Larval zebrafish have transparent bodies that allow non-invasive visualization of whole cells of single spinal motor neurons, from somas to the neuromuscular synapses. This unique feature, combined with its amenability to genome editing, pharmacology, and optogenetics, enables functional analyses of ALS-associated proteins in the spinal motor neurons in vivo with subcellular resolution. Here, we review the zebrafish skeletal neuromuscular system and the optical methods used to study it. We then introduce a recently developed optogenetic zebrafish ALS model that uses light illumination to control oligomerization, phase transition and aggregation of the ALS-associated DNA/RNA-binding protein called TDP-43. Finally, we will discuss how this disease-in-a-fish ALS model can help solve key questions about ALS pathogenesis and lead to new ALS therapeutics.Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord. Spinal motor neurons align along the spinal cord length within the vertebral column, and extend long axons to connect with skeletal muscles covering the body surface. Due to this anatomy, spinal motor neurons are among the most difficult cells to observe  TARDBP gene, in the degenerating motor neurons  is a fatal neurological disorder in which motor neurons in the brain and spinal cord are selectively degenerated, leading to progressive muscle weakness. Approximately 1–2 individuals per 100,000 are diagnosed with ALS each year, with motor symptoms typically appearing in mid-adulthood (average age 55) . About 1 neurons . Better TARDBP mostly occur in the IDR has led to the idea that pathological phase transitions of TDP-43 mediated by altered homotypic and heterotypic interactions through IDR contribute to ALS pathogenesis. Cytoplasmic aggregation of TDP-43 has been observed by overexpressing wild-type and mutant TDP-43 in cellular and animal models of ALS  that self-associates upon blue light exposure (PHR (PHR or a point mutant version of Cry2 (Cry2olig) exhibiting enhanced clustering (The optoDroplet approach, recently introduced by exposure  to inducure (PHR . This teure (PHR , and hasustering  was fuseustering . These oustering , suggestustering . The ligustering . This reustering .in vivo contexts (A key step toward understanding of motor neuron degeneration in ALS is to induce TDP-43 oligomerization and phase transition in motor neurons and evaluate their cellular outcome in contexts . In zebrcontexts , can be contexts , enablincontexts . opTDP-4contexts . Live imcontexts . The precontexts . This obcontexts . KnowledA major advantage of optogenetics is that it allows precise temporal control of a photo-responsive probe. In zebrafish larvae expressing opTDP-43 in most of the spinal motor neurons, the extension of the photostimulation duration from 3 h to 3 days resulted in the accumulation of cytoplasmic opTDP-43 aggregates in the motor neurons . The cytin vivo in motor neurons. The accumulation of cytoplasmic TDP-43 aggregates is a hallmark of degenerating neurons in ALS. However, how TDP-43 is localized in the nucleus under normal conditions and the mechanisms by which it gradually loses its normal state and forms cytoplasmic aggregates are not clear. Thus, it is tempting to speculate that light-dependent opTDP-43 phase transition is a fast-forward replay of the changes occurring in TDP-43 in ALS, especially as TDP-43 dynamics is currently anatomically inaccessible in human motor neurons. Uncovering intermediate stages of the opTDP-43 phase transition would provide mechanistic insights into TDP-43 cytotoxicity and potential therapeutic targets. The illumination time-dependent formation of cytoplasmic opTDP-43 aggregates suggests that oligomerization and phase separation of opTDP-43 is tunable and can be used to reveal the temporal sequence of downstream events driven by pathological TDP-43 phase transition in the spinal motor neurons. This opTDP-43 phase transition can be targeted with subcellular resolution to explore spatial sources of TDP-43 toxicity, such as the nucleus, axon, and pre-/post-synaptic terminals. The ability to observe diseased motor neurons in a transparent animal also allows us to directly investigate systemic responses of neighboring cell types, including interneurons, myofibers, glial cells, and immune cells, to aberrant TDP-43 transitions occurring in the motor neurons. Moreover, since the IDR of TDP-43 is classified as a prion-like domain and TDP-43 has a prion-like aggregation-seeding ability (The optoDroplet approach has explicitly demonstrated the causal relationship between TDP-43 phase transition and neurotoxicity  ability , potentiKA wrote the manuscript and generated the figures, with inputs from HH and KK. All authors 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.


The mean RALA town-wide assessment score across communities was 56.5 + 15.6 out of a possible 100 points. The mean RALA program and policy assessment score was 40.8 + 20.4 out of a possible 100 points. While grocery stores and schools are important for enhancing food and physical environments in rural areas, many opportunities exist for improvements to impact behaviors and address obesity.The built environment contributes to an individual’s health, and rural geographies face unique challenges for healthy eating and active living. The purpose of this descriptive study was to assess the nutrition and physical activity environments in rural communities with high obesity prevalence. One community within each of six high obesity prevalence counties in a rural Midwest state completed the Nutrition Environment Measures Survey for Stores (NEMS-S) and the Rural Active Living Assessment (RALA). Data were collected by trained community members and study staff. All communities had at least one grocery store and five had at least one convenience store. Grocery stores had higher mean total NEMS-S scores than convenience stores (26.6 vs. 6.0,  Obesity is a multifaceted public health issue and adult obesity continues to impact much of the United States (U.S.). Data from the National Health and Nutrition Examination Survey (NHANES) indicate that age-adjusted obesity prevalence among U.S. adults aged 20 years and older is 41.9% . AmericaEnvironmental features can hinder or support active living and healthy eating environments. The built environment is defined as manmade surroundings that include buildings, public resources, land use patterns, the transportation system, and design features . The buiThe built environment contributes to an individual’s health, and rural geographies face unique challenges for active living and healthy eating, including isolation, transportation distances, and lack of facilities ,15. UnheIn 2014, the Division of Nutrition, Physical Activity and Obesity (DNPAO) at the CDC funded several land-grant universities under the High Obesity Program (HOP) cooperative agreement to reduce obesity prevalence in high-obesity prevalence counties using a community-based, policy, systems, and environmental intervention approach . RecipieSix counties with an obesity prevalence >40% in a rural Midwest state (South Dakota) were identified as part of the CDC HOP funding aimed at improving the nutrition and physical activity environment to support healthy behaviors . One comhttps://nems-upenn.org/tools/, accessed on 29 November 2021). While higher scores are better, there are no cut points for what constitutes an ideal score on NEM-S; rather, the tool can be used to track changes pre/post intervention or examine associations between store environments and eating behaviors [To examine the nutrition environment of each community, the Nutrition Environment Measures Survey for Stores (NEMS-S)  was utilTo examine the physical activity environment of each community, the Rural Active Living Assessment (RALA)  was utilT-tests were used to identify differences in the mean scores for availability, price, and quality of food between grocery stores and convenience stores. Chi-squared tests were used to assess differences in the frequency of availability of healthful food items between grocery and convenience stores. T-tests were also used to compare overall RALA TWA and PPA scores. For all analyses, statistical significance was set at p ≤ 0.05. Data are presented as mean ± standard deviation.Analyses were conducted utilizing IBM© SPSS Statistics Version 24. Descriptive statistics were utilized to compile data on total scores and section scores for the NEMS-S and RALA tools. p < 0.001), as well as higher scores for availability , and quality  of healthful foods. Mean price scores did not vary between grocery stores and convenience stores (p = 0.07). Availability of healthful options in both grocery and convenience stores varied by food type (p < 0.05).All communities had at least one grocery store and five of six had at least one convenience store. Overall NEMS-S scores, in addition to sub-scores for availability, price, and quality for grocery stores and convenience stores by community are presented in ood type . HealthfIn terms of pricing of healthful options compared to regular options among the seven grocery stores, healthful options for 100% juice and whole-grain bread were priced lower than regular options in only 14.3% of stores see . HealthfThe mean RALA town-wide assessment score across communities was 56.5 ± 15.6, ranging from 30 to 74 out of a possible 100 points. Communities scored highest in school location and parks/playground sections, and lowest in the trails section. Recreational facilities and water activities fell in the middle range .The mean RALA program and policy assessment score was 40.8 ± 20.4 ranging from 3 to 56, out of a possible 100 points. Communities strongly supported physical activity through school-based policies and school programming. Town programming scored lower. Town policy held the lowest scoring subscale . Total TThe physical environment in which people live plays a role in shaping both diet and physical activity behaviors, thus ultimately impacting obesity ,24. The p < 0.001), it should be noted that these scores are on a scale of −9 to 54, and grocery stores received only 49% of total possible points. Therefore, although grocery stores are better nutrition environments in these rural areas, they are not necessarily ideal. These findings appear to be in line with others in rural areas. A study examining NEMS-S scores among counties in Montana with varying degrees of rurality based on rural–urban continuum (RUC) codes ranged from 28.7 in the most metro county (RUC code 3) to 21.8 in the most rural county (RUC code 9) with a mean among all counties surveyed of 23.8  [n = 1) to 8 (n = 1) to 9 (n = 4). For comparison, a study using the NEMS-S in urban Pennsylvania found supermarkets to have total scores approaching 40 points [Nutrition Environment. All communities had a minimum of one grocery store and all but one community had a minimum of one convenience store. Differences were observed, however, between the two types of retail environments in terms of availability  of healthy foods. While this was reflected