 nM) in water.Human BT-549 and MDA-MB-231 (TNBC) and BT-474 (TPBC) cell lines  were grown as previously reported ,30.5 cells/well, 6-well plates) were transfected for 5 h at 37° C with PD-L1 siRNA or control siRNA (siRNA ctrl), purchased from Qiagen , by using Lipofectamine RNAiMAX Reagent , according to the manufacturer’s instructions. At 48 h post-transfection, cell lysates were prepared and analyzed by immunoblot.BT-549 and MDA-MB-231 cells  were treated with empty@PNPs, empty@PNPs-SCR, or empty@PNPs-sTN145 (0.5 and 1 mg/mL dry matter) for 24 h, and cell viability was assessed by Thiazolyl Blue Tetrazolium Bromide , according to the manufacturer’s protocol.MDA-MB-231 and BT-549 cells (5.0 × 104 cells/well) were seeded on glass coverslips placed in a 24-well plate one day prior to the experiment. Subsequently, the medium was removed and the cells were stained live with LysoTracker Red DND-99  in Roswell Park Memorial Institute-1640 medium  supplemented with 10% fetal bovine serum (Sigma-Aldrich) for 1 h at 37 °C. After three washes with Dulbecco’s phosphate-buffered saline solution , the cells were incubated for 0.5, 1, 2, and 5 h at 37 °C with FAM-siPD-L1@PNPs, FAM-siPD-L1@PNPs-SCR, or FAM-siPD-L1@PNPs-sTN145  diluted in RPMI-1640 supplemented with 0.1 mg/mL yeast tRNA and 0.1 mg/mL ultrapure™ salmon sperm DNA (Invitrogen), as nonspecific competitors. After washing, cells were fixed with 4% paraformaldehyde in DPBS for 20 min and incubated with 1.5 µM 4’,6-Diamidino-2-phenylindole  in DPBS for 5 min, and coverslips were mounted with glycerol/DPBS.Cell internalization experiments were performed as previously reported . Briefly4 cells/well, 24-well plate) incubated for 1 h at 37 °C with FAM-siPD-L1@PNPs, FAM-siPD-L1@PNPs-SCR, or FAM-siPD-L1@PNPs-sTN145 as above served as the nontarget cells.TPBC BT-474 cells  for the silencing power of the PD-L1 gene by cell transfection experiments in both TNBC MDA-MB-231 and BT-549 cells. These cell lines, which express a high level of PD-L1 . A fluorPNPs systems were prepared according to the protocol already described ,22 with DLS revealed FAM-siPD-L1@PNPs-sTN145 and siPD-L1@PNPs-sTN145 diameters, respectively equal to 113.1 ± 0.96 and 98.08 ± 0.49 nm, a low polydispersity index (PDI = 0.172 and 0.159), and a negative ζ-potential value of −16.7 mV and −23.6 mV, due to unreacted carboxylic acid groups, which derive from PEG chains and remain free even after the conjugation protocol, ensuring thus a slightly negative surface charge, which guarantees an optimal and efficient dispersion of the entire nanosystem in water and physiological solution. On the other hand, the particle size of the SCR-labeled system was 100.1 ± 0.67 nm (FAM-siPD-L1 loaded) and 98.43 ± 1.88 nm (siPD-L1 loaded), with a PDI average equal to 0.165 and a negative ζ-potential value around −20 mV. In order to confirm whether all nanosystems remain stable and do not undergo any physicochemical alterations, a certain amount of PNP solutions was collected, suitably diluted, and stored at 4 °C for four weeks. No significant variation was noted in the three values of the above parameters . Furthermore, the macro morphological aspect of the colloidal solution was still clear, opalescent, free of precipitates, and homogenous. The final overall concentration of siPD-L1@PNPs-sTN145 or SCR in solution was determined by gravimetric analysis by drying 100 µL of solution at 120 °C for 24 h and then carefully weighing the amount of residual dry matter. The results showed a final concentration of 4 to 10 mg/mL for all samples; the amount of siPD-L1 (or FAM-siPD-L1) entrapped in the PNPs was calculated  in the cells after 0.5 h of incubation with the targeted FAM-siPD-L1@PNPs-sTN145, but not the non-targeted ones, both unconjugated (FAM-siPD-L1@PNPs) and decorated with the scrambled aptamer (FAM-siPD-L1@PNPs-SCR) A. FurtheConversely, siPD-L1-associated signals were undetectable with SCR-decorated nanovectors for up to 1 h and slightly visible from 2 h incubation . AccordiOverall, these results indicate that the nanoparticles’ cell uptake is driven by the sTN145 aptamer, which retains its binding-competent folding after conjugation onto the outer surface of siRNA-loaded PNPs.As a next step, we asked whether the siPD-L1 cargo delivered into TNBC cells by the sTN145-decorated nanoparticles reduces the level of PD-L1. To this aim, we first assessed that the functional activity of siPD-L1 entrapped in the nanoparticles was not affected by the nanovector preparation procedure. Therefore, siRNA molecules were extracted by siPD-L1@PNPs and transfected into MDA-MB-231 cells (30 nM siRNA concentration) via the Lipofectamine RNAiMax transfection