in a higher total NEMS-S score for grocery stores vs. convenience stores  . In the 0 points . While p0 points ,30.As indicated by previous research, the healthfulness of individual’s diets is correlated with the availability of healthful food in stores within the same zip code , and rurPhysical Activity Environment. The RALA town-wide assessment and program and policy assessment scores across communities were 57 and 41 out of 100 possible points, respectively. In the present study, the town-wide assessment ranged from 30 to 74 and the program and policy section ranged from 3 to 56, highlighting wide variation in the amenities, programs, and policies that support physical activity among communities. These data are comparable with two other studies that have previously utilized the RALA to assess the physical activity environment in rural areas. Robinson et al.  assessedThe amenities within the community that emerged as strengths were linked to schools, such as school location within the community and parks and playground availability, as both sections scored ≥78%. The parks and playground section was the least variable (±20%), with all communities scoring a minimum of 15/25 (60%). These data highlight the key role schools play in supporting physical activity within small, rural communities through active transportation to and from school, and the parks and playgrounds associated with the school. The availability of recreational facilities and water activities was less common of an amenity, with the least common amenity section being trails. Only two of the communities had trails, scoring 16/20 with the other four communities earning zero points in this category. These lower scoring sections highlight the opportunity for improvement. While the availability for water activities and trails can be costly to amend, the availability of recreational facilities could be addressed through shared use agreements between the community and the school district to utilize gymnasiums outside of school hours for other recreational needs within the community.Schools had stronger policies and programs to support physical activity when compared with the community. Although the highest scoring section, the average score in the school policies section was 50%. Only one community earned all points in this category. Since there are national standards for school wellness policies, it was expected to see this section emerge as the highest scoring section but concerning to note the average was only 50%. School programs was the second highest section, highlighting the school as a strength within these rural communities.Similar to the present findings, school location, parks, and playground amenities were shown to be the highest scoring town-wide assessment sections, and school policy was shown to be the highest scoring program and policy assessment section when assessed in eight communities across Alabama and Mississippi , as wellThis study is not without limitations. While the NEMS-S is a validated tool, it captures information only about certain locations within communities , and other places where food is available  were not assessed, nor was the overall availability of food within the community. The RALA is also a validated tool, but it was completed by only one group of stakeholders within the community and could be biased to the information those individuals were aware of and willing to report. Therefore, it is unknown what information remained uncaptured in the assessment. Additionally, while NEMS-S data were collected by Extension professionals, RALA data were collected by community members, which may impact quality of data collection and reporting. Finally, funding limited the type and location of work possible; thus, there remains a small number of communities to assess, and the study is descriptive in nature. The findings presented are specific to rural communities and are unlikely to be generalized to other geographic locations.Conducting assessments on the nutrition and physical activity environments within rural communities is helpful in identifying existing supports that can be enhanced, and in identifying barriers and challenges to overcome. This facilitates interventions that are feasible and impactful for communities while optimizing the use of existing resources. In the rural communities examined in the present study, nutrition environments are enhanced by grocery stores—although price and overall availability of healthful food requires further improvement—and schools serve as an important physical activity hub. This information will be used for developing and implementing community-based, obesity prevention interventions, tailored for rural communities.


Polymer electrolyte fuel cells hold great promise for a range of applications but require advances in durability for widespread commercial uptake. Corrosion of the carbon support is one of the main degradation pathways; hence, corrosion-resilient graphene has been widely suggested as an alternative to traditional carbon black. However, the performance of bulk graphene-based electrodes is typically lower than that of commercial carbon black due to their stacking effects. This article reports a simple, scalable and non-destructive method through which the pore structure and platinum utilisation of graphene-based membrane electrode assemblies can be significantly improved. Urea is incorporated into the catalyst ink before deposition, and is then simply removed from the catalyst layer after spraying by submerging the electrode in water. This additive hinders graphene restacking and increases porosity, resulting in a significant increase in Pt utilisation and current density. This technique does not require harsh template etching and it represents a pathway to significantly improve graphene-based electrodes by introducing hierarchical porosity using scalable liquid processes. Polymer electrolyte fuel cells (PEFCs) are an electrochemical energy conversion technology that is key to helping decarbonise the transport, portable, and stationary power sectors ,2,3. How2 and H2) transport and water management [2 g−1 [Fuel cell CLs are typically 2–20 μm thick and consist of electrically-conducting carbon supports decorated with Pt-based nanoparticle catalysts. They utilise proton-conducting polymers (referred to as ionomer) as both binder and electrolyte, which in turn are in contact with the proton-conducting polymer membrane ,15. To mnagement ,17. Thusnagement ,20,21,22t [2 g−1 ,24. Hight [2 g−1 ,26. Carbt [2 g−1 ,27.2-bonded framework, which is significantly more resistant to oxidation than carbon black [2 g−1), in practical bulk systems, the ‘graphene’ used is rarely fully exfoliated. Graphene-based CLs are typically formed from dispersions of few-layer graphenes (FLGs), which have surface areas of only a few hundred metres squared per gram. Furthermore, 2D sheets are prone to restacking during deposition, which dramatically reduces their accessible surface area and Pt utilisation, due to the catalyst particles becoming trapped between the layers [2 diffusion highways as they are generally regarded as being key to low-tortuousity gas diffusion, particularly in thicker CLs [Graphene has been widely proposed as an attractive catalyst support material, mainly due to its spon black ,17,28,29e layers ,31,32. Te layers ,33,34,35e layers ,36. Micre layers  and mesoe layers ,24,36. Mcker CLs ,38. The cker CLs ,40,41,42cker CLs . One of While attempts have been made to manufacture porous graphene electrodes by novel techniques, such as freeze-drying, they typically use the less conductive and more defective variant of graphene, (reduced) graphene oxide, or they rely on a non-scalable manufacturing route ,31,44,453Ni aerogel as a CL, in which K2CO3 additives were found to induce changes in the CL morphology, modifying the porosity and improving mass transport.The most widely reported technique for mitigating the propensity of graphene inks to re-stack and form low-porosity CLs when deposited is to introduce a second electrically-conducting carbon support material to act as a spacer ,44,46,47In this study, a simple, industrially-scalable and non-damaging method was developed that improved the pore structure and Pt utilisation. This method uses urea as a sacrificial additive to the standard aqueous-based CL ink, which, when deposited, forms water-soluble organic crystals physically separating the FLG sheets during deposition. Urea was chosen as it is low-cost, environmentally benign, and widely available; and unlike salt additives, it was found to not destabilise graphene ink suspension. The urea can then be removed by simple soaking in water, leaving voids between the FLG particles. This process maintains the structure of the graphitic component while introducing meso- and macroscale porosity to the CL, dramatically increasing current density. Due to the mildness of the treatment, the benefits are obtained without damaging the graphene sheets or disrupting the continuous ionomer network.2 g−1), sodium metal , naphthalene (99%), dimethylacetamide , 4-bromothioanisole (97%), chloroplatinic acid hydrate , sodium borohydride (≥98.0%), and urea  were supplied by Sigma Aldrich . The acetone (99.8%) and propan-2-ol (99%) were supplied by VWR Ltd. . The Nafion® Dispersion  was obtained from DuPont Fluoroproducts . The carbon black (Vulcan XC72R) and Freudenberg H23C7 was supplied by Fuel Cell Stores . The argon (99.95%) was supplied by BOC gases .The graphene nanoplatelets  before use. All the other chemicals were used as supplied.The water was deionised using an Elga PureLab Chorus on site. The DMAc was dried over 4 Å molecular sieves  for 48 h before use. The graphene nanoplatelets were dried under vacuum  with a glass stirrer bar until all the sodium dissolved, leaving a deep green solution (ca. 4 h) of sodium naphthalide. Graphene nanoplatelets  were added and stirred (12 h) to form a black solution. 4-Bromoanisole  was added to the solution (12 h) before removing the mixture from the glovebox, filtering it over filter paper and washing it with water and acetone to produce a black powder of functionalised few-layered graphite .FLG Functionalisation. The procedure used was adapted from Clancy et al. . In an aPlatinum Nanoparticle Deposition. The chloroplatinic acid hydrate (1.08 g) and f-FLG (500 mg) were mixed with water (500 mL) and briefly bath-sonicated  to produce a metastable suspension. The mixture was vigorously stirred and sodium borohydride solution  was rapidly added and left to stir (16 h). The mixture was then filtered to produce Pt@FLG.Catalyst Ink Formulation. The graphene-based catalyst inks were prepared by mixing Pt@FLG , Nafion solution ., water (30 mL) and propan-2-ol (30 mL) in a 100 mL Mason Jar. The commercial Pt/C catalyst ink consisting of Pt/C , Nafion solution 11 wt% were supplied by DuPont Fluoroproducts ., water (30 mL) and propan-2-ol (30 mL) were likewise prepared in a 100 mL Mason Jar. The ratio between the ionomer and the carbon support was 0.8:1. In the sacrificial templated samples, urea 120 mg  was also added to the mixture. The mixture was bath-sonicated (30 min) and used immediately after dispersion.−1, with an offset serpentine spray pattern. A loading of 0.4 mgPt cm−2 was calculated from the mass change on the MEA due to spraying. The GDEs sprayed with urea additives in the ink were soaked face-down in a Petri dish filled with distilled water, after 24 h the GDE was removed from the water and dried. The graphene GDE was assembled together with the Gore Select membrane and a 0.4 mgPt cm−2 HyPlat GDE to act as an anode. The MEAs were hot-pressed at 150 °C and 360 psi. The cell assembly was sealed with a torque of 4 Nm applied to the 12 bolts of the MEA assembly.Membrane Electrode Assembly. The catalyst ink was sprayed directly onto the gas diffusion layer (GDL) H23C7 were suppliedFreudenberg  using an ultrasonic spray system . The bed of the spray coater was heated to 90 °C during spraying. The flow rate of the Exactacoat was set to 0.4 mL min2 with no back pressure and at 100% RH, 80 °C; 1.5/3 was used as the H2/O2 stoichiometric flow rate. The cell was conditioned by purging with Ar/Ar for 10 min, followed by Ar/H2 for 10 min, then air/H2 held at OCV for 5 min. The cells were held at 5 A for 1 h, followed by an Ar/H2 purge; 25 CV ‘cleaning’ scans were run between 0.06 and 1 V at 20 mV s−1. Cyclic voltammetry (CV) measurements were performed between 0.06 and 1 V at 20 mV s−1. ‘Fast’ polarisation curves were run at 0.05 V/pt and 10 sec/pt until the current density stabilised. ‘Slow’ polarisation curves were run for data collection purposes at 0.025 V/pt and 30 sec/pt.The fuel cell testing was performed on a Scribner 850e test system under air/H−1) and drop-cast on lacey carbon copper grids .TEM was performed on a JEOL 2100 with an accelerating voltage of 100 keV. The samples were briefly bath-sonicated in HPLC-grade ethanol  was run on a K-Alpha XPS system. The MEA samples were mounted on conductive carbon tape with the top of the GDE pointed upwards and a charge-compensating flood-gun applied to avoid charging effects.The CL microstructure was analysed by scanning electron microscopy (SEM). Small pieces of the GDEs were embedded in epoxy resin, then the GDE cross-sections were mechanically polished to achieve a mirror-like surface. The observations were performed using a ZEISS MERLIN  field emission gun scanning electron microscope (FEG-SEM) equipped with two X-ray energy dispersive spectrometry (EDS) detectors. The backscattered electron images and the platinum and fluorine EDS elemental maps were recorded at an accelerating voltage of 4 kV.The surface areas and pore size distribution were measured through the application of Brunauer, Emmett and Teller (BET) theory, on a Quantachrome Quadrasorb Evo  using nitrogen gas.The pre-exfoliated FLG nanoplatelets were further exfoliated in order to maximise the exposed surface area by using a previously established reduction methodology . The exfAlthough spraying the CL directly onto the electrolyte membrane has been reported to have improved fuel cell performance, in this work the ink was directly sprayed onto the microporous layer (MPL) of the gas diffusion layer (GDL) . This apThe elemental composition of the CLs were estimated via XPS. The standard CL formulation, composed of carbon black/graphene, Nafion and Pt, was found to possess a small nitrogen content, which was likely due to residual DMAc solvent. Urea contains 47 wt% nitrogen, so any significant change in the quantity of urea within the CL was identifiable from the nitrogen content. The nitrogen content in the CL after adding urea to the catalyst ink increased from 0.5 wt% to 2.3 wt%, decreasing to 0.9 wt% after washing , indicat2 transport. The addition of the urea thus significantly changed the pore structure of the catalyst layer, introducing meso- and macropores and improving accessibility to the active Pt NPs.Cross-sectional SEM images of the graphene-based CL without urea a,b revea2 diffusion limitations [2 mass transport resistances.itations . The MEA2 g−1 to 11.9 m2 g−1 due to the addition of urea at the fabrication stage  of cycles had to be performed before the power density stabilised. This instability was found to be significantly worse if the MEA was not soaked in water before assembly, requiring hundreds of polarisation curves to reach a stable performance. We suggest that the initial inability of the MEAs to reach a stable performance was due to the in situ removal of the excess urea from within the CL in the high-humidity environment. It was found that the initial performance reached equilibrium faster during high-humidity conditioning procedures.2, or low surface energy solvents; or direct sublimation, in order to allow a greater quantity of the sacrificial additive to be incorporated into the CL. Furthermore, different additives would be expected to have different morphological effects on the CL; moreover, combinations of different additives may allow more precise control over the PSD and porosity of CLs.This article reports a scalable, non-destructive and simply-implemented method for controlling the internal morphology and pore structure of the micro- to mesoscopic length scales of a graphene-based CL. The use of urea additives allowed the one-step fabrication of the CL, followed by a simple and environmentally friendly single-additive removal step. The addition of the urea to the CL ink mitigated the graphene sheets’ (re)stacking during spraying, creating a more porous morphology, increasing the efficiency of Pt utilisation and improving mass transport, leading to significantly higher current densities. The addition of the urea was only effective on graphene-based CLs; traditional Pt/C based on bulk carbon supports did not benefit from the addition of urea. Understanding how the sacrificial additive affects fuel cell performance, including the power density difference between graphene-based CLs and those based on Pt/C, is key to further improving nanomaterial-based electrodes. The technique reported here could be improved by modifying the process for the removal of the additive, as this step often leads to the mechanical breakdown of the CL. Other potentially scalable approaches to urea removal include the use of supercritical CO


Tendon is a major component of musculoskeletal system connecting the muscles to the bone. Tendon injuries are very common orthopedics problems leading to impeded motion. Up to now, there still lacks effective treatments for tendon diseases.Tendon stem/progenitor cells (TSPCs) were isolated from the patellar tendons of SD rats. The expression levels of genes were evaluated by quantitative RT-PCR. Immunohistochemistry staining was performed to confirm the presence of tendon markers in tendon tissues. Bioinformatics analysis of data acquired by RNA-seq was used to find out the differentially expressed genes. Rat patellar tendon injury model was used to evaluate the effect of U0126 on tendon injury healing. Biomechanical testing was applied to evaluate the mechanical properties of newly formed tendon tissues.In this study, we have shown that ERK inhibitor U0126 rather PD98059 could effectively increase the expression of tendon-related genes and promote the tenogenesis of TSPCs in vitro. To explore the underlying mechanisms, RNA sequencing was performed to identify the molecular difference between U0126-treated and control TSPCs. The result showed that GDF6 was significantly increased by U0126, which is an important factor of the TGFβ superfamily regulating tendon development and tenogenesis. In addition, NBM (nonwoven-based gelatin/polycaprolactone membrane) which mimics the native microenvironment of the tendon tissue was used as an acellular scaffold to carry U0126. The results demonstrated that when NBM was used in combination with U0126, tendon healing was significantly promoted with better histological staining outcomes and mechanical properties.Taken together, we have found U0126 promoted tenogenesis in TSPCs through activating GDF6, and NBM loaded with U0126 significantly promoted tendon defect healing, which provides a new treatment for tendon injury.The online version contains supplementary material available at 10.1186/s13287-021-02679-x. Tendon is a major component of the musculoskeletal system which connects the muscles to the bone. Injuries to tendon including tendinopathy and tendon rupture are very common orthopedics problems. Tendon disease is one of the most common diagnoses of people engaged in sports professions, accounting for 30% of the total number of injuries diagnosed . Althougβ (TGFβ) family of growth factors, GDF6/7, Scleraxis (Scx), Egr1, Mkx, etc. [ β-catenin/ERK signaling pathway, which could be partially rescued by U0126 [The arising of tissue engineering shows promising prospects for tendon tissue regeneration. Many studies have shown that mesenchymal stem cells (MSCs) or tendon stem/progenitor cells (TSPCs) have been successfully applied in tissue engineering to treat tendon injuries –6. MSCs kx, etc. . Howeverkx, etc. . The inhkx, etc. . In our by U0126 .ε-caprolactone (PCL) scaffolds fabricated by electrospinning have been widely used in soft tissue regeneration as well as tendon engineering. PCL is a low-cost aliphatic linear polyester characterized by its good biocompatibility and bioresorbablity. It has been approved by the U.S. FDA for clinical use [As we know, Type I collagen is the main functional component of the tendon. The other components are a small amount of proteoglycans, glycoproteins and minor collagens , 15. Theical use . Gelatinical use –21. Compical use . And it ical use . Using gical use . In the The NBM was generated according to the previously published protocol by Neo Modulus (Suzhou) Medical Sci-Tech Co., Ltd. . The surThe animal experiments were approved by the Animal Research Ethics Committee of Guangzhou University of Chinese Medicine (No. TCMF1-2019053). 