reagent, and the PD-L1 protein levels were determined after 48 h by immunoblot. As shown A, the exNext, in order to compare the silencing effect of siPD-L1 delivered by the sTN145-equipped PNPs with that of the same amount of transfected siRNA, MDA-MB-231 cells were treated for 5 h with siPD-L1@PNPs-sTN145  or transfected with siPD-L1, washed, and left untreated for a further 48 h. As shown in These results indicate that sTN145-conjugated nanoparticles effectively deliver a functional PD-L1 siRNA into TNBC target cells.Using cell-SELEX, we recently generated and characterized an aptamer, termed sTN145, which targets a membrane protein uniquely expressed on the surface of TNBC cells with high affinity and selectivity ,29. HereAptamers targeting cancer cell surface proteins have emerged as a viable alternative or complement to conventional antibodies for active cancer targeting due to their small size, which improves tissue penetration and their easy production through chemical synthesis . AptamerVarious delivery systems, differing in composition, size, and chemical properties, have been developed so far, in an attempt to overcome major barriers to the use of siRNA therapies. Nevertheless, tumor-targeted delivery systems ,38,39 stAmong the different nanocarriers, polymeric nanoparticles have gained increasing and constant attention in recent decades due to their effectiveness in cargo delivery, stability in different physiological media, and reliability of their behavior, release profile, and above all, safety. In particular, PLGA is probably the most-studied polymer as it has complete biodegradability, is FDA approved for medical application, and exhibits an extraordinary ability to assemble into nanometer-sized micelles, capable of trapping small molecules, such as drugs, and releasing them into the body in a time-dependent manner. Despite the excellent qualities, the use of PLGA polymeric nanoparticles for drug delivery applications still remains problematic due to the fast removal of these nanocarriers from the bloodstream by the liver and spleen, thus drastically reducing drug availability to the tumor tissue. To overcome these issues, PEG was employed, because, thanks to its “stealth behavior”, which inhibits the rapid recognition of a foreign agent by the immune system (opsonization), it is able to significantly increase the blood circulation time of the nanocarrier. Moreover, PEG is hydrophilic and able to stabilize nanoparticles through steric effects, especially in water. The PLGA-PEG block copolymer is thus an excellent system, and in the last decade, it has emerged as one of the most-promising systems for nanoparticle formation, drug loading, and drug delivery applications in vivo . FurtherIn vitro characterization of multicomponent nanovectors clearly demonstrates that the targeting agent  and therapeutic cargo  retain their functional activity when integrated into the nanoparticle bioconjugates. This leads to the selective release of siPD-L1 in TNBC cells, due to the high binding sensitivity and specificity of sTN145 ,29 and tOur results strongly encourage further in vivo studies with animal models to confirm the therapeutic utility of our nanovectors. Due to the capability of sTN145 to recognize its target on murine 4T1 TNBC cells , studies in orthotopic 4T1 xenografts in syngeneic mice are ongoing in our laboratories to evaluate the role of the immune system in the response to targeted delivery of therapeutic nanoparticles to tumor. Moreover, the identification of the surface protein that is enriched on mesenchymal TNBC MDA-MB-231 and BT-549 cells and is specifically recognized by the sTN145 aptamer will allow researchers and clinicians to apply our unique drug-loaded nanoparticles to humanized mice obtained from patient-derived TNBC xenografts and to use them as a future valuable tool for personalized treatments.After an initial response to cytotoxic chemotherapy, a large proportion of TNBC patients often develop recurrent tumors, which are chemoresistant and highly metastatic ,30,45. TIn summary, this study reports the development of a new system for active delivery of siPD-L1 to TNBC cells. In the designed nanosystem, the nanocore was assembled with siPD-L1 and PDL through charge interaction, while the outer shell was conjugated with the sTN145 aptamer for selective targeting of TNBC cells.The aptamer-conjugated and siRNA-loaded nanoparticles were efficiently taken up by target cells and reached the cytoplasm for suppression of PD-L1 expression.Our aptamer-based approach provides a safe and efficient tool to target and deliver therapeutic siRNAs to cancer cells with promising potential for targeted applications.