10-week-old male SD rats or GFP-transgenic SD rats were used in this study. The details of TSPCs isolation and culture have been described previously . Briefly3 per well) were subcultured in a 96-well plate. After 24 h of incubation, the medium was changed into U0126 containing medium at different concentrations. Cells were incubated at 37 °C for 1 and 3 days. The cell proliferation was determined using methyl thiazolyl tetrazolium (MTT) reduction assay. After incubation, cells were treated with the MTT solution  for 4 h at 37 °C. The dark blue formazan crystals formed in intact cells were solubilized with 100 μL DMSO and the plate was shaken for 10 min. The absorbance at 570 nm was measured with a microplate reader.The cells (3 × 105 cell/well) and cultured until the cells reached confluence. Then the medium was changed to the tenogenic induction medium with ascorbic acid (25 μM) and CTGF (25 ng/ml) at 37 °C, 5% CO2. The medium was changed every 2 or 3 days. Two weeks later, the expressions of tendon-related markers were evaluated by quantitative real-time RT-PCR (qRT-PCR). At 2 weeks after tenogenic induction, the cells were stained with Sirius Red (0.1%). For quantification, Sirius Red was eluted with 0.1 N sodium hydroxide, and the optical density at 540 nm was determined using a spectrophotometer.The TSPCs were plated in 6-well plates . Real-time PCR was performed using the CFX96 Real-Time PCR Detection System . Amplification conditions were as follows: first at 95 °C for 5 min, and then 45 cycles of 95 °C for 15 s and 60 °C for 60 s. Primer sequences were listed in Additional file Total RNA was obtained from the TSPCs treated with or without U0126 using TRIzol Reagent . The quality and integrity of total RNA samples were assessed using a 2100 Bioanalyzer or a 2200 TapeStation (Agilent Technologies) according to the manufacturer’s instructions. The preparation of whole transcriptome libraries and deep sequencing were performed by the Sangon Biotech Corporation . The differential genes (log2ratio ≥ 1 or ≤ − 1) identified by RNA-seq have been uploaded in Additional file In order to demonstrate the formation of neo-tendon tissue in vivo, the cell sheets formed by TSPCs treated with or without U0126 were transplanted to the dorsal sites of nude mice. Briefly, a total of 8 mice were used; after anesthetized with ketamine and xylazine, an incision was made on the dorsum and a subcutaneous pocket was created to expose the posterior midline. The cell sheet was sutured to the posterior midline at both ends, there was tensile strength on the tendon graft with the mice movement. Six weeks later, the tissues were harvested and subjected to histological analysis.Forty SD male rats  were used in this study. A 2.0 mm wide tendon defect was created from the distal apex of the patella to the insertion of the tibia tuberosity with two stacked sharp blades according to our well-established protocol. U0126 was dissolved in DMSO and diluted with PBS. Then NBM scaffold was immersed in the U0126 solution overnight at 4 °C. The operated rats were divided into four groups: (a) Blank control group; (b) NBM-PBS group; (c) NBM-U0126 (100 mΜ) group; and (d) NBM-U0126 (200 mΜ) group. The NBM loaded with PBS and/or U0126 was placed in the tendon defect and sutured to the patellar bone and tibia tuberosity using Ethicon 6-0 suture. At 4 weeks after surgery, the animals were sacrificed by an overdose of ketamine and xylazine, and the patellar tendons were harvested for histological examination and biomechanical test.2) was calculated based on the ultimate load divided by the cross-sectional area at the breaking point measured by a high-resolution Vevo 770 animal ultrasound system  with images taken immediately prior to the biomechanical test. The Young’s modulus (N/mm2) was calculated from the linear slope of a stress strain curve.The procedure of biomechanical testing was described in a previous study . GeneralThe regenerated patellar tendon tissues were washed with PBS, fixed in buffered formalin, embedded in paraffin and sectioned for histological examination. Immunohistochemistry was done as described previously . Brieflyp < 0.05 was considered statistically significant. At least three sets of independent experiments were performed for each assay.All data were presented as mean ± SD. The statistical analysis was performed using one-way analysis of variance (one-way ANOVA). A value of Scx, Col1, Tnmd, Fmod and Mkx. While, PD98059 did not show an obvious promoting effect. The Sirius Red staining at 2 weeks after tenogenic induction also demonstrated that U0126 significantly promoted the tenogenesis of TSPCs  did not affect the cell viability of TSPCs  analysis revealed that many signaling pathways were enriched, the Top 30 of which were shown in Fig. GDF6, an important member of TGF-β signaling which has been well known as an indispensable factor for tenogenesis and tendon development, was significantly increased by U0126 which was confirmed by qRT-PCR and immunocytochemical analysis  in four-week-old male mice, which has direct functional consequences for the mechanical integrity of the tissue  [GDF6 has promoting effect on the tenogenic differentiation of BMSCs [U0126 has been used as a specific inhibitor of the ERK1/2 signaling pathway by selectively inhibiting MEK-1 and MEK-2 . It has  in vivo . And the in 2009 . They haperties) . Our preof BMSCs .The NBM, composed of electrospun gelatin and polycaprolactone nanofiber nonwovens, mimics the ECM of tissues. It was fabricated by electrospinning of in situ cross-linked GE nanofiber nonwovens and subsequent lamination with electrospun PCL nanofiber nonwovens by heating the layer assemblies above the melting temperature of PCL. It has been used to treat soft tissue defects . The preGDF6 expression. Most importantly, we also developed a novel combination of NBM and U0126 which could be used to promote tendon injury healing in a rat tendon defect model.In this study, we have demonstrated that U0126 could promote tenogenesis of TSPCs by activating Additional file 1. Fig. S1. Release kinetics of U0126 from NBM scaffold in vitro.Additional file 2. Table S1. Sequences of primers for real-time PCR.Additional file 3. Table S2. Differentially expressed genes (log2ratio ≥ 1 or ≤ -1) in U0126-treated TSPCs.


Various techniques such as scanning electron microscopy, UV-vis absorption spectra, tensile testing, Shore A hardness, surface resistance, thermal conductivity, and thermogravimetric analysis were conducted to characterize the microstructure and properties of the obtained composites. The results showed that the π–π stacking interfacial interaction between phenyl groups of SIS and graphene play an important role in the properties’ improvement, and the effect of interfacial interaction on the properties was revealed.Interfacial interaction is one of the most important factors in the construction of high-performance graphene-based elastomer composites. In this paper, graphene/poly (styrene- Polymer composites based on nanofillers such as fullerene, carbon nanotubes, nano-diamond, and layered silicates have attracted much attention in recent years, because of their prominent properties and numerous applications ,2,3. NowThe other important factor that influence the properties of graphene-based polymer composites is the interfacial interaction, which determines the stress dispersion and transport performance of the obtained nanocomposites ,12,13,14b-isoprene-b-styrene) (SIS) is a triblock copolymer belonging to the family of thermoplastic elastomers. The SIS composites with carbon nanotube or graphene have applications in photomechanical actuation [Styrene-based block copolymers are basically thermoplastic elastomers, with good performance for a wide range of applications, such as toys, packaging, adhesive, and medicinal materials . Poly  (Melt flow rate = 23 g/10 min) was purchased from Xingzhi New Materials Co. Ltd. . Chloroform was obtained from Sinopharm Chemical Reagent Co., Ltd.  without purification.Graphene was purchased from Suzhou TanFeng Graphene Tech Co., Ltd. . Poly (styrene-Graphene/SIS nanocomposites with different graphene concentrations (0~2.0 wt%) were prepared by a solution mixing method. In a typical procedure, the graphene was dispersed into 10 mL chloroform using a bath sonicator for 1 h. Then, 4 g of SIS was added into the dispersion with vigorous stirring for 2 h, and the mixture was sonicated for 1 h. Finally, the obtained mixture was poured into a polytetrafluoroethylene mold, the graphene/SIS films were obtained with an evaporation-induced self-assembly process, and the obtained films were dried at 60 °C to a constant weight.Scanning electron microscopy  was used to examine the morphology of the graphene sheets. UV-Vis absorption spectra were conducted with a S 3100 spectrophotometer . The tensile properties of graphene/SIS composites were measured by a universal testing machine ; at least five samples were tested to obtain average values. Measurements of Shore A hardness consisted of vertical immersion of the indenter into the composite surface with a Shore hardness tester . The surface resistance of the obtained composites was measured with an ultra-high resistance micro-current tester . The thermal conductivity of the composites was measured using thermal conductivity test equipment . Thermogravimeter (TG) analysis was performed with a TG-209-F3  under the nitrogen atmosphere at a heating rate of 10 °C/min from 30 to 650 °C.Scanning electron microscopy (SEM) of the graphene powder is given in Solution mixing has been found to be an effective method for the preparation of graphene-based polymer composites, as the polymer can be absorbed on the surface of graphene through interfacial interaction, resulting in a good dispersion state ,12. The Tensile testing was used to evaluate the mechanical properties of pure SIS and the graphene/SIS composites. The stress–strain behavior of all samples is presented in The investigation of Shore A hardness of SIS and corresponding graphene nanocomposites as a function of filler content are shown in According to the electrical percolation theory, graphene sheets provide percolated pathways for electron transfer, which imparts electric conductivity to the nanocomposites. However, the improvement efficiency of graphene sheets dramatically depends on the degree of sheets’ dispersion and interfacial interaction ,33. The Thermally conductive polymer composites are attracting considerable attention, especially in recent years because increasingly more powerful electronics are being developed . The theIncreased thermal stability is typical of polymer-layered nanocomposites, usually attributed to heat and mass barrier effects of layered nanocompounds, which delay heat and pyrolysis products’ diffusion. The thermal stability of graphene/SIS composites is presented in In summary, the graphene/SIS composites were prepared through solution mixing followed by an evaporation-induced self-assembly process, enhanced mechanical, electrical, thermal properties were obtained through π–π stacking interfacial interaction, and the effect of interfacial interaction on the properties was revealed. The results of UV-vis absorption spectra revealed that π–π stacking interaction between phenyl groups of SIS and graphene can be formed in the process of evaporation-induced self-assembly. The π–π stacking interaction can serve as a sacrificial bond to dissipate energy, leading to the effective dissipation of stress, and the tensile strength of the 0.5 wt% graphene/SIS composites is increased by 26.4% from 1.4 to 1.77 MPa. The π–π stacking interaction benefits from the formation of a conductive network; the surface resistivity of 2.0 wt% graphene/SIS composite decreased by four orders of magnitude. The high specific surface area and π–π stacking interaction provide more sites that can scatter phonons and damp the vibrational amplitude at the interface, inducing a higher thermal resistance. The π–π stacking interaction is benefits for the effective obstruction of low molecules from the degraded SIS and the shield function of the heat; thus, the obtained composites exhibit improved thermal stability in comparison to pure SIS.