A persistent purpose for self-powered and wearable electronic devices is the fabrication of graphene-PVDF piezoelectric nanogenerators with various co-solvents that could provide enhanced levels of durability and stability while generating a higher output. This study resulted in a piezoelectric nanogenerator based on a composite film composed of graphene, and poly (vinylidene fluoride) (PVDF) as a flexible polymer matrix that delivers high performance, flexibility, and cost-effectiveness. By adjusting the co-solvent in the solution, a graphene-PVDF piezoelectric nanogenerator can be created . The solution becomes less viscous and is more diluted the more significant the concentration of co-solvents, such as acetone, THF, and EtOH. Additionally, when the density is low, the thickness will be thinner. The final film thickness for all is ~25 µm. Furthermore, the- crystal phase becomes more apparent when graphene is added and combined with the four co-solvents. Based on the XRD results, the peak changes to the right, which can be inferred to be more dominant with the β-phase. THF is the co-solvent with the highest piezoelectric output among other co-solvents. Most of the output voltages produced are 0.071 V and are more significant than the rest. An attractive possibility for producing electricity is to capture mechanical energy from the vibrations in the surroundings that would otherwise go to waste. Piezoelectric , triboel2–CF2)n is called PVDF [A polymer with a repeating unit of  [3O4-graphene oxide/hybrid nanocomposites [In addition, several previous studies have looked at adding other materials to increase further the electrical behavior of PVDF, including PVDF-TrFE , carbon es (NPs) , polytetes (NPs) , Fe3O4-gmposites , graphenmposites ,16,24. Amposites ,26,27. Dmposites ,29. Grapmposites ,31.N,N-dimethylacetamide (DMAc) [N-methyl-2-pyrrolidone (NMP) [More than adding other materials is needed to increase the piezoelectric properties of PVDF. There are previous studies that have examined the effects of solvent and cosolvent on polymeric PVDF/graphene films; for example, using NMP ,33, DMF e (DMAc) , dimethye (DMAc) , and N-mne (NMP) ,44 are sIn this study, we constructed a piezoelectric generator using a PVDF film that contained a combination of graphene. We incorporate particular co-solvents into the NMP solution, such as acetone, tetrahydrofuran (THF), water, and EtOH. The spherulitic size of the PVDF was reported to have decreased, and the crystallization kinetics to have been affected by the addition of a co-solvent. The nucleation of the PVDF phase was also shown to be more successfully promoted by graphene with a negative surface charge. Two other efficient ways to boost the piezoelectric performance of PVDF nanofibers for energy harvesting applications include using polar solvents to dissolve and recrystallize the material, and adding additions such as graphene.N-methyl pyrrolidone , and acetone were acquired from Echo Chemical Co., Ltd. Taiwan, China; tetrahydrofuran  and ethanol ; graphene , DI water, and a glass plate.Polyvinylidene fluoride , N-methyl pyrrolidones (NMP), the first thing to do is mix 5.4 g of water and 48.6 g of N-methyl pyrrolidone. Stir well with ultrasonic vibration for 30 min. Next, take 10 g of the mixed solvent and add 6 g of polyvinylidene fluoride (PVDF). Heat all the solutions and stir until dissolved using a magnetic hotplate stirrer. Repeat the above steps by replacing the aqueous solution with different co-solvents, such as acetone, tetrahydrofuran (THF), and EtOH in the NMP solution.Making 10 wt% water/Using a dry technique, the PVDF–graphene composite film was created . In a nuTo study the surface composition of the composite film, the composites were characterized using attenuated total reflectance—Fourier transform infrared spectroscopy (ATR-FTIR)  and Raman spectroscopy . For morphological and microstructural studies, scanning electron microscopy (SEM)  and X-ray powder diffraction analysis  were carried out. Water contact angle (WCA)  measurements were conducted to evaluate the composite film surface hydrophilicity or hydrophobicity. The viscosity of graphene–PVDF solutions with different cosolvent concentrations (ranging from 10 to 40 wt%) was measured at 25 °C using an SV-100 Vibration Viscometer .In this study, the piezoelectric performance of the composite film will be investigated by applying the reciprocating 1 Hz impact. PVDF/graphene will be cut into 5 cm × 8 cm and placed in front of a linear motor after attaching the electrode. The charge will be generated to the two side surfaces of the composite film due to the piezoelectric effect, and was measured by connecting the composite film to the charge amplifier and dynamic signal acquisition module .Generally, the fabricated graphene–PVDF composite film morphology and topology depend on the solubility properties of the solvent and nonsolvent. To prove the viscosity of a solution mixed with graphene, we carried out a viscosity test. The results of the viscosity test are shown in For the use of piezoelectricity, the microstructure is essential. It is not easy to evenly distribute much graphene in a PVDF composite sheet. With a 10 wt% graphene load in a PVDF composite film, we demonstrate in this study how to use a powerful shear force to overcome a challenge that was once unsurmountable. Photographs of the PVDF and graphene–PVDF composite films are shown in The following data are the SEM characterization of graphene–PVDF with co-solvent variations. In Furthermore, in relation to graphene–PVDF with a mixture of NMP solvent and co-solvent acetone, the cross-section will decrease with the increasing concentration of the co-solvent. This trend is also shown in the co-solvent THF and EtOH. Solvent/co-solvent mixtures such as NMP/acetone and NMP/THF show that the viscosity will decrease or become thinner when the co-solvent concentration increases. Therefore, the dilute mixture will be easier to spread on the glass plate, causing the thickness of the composite film to decrease and become thinner. Co-solvent, polymeric additive, polymer, and solvent will influence the composite film thickness and viscosity change for a given system .−1, the penetration depth ranges from 2.00–2.86 μm. The top surface’s crystalline structure mostly matches the bulks for all the graphene–PVDF composite films, as seen in −1 and 1274 cm−1 were attributed to the β-phase structure, the transmittance bands that characterize the pure PVDF at 874, 1071, 1168, 1231, and 1041 cm−1 correspond to the α-phase. According to a report, higher polymer concentration favored a better-orientated packing of CH2–CF2 dipoles and the TTTT confirmation of the β-phase that followed. When the graphene content was barely 10 wt%, almost all the β-phase was generated.Since the crystallization environment differs depending on the sample thickness, variations in crystalline structure may be present. Attenuated total reflectance (ATR) is the most widely used sampling methodology for Fourier transform infrared (FTIR) spectroscopy. The FTIR-ATR spectroscopy method was used to examine the crystalline structure of the composite film top surfaces. Between 1600 to 650 cmThe XRD data (17 to 23 degrees) of the final composite films cast from various co-solvent systems are shown in −1 and the D band at ∼1355 cm−1, which are caused by disordered graphitic structures in sp2 hybridized carbons, respectively. The ID/IG ratios in the figure show where the D and G bands are located. The greater strength of the D bands compared to the G bands suggests that the disordered phase of graphene is increasing. The average cluster size, sp2, and the ID/IG ratio, which quantifies the degree of disorder, are inversely correlated [Raman spectroscopy was used to further study the interfacial interactions occurring in the nanocomposites to support the data obtained from FTIR. rrelated . The ID/rrelated .The press-hold-release method was used to investigate the conversion of mechanical energy to electrical for a liner motor action to show how the produced graphene–PVDF may be used as piezoelectric nanogenerators a. The ouIn this study, we wanted to see the effect of adding co-solvent in the PVDF/Graphene/NMP solution. In the co-solvent acetone, the average output voltage is about 0.059 V. At the same time. THF was higher, at about 0.073 V, followed by EtOH at 0.061 V. However, the co-solvent water had a poor piezoelectric yield. Because the mixture of water/NMP causes the viscosity and graphene dispersion in the solution, which it needs to be better, all the co-solvent combinations increase the output voltage compared to before being given a co-solvent. The generated voltage is only 0.048 V . TherefoWe discovered that liner motor motions are well-synchronized using graphene–PVDF nanogenerators. The mechanical strain caused by the pressing movement along the thickness of the composite film necessitates the installation of an external circuit to carry free electrons across the positive and negative electrodes. These free electrons will accumulate at the electrode mixed film contact and create a positive voltage peak. The piezo potential balances free electrons via an external circuit during the holding action. No voltage peak was observed. However, during the release action, the piezo potential vanishes, and the accumulated free electrons are sucked back into the electrode, causing the observation of a negative signal that is typical of the activity of composite film generators.In the early stage, as shown in This research created a graphene–PVDF piezoelectric nanogenerator by varying the co-solvent in the solution . The higher the co-solvent acetone, THF, and EtOH concentration, the lower the resulting viscosity and the more dilute the solution. In addition, the thickness will be thinner when the density is low. For all, the resulting composite film thickness is ~25 μm. Furthermore, the β-crystal phase is more evident when graphene is added and mixed with the four co-solvents. Judging from the XRD results, the peak β-phase shifts to the right, which can be assumed to be more dominant with the β-phase. Of the four co-solvents used, THF has a significant piezoelectric output. Almost all of the resulting output voltages are 0.071 V and more significant than the others.


In recent years, graphene aerogel (GA) has been widely used as a 3D porous stable network structure material. In order to identify the main research direction of GA, we use the bibliometric method to analyze its hot research fields and applications from the Web of Science database. First, we collected all relevant literature and analyzed its bibliometrics of publication year, country, institution, etc., where we found that China and Chinese Academy of Sciences are the most productive country and institute, respectively. Then, the three hot fields of fabrication, energy storage, and environmental protection are identified and thoroughly discussed. Graphene aerogel composite electrodes have achieved very efficient storage capacity and charge/discharge stability, especially in the field of electrochemical energy storage. Finally, the current challenges and the future development trends are presented in the conclusion. This paper provides a new perspective to explore and promote the related development of GA. Graphene was successfully extracted from graphite using mechanical exfoliation by Geim and Novoselov in 2004 . As the Due to the wide range of GA applications, reviews that summarizes its application have been widely reported ,25,26,27The data used for this review were retrieved from Web of Science on 4 October 2022. The preliminary results were obtained by advanced search “TS = Graphene Aerogel* not AK = (Foam* or Sponge*),” and then the keywords and abstracts of the retrieved literature were manually filtered to obtain the full WOS core collection of 5705 papers and 234 reviews.It should be noted that the combination of the word Graphene aerogel first appeared in a paper published by Wang et al. in 2009 . Since tAfter 13 years of research and development since 2009 (2009–2021), the number of published articles has grown to more than 900 in the last three years, as shown in After common bibliometric analysis of GA research performance, we used VOSviewer to analyze the keywords of the articles to determine the hotspots in the GA research field, and selected keywords with more than 20 occurrences. We discarded some keywords like Graphene, Aerogel, Graphene aerogel, etc., which did not show any useful information in The fabrication of GAs are mainly classified according to the nodes presented in the green clusters. By employing different methods, diverse properties of GA could be excavated and obtained. At present, the main fabrication methods involved in the formation of graphene aerogels include self-assembly methods, template methods, and 3D printing methods.The in-situ assembly method is usually divided into two types: hydrothermal reductive assembly method and chemical reductive assembly method. Graphene oxide (GO) is used as a precursor, which is uniformly and stably dispersed in solution by the electrostatic repulsion of the surface