Gated communities are the most popular residential pattern in the urban areas of China. However, along with the increasing population density in urban areas, this pattern may have negative influences on people’s daily lives, such as traffic jams. To avoid the negative influences, the government has encouraged residents to open their gated communities; however, few positive actions have been taken. With this background, this study aims to explore the key factors in residents’ willingness to open their gated communities. To start with, a total of 26 potential factors were identified based on a comprehensive literature review. Then, a questionnaire was designed and distributed to collect empirical data. Furthermore, logistic regression was employed to analyze the collected data. Based on the derived results, it was revealed that concerns are different between male and female residents. Male residents regarded “community safety” and “property management” as having a significant impact on their decision to open a gated community, while female residents paid more attention to the factor of “proprietary equity”. The results of this study could provide valuable references that enable the government to better understand residents’ underlying concerns and to make relevant policy decisions. In recent decades, China has been experiencing a rapid urbanization process ,2,3,4. NDuring the long period of China’s history, the gated community has been regarded as the main residential pattern as it has several advantages, such as guaranteeing people’s living safety, privacy and living environment . In the Nevertheless, with the rapid urbanization process and the drastic increasing number of gated communities in recent decade, a number of urban problems has emerged in many cities, such as urban traffic congestion, public facilities division and neighborhood environmental quality ,14,15. IBearing this research question in mind, this study aims to identify the most significant factors that influence residents’ willingness to open their closed communities. A comprehensive review was conducted to collect the suggested advantages and disadvantages of gated communities from the existing literature. Then, follow-up interviews were carried out to further formulate a formal questionnaire. Based on the data collected through questionnaire surveys, the research findings are presented and discussed at the end of this paper.In the existing literature, researchers have explored gated communities from different perspectives. For example, Liao et al.  employedVarious studies have been conducted to discuss the side effects of gated communities. For example, Sun et al.  recordedDifferent community patterns can also have impacts concerning social perspectives. As income inequality has become more and more visible in big cities, Lestari Olivia et al.  argued tResearchers also investigated the barriers to opening the gated communities from different perspectives, such as property law, privacy and employment pressure . HoweverTo identify the potential factors that affect the opening of gated community, a literature review was conducted. Through the literature review, a total of 26 potential factors were identified, as shown in Based on the identified factors listed in In the designed questionnaire, the classical five-point Likert scale was used to measure respondents’ agreement with the identified factors, in which “1” refers to “strongly disagree”, “2” represents “disagree”, “3” means “neutral”, “4” stands for “agree” and “5” is “strongly agree”. The “neutral” option indicates that the respondent does not have an obvious tendency concerning both gated and opened patterns.The questionnaire was distributed and collected through a field investigation method. Several rounds of field investigations were conducted in Shenzhen during the period from 6 January 2019 to 26 March 2019. Respondents were randomly selected on streets based on their willingness to fill out the questionnaires. Finally, a total of 312 questionnaires were collected for further data analysis.SPSS  software was used to test the reliability of the collected data. The coefficient of Cronbach’s α was derived to judge the reliability. Chan et al.  suggesteThe maximum likelihood (ML) method was used to estimate the parameters in the logistic regression models after transforming the dependent variable into a logit variable . As suchp value derived from the Hosmer–Lemeshow test was less than 0.05, which indicated that the model does not adequately fit the data. The respondents were divided into different groups according to personal information. Results reveal that, except for the gender groups, the p values of the Hosmer–Lemeshow tests of other groups were less than 0.05; thus, two independent logistic regression models  were used to test the identified factors. p values of the Hosmer–Lemeshow tests were 2.869 and 8.229, respectively, indicating good data fit in the two models.In this study, logistic regression was employed to investigate the key factors of opening gated communities. Firstly, the collected data of all respondents were analyzed using logistic regression. Results showed that the To test the empirical results, three professionals who are experienced in this field were invited to provide their comments. The three invited interviewees included a government officer, a real estate developer and a scholar. The government officer advocated opening gated communities. From her perspective, the advantages of this measure outweigh the disadvantages; more specifically, the opening of gated communities can effectively solve social problems such as traffic jams. Unlike the government officer, the real estate developer was more concerned about his own economic benefits. In the current situation, gated communities are favored by the public, so the developer believed developing gated communities could attract more customers and improve profits. From the scholar’s point of view, whether to open the gated community should be considered according to the local conditions. This measure only makes sense under the premise that the opening of a gated community could bring more benefits.It is not surprising that community safety is an important factor that influences residents’ willingness to open gated communities. During the process of national development, social stability is an essential prerequisite. Fences and walls can physically isolate a community’s residents from the public; thus, the gated-community pattern can prevent potential crimes to a large extent. In this regard, residents prefer to live in a gated community, and the developer generally adopts the gated-community pattern for new residential buildings to improve sales. However, the governmental officer held the opinion that, with economic and social development, crime rates in urban areas have decreased to a large extent. In these circumstances, the function of guaranteeing safety should not be obvious in the future. However, the officer also agreed that it takes time for the public to become aware of such changes. In previous studies, the relationship between safety and gated communities was investigated; however, the research findings showed some unexpected conclusions. For example, Breetzke et al.  found thIn addition to community safety, proprietary equity is also emphasized when considering opening gated communities. In China, housing is the main asset of the Chinese people. In general, communities with high housing prices have more recreation facilities and prettier garden landscapes. The residents living in such communities are unwilling to open their gated community because they believe that they have paid for these privileges and they do not want to share the facilities and landscapes with the public. In these circumstances, it is understandable that gated communities are preferred. This phenomenon was also observed by the invited academic scholar, and he suggested that this problem may be solved along with the social and economic development which will bring more facilities and more beautiful landscapes to public areas. This research finding is echoed by Salah and Ayad . They coProperty management is another critical aspect when residents consider opening their gated communities. This aspect is mainly focused on the economic perspective. Opening a gated community can reduce property management fees and increase the income channels of property management companies. However, property management is more difficult after a community is opened. Thus, residents are ambivalent to opening their gated communities from this perspective. This is also an important issue which requires a series of comprehensive and thoughtful guarantee measures to remove people’s concerns. Soyeh, et al.  argued tThe social development aspect was found to be insignificant based on the investigation results. From a historical perspective, the gated-community pattern has been there for thousands of years. In the ancient times, gated communities had the function of both guaranteeing residents’ safety and strengthening social control. In addition, China’s traditional culture emphasizes distance and hierarchy among people. In this context, even in modern times, a neighborhood’s impact on housing segregation attracts little attention, and ordinary citizens are more concerned with protecting themselves than with social interaction.This study investigated the key factors of opening gated communities in the urban areas of China. A total of 312 questionnaires were collected and the logistic regression model was used for data analysis. Results showed that community safety, proprietary equity and property management were the three key factors that influence residents’ willingness to open their gated communities. In the meantime, it was found that residents care less about the social development aspect of opening a gated community. From the survey results, the number of residents who are unwilling to open their own communities was much more than the number of residents who are willing to. This result is mainly because the residents have paid too much money for their residence, and they do not want to share the privileges with the public. Based on the above research findings, this study suggested that it may be inappropriate to compulsorily require the gated communities to be opened immediately. In other words, the government should solve the problems that the residents care about the most before the compulsory requirement of opening gated communities.This study has made contributions to the existing knowledge, as there was no such study in Shenzhen; the research findings could aid relevant policy makers in formulating more effective and appropriate measures to design the city better. However, this research also has some limitations. For example, the majority of the respondents was under the age of 40. It is recommended that in future research the questionnaire be distributed to more respondents, covering all age groups. In addition, the vignette approach is suggested as it can be used to deal with sensitive topics and to capture the context of decision making.