negatively charged functional groups such as epoxy groups and carboxyl groups, which are shown in ,30 Figur+ concentration in the solution, the adsorbent is less protonated, causing a weakening of the adsorption capacity [Hydrothermal assembly utilizes high temperature and pressure conditions in closed conditions where graphene sheets in solution associate with each other to form a stable three-dimensional structure ,30,32 Ficapacity ,34. Secocapacity . TherefoCompared to the harsh conditions of hydrothermal assembly, the synthesis of graphene hydrogels using chemical reductive assembly requires the addition of reducing agents  to chemically reduce GO ,37,38, w+, Na+, Co3+, etc.) and organic compounds containing functional groups  can be able to act as cross-linking agents to promote gel formation. Graphene aerogel materials with different properties can be achieved by adding different kinds of cross-linking agents. For example, Cong et al. reported the use of α-Fe(OH)3 nanorods with magnetic Fe and other metal oxides to induce the assembly of graphene sheets into three-dimensional graphene hydrogels used as adsorbents for water purification. The combination of electrostatic attraction, ion exchange between metal hydroxides and heavy ions, surface complexation, and oxygen-containing functional groups showed a strong adsorption capacity [The prominent feature of cross-linker-induced self-assembly lies in the addition of chemical cross-linkers during the synthesis process, and the surfaces where GO and cross-linkers are bound form bond bridges could interlink into a three-dimensional network structure ,40. The capacity . In addicapacity . Differecapacity , and the3)3 and (NH4)2S2O8 to remove the foam Ni, which shows gentler and less bubbles during etching. As a result, better structural integrity was obtained with better and less defects [Template-assisted chemical vapor deposition (CVD) is another widely used method to catalyze the synthesis of three-dimensional GA . The GA  defects . Finally defects . In this defects . Ice templates, also known as freeze casting, is another general method for the preparation of inorganic materials. It mainly refers to the solid phase (ice) generated by the phase separation that occurs during the solidification of the solution as a template . As the 3D printing, as a new and unique preparation method, has obvious advantages in GA field , as showSynthesis of GA is an important part of GA-related research, and different synthesis methods are used to obtain materials with specified properties to meet the needs of researchers. Therefore, as the first step in GA research, synthesis occupies a fundamental and important role.According to the red clusters, we can see that the application of graphene aerogel in the whole energy storage field is mainly focused on functions as an electrode material or a framework for electrode material, as evidenced by energy-storage, electrode, supercapacitors, and batteries. The main research hotspots include battery electrodes and supercapacitor electrodes . For batLithium battery is the most widely used battery system in the field of electrochemistry. According to the types of electrodes, lithium batteries are mainly divided into two kinds of lithium-ion batteries (LiBs) and lithium metal batteries , as showCathode: Anode: The anode plays the role of energy storage and release by absorbing or shedding ions during battery charging or discharging , and is The most prominent anode materials for lithium-ion batteries include graphite  and lithium titanate . GraphenGraphene aerogel-containing anode materials are classified into two categories based on the involvement of GA: pure graphene aerogel anodes and hybrid graphene aerogel composite anodes. Firstly, in the fabrication of pure graphene aerogel anode materials, a good structural design can enhance the electrode performance while ensuring a stable structure. In order to prevent the overlapping of layers as in the case of two-dimensional graphene, graphene oxide can be hydrothermally reduced and annealed under treated filter paper to obtain three-dimensional graphene aerogel electrodes. During the synthesis process, the filter paper acts like a rope to weave the dispersed graphene oxide flakes into a well-connected porous 3DGA. At the same time, the graphene flakes covered with folds are cross-linked with each other to form a porous structure for easy lithium ion embedding . This elThe use of graphene aerogels hybridized with common anode materials including metal/alloys, metal compounds, and silicon-based materials to form composites for use on lithium-ion battery electrodes is another hot research topic in recent years. These materials have good theoretical performance when used as anode alone, but suffer from various shortcomings during use. For example, these materials in ion batteries metal/alloys can charge and discharge by achieving an alloying reaction or a reverse alloying reaction, both of which theoretically have a high specific capacity . However2@few-layered MoS2 covered by S-doped graphene aerogel (MO/MS/SGA) prepared by Zhang et al. can not only effectively reduce the aggregation of MoO2, but prevent the MoS2 stacking. The graphene aerogel structure acts as a flexible matrix and conductive network while effectively relieving the volume buffering of Mo. The performance is good: the capacity is 683 mAh/g when cycled 100 times (current density of 0.1 A/g) [Metal compound electrodes, mainly consisting of two types of metal oxide (MO) electrodes and metal sulfide (MS) electrodes, utilize the Faraday reaction of metal ions to store charge and thus have the potential to achieve ultra-high energy density and large theoretical capacity (500–1000 mAh/g) . It also0.1 A/g) . The int1/rGO2 composite consisting of a 3D conducting graphene backbone and a flexible pre-wrapped graphene coating as well. The special structure of graphene aerogel wrapped with silicon particles is utilized, which can improve the electrical conductivity and has a stable structural buffering volume effect. The bilayer graphene layer Is able to separate the electrolyte and silicon, thus generating a thin and stable SEI film to avoid reversible capacity loss [Silicon is another material system with the highest theoretical specific capacity in the negative electrode of lithium-ion batteries, which is considered as one of the substitutes for carbon materials in commercial batteries . Howeverity loss .Cathode: Anode: Li-S batteries are expected to replace lithium metal batteries as a new generation of efficient secondary batteries due to their high theoretical specific capacity (1675 mAh/g) and high energy density (2600 Wh/kg) . Besides2S; (2) the polysulfide (LiPS) produced during charging and discharging is easily dissolved in the electrolyte to form a “shuttle effect” to reduce the battery performance; (3) the large change in electrode volume during cycling, which causes damage to the structure and affects the battery life [2/g) and a high electrical conductivity (67 S/m). The porosity is also able to act as a channel for ion diffusion and absorption of LiPS [However, the current development path has been hampered by some obstacles. The main problems include the following: (1) the poor electrical conductivity of cathode sulfur and discharge product Liery life ,85. Grapery life . For exa of LiPS . Meanwhi of LiPS .2 SnS2 nanoparticles embedded in graphene aerogel acting as sulfur hosts to form electrodes SnS2-Nano Dots@GA, which can effectively ensure a small amount of SnS2 in graphene aerogel with uniform deposition of SnS2 nanoparticles while still maintaining a high positive sulfur content (75 wt%). Using the adsorption ability of 3D graphene and SnS2 on LiPS, the material showed chemical stability as well as excellent performance with a capacity of up to 1016 mAh/g after 300 cycles [2 with abundant defects wrapped by graphene aerogels also exhibit adsorption properties on LiPS and act as catalysts to accelerate their redox reactions [3O4/porous carbon/graphene aerogel (Fe3O4/NC/GA). The large specific surface area and porous structure of the graphene aerogel provided the adsorption capacity for LiPS, and the highly conductive Fe3O4 embedded in the nanowafer could play a catalytic role to accelerate the conversion of LiPS [2-decorated graphene aerogels in the separator for the “shuttle effect” of polysulfides, while the cathode obtained in combination with I/N-doped graphene aerogels was able to effectively immobilize and capture LiPS with long term stability—no sulfite was detected in 200 cycles substances, with intact electrode performance [The use of graphene aerogel-encapsulated nano MS/MO as the positive electrode material and the layered porous structure can promote ion diffusion and electron transfer, and also treats LiPS for performance enhancement by the dual effect of adsorption through van der Waals interactions and catalytic conversion of LiPS by MS/MO0 cycles . In addieactions . Similar of LiPS . Tan et formance .2 batteries have received widespread attention for their high energy density as well as low cost [Cathode: Anode: As a new electrochemical energy storage device proposed in recent years, Li-Olow cost . Since tlow cost . The rea2O2 need to find a suitable method to solve. The cathode of Li-oxygen battery, as the main research object, is the support for the reaction sites of the three phases . Therefore, the following advantages are required: (1) good electrical conductivity (to facilitate electron transfer); (2) good gas diffusivity (to ensure oxygen concentration at the reaction active site); (3) porous structure, large pore capacity, and high specific surface area (to facilitate the deposition of discharge products); and (4) stable non-decomposition during charging and discharging. Based on these requirements, GA is widely used as the cathode material for lithium-oxygen batteries because of its good electrical conductivity, porosity, light weight, and relatively easy preparation. The special three-dimensional conductive network structure and high specific surface area of GA can accelerate OER and ORR, and its synthesis does not involve other superfluous substances such as adhesive, which can cause further damage to the battery performance [2O2 to reduce deposition and increase the capacity of Li-Ox batteries. For example, Zhao et al. used carbon nanocages prepared from polystyrene sphere@polydopamine (PS@PDA) and then hydrothermally self-assembled coupled into a 3D porous N-doped graphene aerogel (NPGA). The material has a crumpled and loose structure that prevents the stacking of graphene sheets thereby exposing more reactive sites. Compared to the GA without the carbon nanocage structure, NPGA acts similarly as a catalyst, increasing the ORR onset potential and decreasing the OER onset potential with a faster reaction rate [x battery. As a two-in-one material for positive and negative electrodes, it can not only reduce the formation of negative Li dendrites to increase the battery life, but mitigate the bulk effect to increase the battery capacity and ensure the cycle efficiency [Many problems such as low cell cycle efficiency and slow reaction kinetics due to the poor conductivity of the reaction product Liformance , so GA hformance ,96. At tion rate . Not onlficiency . TherefoSupercapacitor, also known as Ultracapacitor, is an energy storage device with a large capacity . Accordi3 S/m) and remains stable after 100 bending cycles [The main reason for that graphene aerogels can be used as electrode materials in double-layer capacitors is due to their good electrical conductivity and structural properties . When thg cycles . While ig cycles . Therefog cycles .2 on RGO, which was also effective in controlling graphene sheet agglomeration and expanding the electrode capacitance. At a current density of 1 A/g, it exhibited a high specific capacitance of 645 F/g [2 for the composite with GA. In the course of performing cyclic stability tests, it was found that the specific capacitance of this NiO/GA material remained almost constant and had excellent stability [Graphene aerogel-related composites also offer superior performance when used as electrochemical double-layer capacitor electrode materials. Typically, the electrical conductivity and mechanical stability of metal oxides can be improved when combined with 3D graphene . Tian et 645 F/g . Howevertability . Composi2 compounded with MnO2 on its surface using 3D printing technology as an electrode material. It was found that, to a certain extent, the electric capacity increased with increasing MnO2 loading content and the weight of the electrode material remained almost constant. This shows the ability to substantially increase the loading of the pseudocapacitor material without sacrificing rate capability and weight capacitance, which is essential for the development of practically useful pseudocapacitors [Compared to EDLC, pseudocapacitor electrode materials transfer electrons and store energy through redox reactions between the electrode surface and the electrolyte, resulting in greater adsorption and thus greater capacitance . Since tpacitors . The inhpacitors . The