TARDBP gene that is involved in RNA splicing, trafficking, stabilization, and thus, the regulation of gene expression. Cytoplasmic inclusion bodies containing phosphorylated and truncated forms of TDP-43 are hallmarks of amyotrophic lateral sclerosis (ALS) and a subset of frontotemporal lobar degeneration (FTLD). Additionally, TDP-43 inclusions have been found in up to 57% of Alzheimer’s disease (AD) cases, most often in a limbic distribution, with or without hippocampal sclerosis. In some cases, TDP-43 deposits are also found in neurons with neurofibrillary tangles. AD patients with TDP-43 pathology have increased severity of cognitive impairment compared to those without TDP-43 pathology. Furthermore, the most common genetic risk factor for AD, apolipoprotein E4 (APOE4), is associated with increased frequency of TDP-43 pathology. These findings provide strong evidence that TDP-43 pathology is an integral part of multiple neurodegenerative conditions, including AD. Here, we review the biology and pathobiology of TDP-43 with a focus on its role in AD. We emphasize the need for studies on the mechanisms that lead to TDP-43 pathology, especially in the setting of age-related disorders such as AD.Transactive response DNA binding protein of 43 kDa (TDP-43) is an intranuclear protein encoded by the  Alzheimer’s disease (AD), the leading cause of dementia, is a heterogeneous neurodegenerative disorder in terms of clinical presentations and the density and distribution of the cardinal neuropathologic lesions. The neuropathologic hallmarks of AD are senile plaques composed of extracellular deposits of amyloid-β (Aβ) and neurofibrillary tangles composed of intracellular aggregates of tau protein with multiple post-translational modifications including phosphorylation. Senile plaques are complicated and heterogeneous lesions that contain not only amyloid deposits and tau positive neurites, but also neurites with degenerating pre- and post-synaptic elements , as well as activated microglia and reactive astrocytes . Aβ depoClinically, the two major presentations of AD can be classified as amnestic and non-amnestic. The former is characterized by deficiencies in short-term memory, recall and learning, which are the most common clinical presentations of typical and limbic predominant subtypes of AD. The latter shows impairment in other cognitive domains, such as language, visuospatial skills, or executive functioning. This is often associated with hippocampal sparing AD.In addition to senile plaques and neurofibrillary tangles, many AD brains have other pathological lesions, such as cerebrovascular pathology, Lewy bodies, argyrophilic grain disease, hippocampal sclerosis, cerebral amyloid angiopathy, and transactive response DNA binding protein of 43 kDa (TDP-43) pathology , 11. ImpTARDBP gene located on chromosome 1 (1p36.22) [TDP-43 is a 43 kDa heterogeneous nuclear ribonuclear protein (hnRNP) composed of 414 amino acids and is encoded by the The function of TDP-43, much like other hnRNPs, is to regulate gene expression and other aspects of RNA processing including RNA splicing, mRNA turnover, RNA trafficking, and microRNA (miRNA) biogenesis –22. TDP-Additionally, TDP-43 plays a role in the cellular stress response , 26–28. TDP-43 has been reported to regularly shuttle between the cytoplasm and nucleus depending on transcriptional needs . InteresThe structure of TDP-43 is composed of an N-terminal domain, a nuclear localization sequence (NLS), two RNA binding domains RBD1 and RBD2), a nuclear export signal (NES), and a C-terminal glycine rich domain (GRD)  and bvFTD are common clinical phenotypes in Type C, but no genetic association has been reported. Type D is associated with frontotemporal dementia (FTD) and Paget’s disease of bone caused by mutations of VCP gene [Based on the morphology, cell type, and distribution of TDP-43 pathology, FTLD-TDP can be classified into four main subtypes –69 Fig . Type A VCP gene . This clPathological progression of TDP-43 varies depending upon the underlying neurodegenerative disease with different progression patterns proposed for FTLD and ALS by Brettschneider et al , 72. In in vitro [in vitro neuronal cell culture models [Ubiquitination, phosphorylation, and truncation modifies the conformation of TDP-43, as well as its size and charge, contributing to the decreased shuttling into the nucleus –75. Ubiqin vitro . It is pe models , 83. Adde models .in vitro study has indicated that soluble TDP-43 is degraded by the ubiquitin-proteasome system, while insoluble TDP-43 aggregates are degraded via the autophagy system [An impairment in the clearance of TDP-43 may also contribute to the pathogenic process. An y system . Other iy system . Interesy system , suggestIn a non-diseased state, a balance between soluble and insoluble forms of RNA binding proteins (including TDP-43) and cell stress granules is maintained in the cytoplasm primarily due to their reversibility during cellular stress response . In ALS in vitro and in vivo [Neuronal death associated with pathological TDP-43 is thought to be caused by a combination of both a toxic gain of function, as well as a loss of physiological function associated with depletion of TDP-43 from the nucleus . Oligome in vivo –95. Addi in vivo , 96–98.  in vivo , 99, 100C9ORF72-linked FTLD, there is loss of nuclear TDP-43 at pre-symptomatic stages [Loss of function of TDP-43 is another mechanism implicated in neuronal loss in ALS and FTLD. Studies in mouse models rarely detect TDP-43 cytoplasmic inclusion bodies; however, neurodegeneration associated with loss nuclear TDP-43 can be evident . In humac stages . Furtherc stages . This obc stages , 104–111Interestingly, nuclear TDP-43 suppresses splicing of non-conserved cryptic exons, reducing the number of frameshift or nonsense mutations in mRNA transcripts , 112. PaTDP-43 pathology is frequently detected in pathologically confirmed AD brains in up to 57% of AD cases , 115–123TDP-43 has been reported to influence the clinical features of dementia, including cognitive deficits and the likelihood of dementia. Josephs and coworkers sought to determine the frequency of TDP-43 pathology across AD subtypes and its effects on cognition . They foOverall, these results suggest that TDP-43 pathology is common in AD, especially in the limbic predominant subtype. These results also suggest TDP-43 pathology is a risk factor for developing dementia of the Alzheimer type independent of pathological subtypes, and TDP-43 pathology increases the rate of hippocampal atrophy in AD.Interestingly, TDP-43 presenting as a secondary comorbid pathology in AD follows its own distinct pathological distribution pattern compared to that of ALS and FTLD. Josephs et al proposed that the progression of TDP-43 pathology in AD occurs in six stages, with stage 1 being characterized by TDP-43 pathology present within the amygdala  resulted in a decrease in Aβ plaque burden [Another study revealed that the overexpression of TDP-43 in the cortex and hippocampus of an APP/PS1 mouse model , a glycoprotein present within the central nervous system and periphery, is an important lipid transporter, especially for cholesterol . The humonset AD –139. Apoonset AD , 140–144reported , 145. A  APOE3/3 . Using aAD cases . AdditioAD cases . Similarhologies . WennberTARDBP 3′ untranslated region has been reported to increase TARDBP mRNA expression in the motor cortex in ALS [Age-dependent demethylation of the x in ALS . Besidesx in ALS –150. Frox in ALS . Additiox in ALS . These dx in ALS . Similarx in ALS .GRN and TMEM106B are risk factors for FTLD [GRN and TMEM106B genetic associations have also been observed in HS in the setting to Lewy body dementia, most of whom have at least some co-existing Alzheimer type pathology [Hippocampal sclerosis (HS) increases in frequency with age and is a distinct process from AD, even though they both are associated with an amnestic clinical syndrome . About 1for FTLD , 157 andfor FTLD , 159, liathology .Given the fact that HS can be associated with degenerative, toxic, and selective hippocampal neuronal loss associated with anoxic-ischemic injury or epilepsy, the term HS has fallen out of favor. An international group of experts proposed a new name for TDP-43 pathology in the elderly, often associated with HS, “limbic-predominant age-related TDP-43 encephalopathy” (LATE) . LATE neTDP-43 has been reported as a co-pathology in other neurodegenerative disorders besides AD, including Huntington’s disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease, DLB, and multiple system atrophy (MSA) , 164–170n vivo study using transgenic mice expressing human TDP-43 mutants found that administration of an autophagy-inducing drug could ameliorate TDP-43 pathology in the brain and spinal cord of the transgenic animals [An i animals . Given t animals –174, dev animals , but alsAPOE4 gene. The mere presence of TDP-43 pathology increases the likelihood of developing Alzheimer-type dementia. These findings provide strong evidence for TDP-43 being an integral part of multiple neurodegenerative conditions, emphasizing the need to better understand the mechanisms of TDP-43 pathogenesis in AD and other age-related disorders.Significant efforts in the past decade have been placed in finding and testing new treatment methods for AD in hopes to prevent or cure this devastating disease. TDP-43 pathology, commonly found in AD brains, has been shown to influence AD pathology and neurodegeneration, whether it be decreasing senile plaque load through overexpression, or increasing amyloid oligomers and synapse loss through depletion. It also shares an important genetic risk factor with AD, the


Background: The prevalence of parkinsonism in developing countries is largely unknown due to difficulty in ascertainment because access to neurologists is often limited.Objective: Develop and validate a parkinsonism screening tool using objective motor task-based tests that can be administered by non-clinicians.Methods: In a cross-sectional population-based sample from South Africa, we evaluated 315 adults, age >40, from an Mn-exposed (smelter) community, using the Unified Parkinson Disease Rating Scale motor subsection 3 (UPDRS3), Purdue grooved pegboard, and kinematic-UPDRS3-based motor tasks. In 275 participants (training dataset), we constructed a linear regression model to predict UPDRS3. We selected motor task summary measures independently associated with UPDRS3 (p < 0.05). We validated the model internally in the remaining 40 participants from the manganese-exposed community (test dataset) using the area under the receiver operating characteristic curve (AUC), and externally in another population-based sample of 90 participants from another South African community with only background levels of environmental Mn exposure.Results: The mean UPDRS3 score in participants from the Mn-exposed community was 9.1 in both the training and test datasets . Together, 57 (18.1%) participants in this community had a UPDRS3 ≥ 15, including three with Parkinson's disease. In the non-exposed community, the mean UPDRS3 was 3.9 (standard deviation = 4.3). Three (3.3%) had a UPDRS3 ≥ 15. Grooved pegboard time and mean velocity for hand rotation and finger tapping tasks were strongly associated with UPDRS3. Using these motor task summary measures and age, the UPDRS3 predictive model performed very well. In the test dataset, AUCs were 0.81  and 0.91  for cut points for neurologist-assessed UPDRS3 ≥ 10 and UPDRS3 ≥ 15, respectively. In the external validation dataset, the AUC was 0.85  for UPDRS3 ≥ 10. AUCs were 0.76–0.82 when excluding age.Conclusion: A predictive model based on a series of objective motor tasks performs very well in assessing severity of parkinsonism in both Mn-exposed and non-exposed population-based cohorts. The Global Burden of Disease Study estimates that the number of people affected by Parkinson's disease (PD) more than doubled from 1990 to 2015 with the highest prevalence in high-income regions and lowest in sub-Saharan Africa and Eastern Europe in 2015 . RelativThe Unified Parkinson Disease Rating Scale motor subsection 3 (UPDRS3) remains the most widely used tool to quantify parkinsonian motor signs in patient and non-patient populations –10. In aThe Washington University School of Medicine Human Research Protection Office  and the University of the Witwatersrand Human Research Ethics Committee  approved this study. All participants provided written informed consent.N = 275, 87%) and test  datasets, respectively. We enrolled the participants in these two groups consecutively (SD = 0.77) from the smelter, and the participants in the test dataset lived a mean of 1.96 Km (SD = 0.74) from the smelter. The participants from Meyerton comprised a subset of participants who were recruited as part of a larger environmental Mn study   smelter in Meyerton, South Africa, we developed and validated a predictive model for UPDRS3, in training (cutively . ParticiMn study . The reclements) . This ish Africa , 12, and N = 40, % dataset2.5-Mn ~20 times lower than at the fixed site in Meyerton (10 ng/m3)  of homes that we visited had at least one age-eligible adult who agreed to participate. Ethembalethu is an industry-free community, with no nearby Mn smelting or mining operations, located ~70 km from Meyerton. Air monitoring in Ethembalethu demonstrated mean concentrations of PM0 ng/m3) . This istivities . We therFor inclusion in the present work, we required participants in both communities to meet the following criteria: (1) be non-ambidextrous (self-report as right-handed or left-handed), (2) complete at least one trial for the dominant and non-dominant hand for each of five motor tasks under the direction of a trained test administrator, (3) have been examined by a movement disorder specialist using the UPDRS3 and had complete UPDRS3 data. Exclusion criteria were: (1) current use of neuroleptic medications, and (2) neurologic co-morbidities that might compromise the accuracy of the UPDRS3, such as stroke or spasticity.In order to include individuals with other conditions that would cause incomplete UPDRS3 data, such as injured/missing limbs or inability to undergo the pull test to assess balance, we used imputation of the respective subscore(s) to obtain complete UPDRS3 data when possible. We included individuals regardless of UPDRS3 score, presence of PD, or occupational Mn exposure, in order to ensure that our original population-based samples remained representative of the respective settlements. The above exclusions, need for imputation, presence of PD, and past or current occupational Mn exposure were all uncommon  in the larger study .All recruitment, testing, and examination occurred in 2016–2020 .TM, Great Lakes NeuroTechnologies, Independence, Ohio)  action tremor—participant alternated touching his/her index finger to his/her nose and to the administrator's finger held an arm's length away from the participant; (3) finger tapping—participant tapped his/her index finger and thumb together while keeping the other fingers stable and the elbow extended; (4) hand rotation—participant rotated his/her hand at the wrist, positioning the arm so that the elbow was flexed and the hand open. Participants were instructed to perform finger tapping and hand rotation tasks with as large an amplitude and as fast, as possible. Participants completed the trials for the right hand first, followed by the left hand, for each of these four tasks. A previous study in PD patients that used a similar Kinesia Motion Sensory device found that in the more parkinsonian hand the test-retest reliability across three 15-s trials, as measured by the intraclass correlation coefficient, was 0.71 for postural tremor and 0.94 for finger tapping speed  and followed published testing procedures . For thee, Ohio) –19 on thng speed .We then developed, validated, and applied comprehensive computer code to process these large datasets . SpecifiOne movement disorder specialist (BR) examined all participants using the UPDRS3 , blindedWe used self-reported handedness to classify UPDRS3 subscores and the five motor tasks as dominant or non-dominant. Participants also provided socio-demographic information, including age, sex, ethnicity, and home language.p < 0.05) and, more importantly, in the expected direction (positive or negative). Greater UPDRS3 scores and subscores indicate greater parkinsonism, which we anticipated would be associated with longer grooved pegboard times, fewer pegs placed, and more pegs dropped. With regard to the kinematic tests, we also anticipated that greater UPDRS3 scores would be associated with lower velocities (or fewer cycles per second) and greater decrement (and hence greater variability as assessed by the coefficient of variation) on the finger tapping and hand rotation tasks. We investigated whether correlations differed according to hand dominance and whether motor task data from only one hand might be sufficient to predict UPDRS3. We also examined the association between summary measures derived from only the first of the three trials to determine if a single trial would be sufficient for UPDRS3 prediction.We performed all data processing and statistical analyses using Stata version MP 14.2  . To helpN = 275). Age and motor task summary measures selected above were our primary a priori predictors of interest, and we initially retained all as continuous measures  and in the independent, external dataset (N = 90), using the receiver operating characteristic (ROC) curve  curve , 23. We C) curve –26, i.e.mination . TherefoSD = 6.3, range 0.0–38.5), but differed markedly between the two communities. Upper limb bradykinesia contributed substantially to UPDRS3 as 16.8% of participants had >6 points in total across the six upper limb bradykinesia subscores, i.e., a subscore of >1 on at least one of these six scores (not shown). In contrast, action/postural tremor was relatively uncommon. Only 20 (4.9%) of the participants had an action/postural tremor subscore of 2, and only three participants had prominent action/postural tremor or only rest tremor (subscore >2). Three participants who had complete kinematic and grooved pegboard data, all from Meyerton, had PD according to this neurological examination.Most (98.5%) of participants were of black/African ethnicity, with a median age of 51 . The meaSeveral summary measures for upper limb bradykinesia correlated with the respective UPDRS3 subscores , with grThe absolute value of the correlation coefficients between the action/postural tremor summary measures and UPDRS3 subscore were all well below 0.20 ; finger tapping kinematic mean velocity; grooved pegboard time; and age, each as linear terms . Finger In our test dataset, performance of the predictive model as measured by the AUC was 0.81  for identifying participants with neurologist-assessed UPDRS3 ≥ 10 . The modWhen we applied our predictive model to the external validation dataset, the AUC was 0.85  when identifying participants with UPDRS3 ≥ 10 . Too fewWhen we explored the effect of using only the motor tasks as predictors, i.e., removing age from the model, AUCs were only modestly attenuated. In our test dataset the AUC was 0.76  for UPDRS3 ≥ 10 and 0.86  for UPDRS3 ≥ 15. In the external validation dataset the AUC was 0.82 .post-hoc exploratory analysis in which we considered how well the predictive model might identify the three individuals with PD in Meyerton (training and test datasets combined), the predicted UPDRS3 scores were 16.3, 17.1, and 21.7, all above the UPDRS3 ≥ 15 cut point.In a In this large study in South Africa we predicted the UPDRS3 score with a limited battery of kinematic and grooved pegboard tests administered by trained non-clinician community members in <10 min per participant. AUCs in two validation datasets demonstrated that performance of this predictive model for identifying individuals with UPDRS3 scores ≥10 or ≥15 was good to excellent. In addition, we confirmed that there was at least a moderate correlation between the predicted UPDRS3 scores and neurologist-assessed UPDRS3. Notably, this UPDRS3 predictive model worked equally well in communities with and without environmental Mn exposure, i.e., in population-based samples with either relatively high or relatively low UPDRS3 scores. Taken together, our findings indicate that this UPDRS3 predictive model likely can be applied to facilitate screening