devThe emergence of GA has brought a big change to the energy storage, making battery systems and supercapacitors show high energy conversion efficiency and good development prospects. With its lighter material and larger energy storage capacity, it is expected to replace the traditional carbon material electrode as the new favorite of scientific researchers. However, the practical application of the whole GA material has not been well solved, and a perfect system to promote the practice is needed. There is still a long way to go to realize the application in energy storage field.According to the keywords presented in the blue clusters, the main research hotspot are Adsorption and Removal, which clearly shows another major application area of GA: environmental protection. Since the 3D porous structure has good adsorption and absorption ability as well as large specific surface area for catalyst loading, GA plays a great role in water pollution and air pollution control methods.The porous structure and large specific surface area of aerogels determine their excellent adsorption of pollutants . MoreoveIn recent years, the number of offshore oil spills has been increasing and the leaked crude oil has seriously affected the balance of the surrounding ecosystem . The eff2 aerogel for crude oil adsorption using solar energy drive. The photoexcitation heat generated by the photothermal material CuFeSe2 to heat the crude oil drives its diffusion into the GA, and oil can be retained in the pores of aerogel after the removal of the light source. The method low cost and in line with the green development concept [In 2016, Liu et al. prepared anisotropic graphene aerogels (AGAs) with lipophilic hydrophobicity and efficient adsorption properties using unidirectional freeze casting. The oxygen-containing functional groups on the surface of GA are quite compatible with many organic polymers and have a good adsorption effect on n-heptane, ethanol, acetone, etc. It can adsorb organic masses more than 120–200 times its own and can be easily recovered by combustion, distillation, and extrusion  Figure 6 concept .2+ up to 408 mg/g [2+, Cu2+, and Cr2+ while achieving remarkable removal rates of 95.4%, 81.2%, and 73.2%, respectively [Pollution caused by heavy metal ions leaking into water bodies when produced by industrial manufacturing can also seriously endanger human health and damage the ecosystem in water . GA with408 mg/g . Pan et ectively . It can ectively .2S, NO, etc.) or greenhouse gases (mainly CO2) and the adsorption-desorption reuse of energy gases .The current applications of GA in gas adsorption include the adsorption of toxic gases  was prepared using Na2CO3 and GA to adsorb and catalyze the oxidation of H2S. The macroporous structure of AGA provides large capacity for adsorption of H2S and the alkaline environment provided by the Na2CO3 attached on the large specific surface area promotes the dissociation of H+ from H2S. Oxygen molecules are activated as superoxide radicals on the surface of graphene sheets, which promotes the catalytic oxidation of HS- to sulfur monomers [2 using oxygen oxidation. HK loading on the GA surface enhanced the adsorption capacity of CO and increased the catalytic efficiency by ≈48.4% [2 emissions are the main cause of the intensification of the greenhouse effect, and reasonable control of CO2 content is of great importance [2 capture at high temperature and pressure. Compared to the two-dimensional MgAl-LDH powder, the adsorption efficiency of the three-dimensional porous structure of MgAl-LDH/RGO aerogel was improved by about 160% [2 gas by condensation, adsorbents containing amine groups can be used to adsorb CO2. Therefore, CS-embedded GO aerogels were used for CO2 adsorption by Hsan et al. The large number of amine groups contained inside the CS attached to the GA surface can have a larger reaction area to react with CO2, much higher than the adsorption content of pure CS [2 should ensure the efficiency and stability of adsorption-desorption. Peng et al. reported the shell-core MgH2@carbon aerogel microspheres for the storage of H2. Thanks to the bonding between Mg and hydrogen atoms and the microsphere structure that inhibits the agglomeration of the material, the material exhibits a very good adsorption capacity and high cycling stability to meet the requirements of long-term use [Leakage of toxic gases is detrimental to both human and environment, so adsorption and subsequent treatment of leaked gases is important for environmental protection . Due to monomers . In addiy ≈48.4% . Apart fportance ,137. Xiaout 160% . Since b pure CS . Leakageterm use . After aterm use .In addition to adsorption, GA photocatalytic carriers are also a good method for catalytic removal of pollutants. 3D GA has a macroscopic ontological structure and is an ideal carrier for photocatalysts . It can 2 with GA-loaded surface catalysts [2/reduced graphene oxide aerogel composite was prepared by Qiu et al. The catalyst TiO2 grows along the glucose on the surface of GA and interacts to form a stable structure, while the large specific surface area provided by GA accelerates the catalytic degradation of methyl orange (MO) by TiO2 (up to 90% degradation rate by sunlight irradiation for 300 min). At the end of degradation, GA and TiO2 can be easily separated by physical manipulation, which is very convenient [4/RGO aerogel/CeVO4 (BGC) degradation of tetracycline (TC) in visible light was proposed by Liu et al. The high electron mobility inside the material under the joint action of the three compounds enables a more efficient separation of photogenerated electric carriers. And compared to pure BiVO4 and CeVO4, the degradation rate was increased by 66% and 69% using GA as the backbone structural material [Generally, organic compounds are removed by degradation to water and COatalysts . For insnvenient . At the material . In addimaterial . Additio2/GA to perform degradation inactivation studies on E. coli and S. aureus. AgBr nanoparticles are effectively combined with TiO2 nanocrystals to accelerate the light-induced charge transfer and separation to achieve the decomposition of E. coli and S. aureus. The backbone structure was provided by GA and the large specific surface area promotes Ag-AgBr light collection and reaction platform [Microbial scavenging mainly uses the products  obtained from redox reactions induced by active substances to cause damage to cell structures to achieve inactivation, and GA as a carrier template provides a large amount of reaction space and contact area with microorganisms . For exaplatform . In addiplatform .In environmental protection, the porous structure and surface functional groups of graphene play an important role. Meanwhile, more and more three-dimensional composites based on graphene aerogels are emerging, demonstrating the performance advantages and wide practicality of graphene aerogels.(1)We firstly discuss the self-assembly method, the template method, and the 3D printing preparation of GA, respectively, and analyze the advantages and limitations of different preparation methods. We found that the assembly method as a commonly used preparation method has the advantages of easy operation and low cost, and is expected to be produced in large quantities on a factory scale. However, the molded GA surface often contains many defects and collapses, which can seriously impair performance. The GA obtained by template method and 3D printing has better results in terms of material structure and uniformity of properties as well as stability in long-term use. However, the relatively high cost of these two methods and the tedious preparation process makes them difficult to meet in industrial scale manufacturing at present.(2)GA with its excellent conductivity and large specific surface area is used as an electrode for batteries and supercapacitors to store energy. In lithium battery systems, GA is usually used as an electrode material alone or in combination with other electrode materials. Its good electrical conductivity and large specific surface area can effectively improve the transfer efficiency of lithium ions during charging and discharging to achieve rapid charging and discharging. In supercapacitors, the porous structure provided by GA can increase the electrode capacity as well as provide electrochemically active sites for electron attachment after doping with heteroatoms to achieve high energy density. The large specific surface area and high electrical conductivity possessed by GA are its advantages, and it has a huge advantage in the development of energy storage.(3)In the field of environmental protection, the hydrophobic properties of the oxygen-containing functional groups on the surface of GA combined with the porous structure enable effective adsorption of organic substances, and its good resilience can effectively achieve multiple cycles of adsorption-desorption under the action of mechanical pressure. Meanwhile the large surface area provides loading for photocatalysts to improve the efficiency of catalytic degradation of pollutants, which will play a very significant role in promoting the improvement of environmental problems. The development and progress of GA is closely related to the development of these three major research fields. As a visualization method, the bibliometric analysis presents objective and comprehensive content. The summary of the key sections will be presented to the reader more clearly and quickly.The development of nanotechnology has greatly contributed to the interest in various fields. Surprisingly, the related three-dimensional material GA has made considerable achievements in the past decade, and its three-dimensional stable structure has shown superiority over two-dimensional graphene in many aspects. The review uses bibliometric methods to analyze three hot areas of GA fabrication methods, energy storage applications, and environmental applications.Currently, more and more studies on GA are being reported in a wider range of applications, for example, as electromagnetic shielding materials to reduce electromagnetic interference and as biosensors for sensitive detection of cells ,152,153.


Symptoms during acute COVID-19 can limit daily activities and delay return to work and school. Little is known about the association between SARS-CoV-2 burden in either the upper airway or plasma and the duration of COVID-19 symptoms.ACTIV-2/A5401 is a platform trial for COVID-19 treatments in non-hospitalized symptomatic adults enrolled within 10 days of symptom onset. We included participants randomized to placebo from August 2020 to July 2021. Participants self-reported severity of 13 symptoms daily from day 0 (baseline) to 28 as Absent 0, Mild 1, Moderate 2, Severe 3; total symptom score was calculated as the sum of all scores. Anterior nasal (AN) and plasma SARS-CoV-2 RNA levels at day 0 were measured with a quantitative qPCR assay. The relationship between day 0 RNA and time to symptom improvement or resolution  was evaluated using proportional hazards regression adjusted for time from symptom onset. Time to resolution of distinct symptoms was also assessed.10 copies/ml  and plasma RNA was detectable in 19% (91/476). Detectable plasma RNA at day 0, but not AN RNA, was associated with more severe symptoms at day 0 . Both high AN  and detectable plasma RNA  at day 0 predicted delayed symptom improvement. High AN RNA at day 0 also predicted a delay in symptom resolution . Both high AN RNA and detectable plasma RNA levels predicted delays in the resolution of cough and shortness of breath. Detectable plasma RNA also predicted delayed body pain resolution.Among 570 participants randomized to placebo, median age was 48 years, 51% were female, and median time since symptom onset at baseline was 6 days; 7% had prior COVID-19 vaccination. At day 0, AN RNA was detectable in 80% with a median of 4.1 logCOVID-19 outpatients with high AN or detectable plasma SARS-CoV-2 RNA at day 0 are more likely to have prolonged symptoms, particularly respiratory symptoms. Additional studies are needed to determine whether the decline in viral load with early treatment impacts symptom duration.Kara W. Chew, M.D., M.S., Merck Sharp & Dohme: Grant/Research Support|Pardes Bioscences: Advisor/Consultant Joseph J. Eron, MD, GSK: Advisor/Consultant|Merck: Advisor/Consultant Eric S. Daar, M.D., Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Merck: Advisor/Consultant|ViiV: Advisor/Consultant|ViiV: Grant/Research Support David A. Wohl, M.D., Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Lilly: Grant/Research Support|ViiV: Advisor/Consultant|ViiV: Grant/Research Support Judith S. Currier, M.D., MSc, Merck: Advisor/Consultant Davey M. Smith, M.D., M.A.S., Arena Pharmaceuticals: Advisor/Consultant|Bayer Pharmaceuticals: Advisor/Consultant|Brio Clinical.: Advisor/Consultant|Fluxergy: Advisor/Consultant|Kiadis: Advisor/Consultant|Linear Therapies: Advisor/Consultant|Matrix BioMed: Advisor/Consultant|Model Medicines: Advisor/Consultant|Signant Health: Advisor/Consultant|VxBiosciences: Advisor/Consultant Jonathan Z. Li, MD, MMSc, Abbvie: Advisor/Consultant|Merck: Grant/Research Support.