programs or research studies in a wide variety of settings, including under-resourced environments with limited access to PD specialists or other neurologists.In all potential applications of this UPDRS3 predictive model, the first step after administering the three-test battery is to calculate the mean velocities from the two kinematic tests. Of the many kinematic summary measures we assessed, the mean velocities are the easiest to calculate, further ensuring the potential usefulness of our model in many situations. Calculation of these summary measures only requires the isolation of the kinematic data from the relevant axis, i.e., x-axis for finger tapping and y-axis for hand rotations, before taking the mean across all sampled time points with the respective movement. In the second step, these two means from kinematic testing, along with the grooved pegboard time and age, are simply inserted into the equation produced by our predictive model. The resulting UPDRS3 score could be used in an epidemiologic study in which it is not possible for a neurologist to examine any participants. Alternatively, in a third step, one would dichotomize the predicted UPDRS3 score according to a pre-selected cut point to identify individuals with a particular UPDRS3, corresponding to the desired sensitivity and specificity. One likely would dichotomize the predicted UPDRS3 scores at the lowest possible cut point so as to achieve the highest sensitivity that is feasible, given that a specialist would need to examine all individuals with predicted UPDRS3 scores above the selected cut point. As part of a tiered screening protocol, maximizing sensitivity would either facilitate clinical care to the largest percentage of people in a community who might benefit from treatment, or alternatively would minimize misclassification of research study participants in terms of a dichotomous outcome of interest. As evidence of the potential utility of this type of protocol, a similar tiered screening approach was adopted in a previous occupational Mn exposure setting in South Africa .We acknowledge some limitations that could affect usefulness of our predictive model. First, the kinematic methods may not be easily incorporated in all settings. We trained our fieldworkers extensively in the use of kinematic devices, and we view this training as essential. Second, kinematic testing generates large amounts of data, requiring expertise in data handling. In addition, a substantial amount of computer storage and computational power would be required to process these data for many individuals at once, which might be a challenge in under-resourced environments. Third, while we used a common motion capture device, such devices have unique operational principles, instrumentation, and type of data produced  so data The strengths of our study include the use of one movement disorder specialist to obtain gold standard UPDRS3 ratings, the relatively large sample size, and a unique study population at risk for parkinsonism paired with a lower risk population, each representative of their underlying communities. These strengths positioned us well to develop and validate the resulting UPDRS3 predictive model, which also has several strengths. First, we were successful in minimizing the time required to complete the included motor testing without materially affecting performance of the UPDRS3 predictive model. Most notably, we confirmed that it was sufficient to conduct only one trial of the selected tests in one hand. Second, all predictors can be assessed objectively, i.e., without subjective assessments that might require clinical expertise. Although some prior predictive models of UPDRS3 score or “parkinsonism” benefitted from the use of objective motor assessment (either a different fine motor task or gait assessment), some of these models required test administrators to make clinical judgements , 31. ThiRestriction of our model to age and a modest battery of objective motor tasks to avoid the above potential challenges did not come at the expense of performance. Previous occupational or community-based cohorts in which investigators attempted to identify people with parkinsonism achieved AUCs ranging from 0.72 to 0.79 –32. The In summary, using selected accelerometry-based kinematic-UPDRS3 tasks and the grooved pegboard, we developed a UPDRS3 predictive model to identify individuals with potential parkinsonism, which demonstrated good predictive ability. The model performed exceptionally well considering that we applied it in non-patient populations and relied on non-clinicians to administer the tests. These findings have important public health applications in screening for or assessing parkinsonism in clinical or research settings in regions of the world with limited clinical neurologic expertise. The proposed screening tool provides an alternative assessment in these low-resourced environments because of ease of administration.racetteb@wustl.edu, and requestors will need to sign a data use agreement.Individual participant data that underlie the results reported in this article, after de-identification, will be made available upon reasonable request, following article publication. Data will be made available to investigators whose proposed use of the data has been approved by an independent review committee identified for this purpose and after approval of the protocol by the University of the Witwatersrand Ethics Committee and the Washington University Human Resource Protection Committee. The analysis performed must be solely to achieve aims in the approved proposal. Proposals should be directed to BR, at The studies involving human participants were reviewed and approved by Washington University School of Medicine Human Research Protection Office  and the University of the Witwatersrand Human Research Ethics Committee . The participants provided their written informed consent to participate in this study.BR, MU, GN, and SN: research project conception, organization, execution, and manuscript preparation review and critique. SN: statistical analysis design. WD and SN: statistical analysis execution. BR, MU, and GN: statistical analysis review and critique. WD: manuscript preparation and writing of the first draft. All authors: approval of 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.


Recently, working memory (WM) has been conceptualized as a limited resource, distributed flexibly and strategically between an unlimited number of representations. In addition to improving the precision of representations in WM, the allocation of resources may also shape how these representations act as attentional templates to guide visual search. Here, we reviewed recent evidence in favor of this assumption and proposed three main principles that govern the relationship between WM resources and template-guided visual search. First, the allocation of resources to an attentional template has an effect on visual search, as it may improve the guidance of visual attention, facilitate target recognition, and/or protect the attentional template against interference. Second, the allocation of the largest amount of resources to a representation in WM is not sufficient to give this representation the status of attentional template and thus, the ability to guide visual search. Third, the representation obtaining the status of attentional template, whether at encoding or during maintenance, receives an amount of WM resources proportional to its relevance for visual search. Thus defined, the resource hypothesis of visual search constitutes a parsimonious and powerful framework, which provides new perspectives on previous debates and complements existing models of template-guided visual search. We spend a large part of our daily lives searching for known objects in dense visual scenes, such as car keys on a cluttered desk or a child’s jacket in a crowded playground. That is, our visual environment comprises an overwhelming amount of information from which we must select a limited quantity that is of interest. To achieve this complex operation, an accurate representation of the relevant features, for instance the shape of the car keys or the color of the child’s jacket, may give a significant advantage in achieving efficient visual search. This theoretical review examines how these two abilities – looking for goal-relevant objects and remembering their features – interact in a way that optimizes behavior.internal attention  commonly refers to the processes that maintain and manipulate representations most needed for ongoing cognitive operations , an electrophysiological correlate of the number of representations maintained in WM  to specify the target’s features prior to visual search. While both types of cue allow setting up attentional templates, search is consistently less efficient with verbal than pictorial cues, as less visual information is available , a von Mises distribution that reflects the precision of responses to the probed item (PSD), and a von Mises distribution that reflects the proportion of responses to the non-probed item (PSwap). In the theoretical interpretation of these parameters, only PSD is assumed to reflect the continuous allocation of WM resources to the corresponding representations .Hollingworth and Hwang  were thesufficient, the allocation of the largest amount of resources may be necessary for WM representations to act as attentional templates. Using single- and dual-target search, studies provided convincing evidence in favor of this assumption. For instance, Rajsic et al. (Protection from interference). Therefore, it would be more appropriate to conclude that representations obtaining the status of attentional template receive an amount of WM resources proportional to their relevance for visual search. In other words, a single attentional template receives the largest amount of WM resources since it is the only relevant representation for visual search, whereas multiple attentional templates receive an amount of WM resources that depends on their relevance for the search task. In any case, by directly assessing the allocation of resources in WM, this line of research provides converging evidence that the precision of attentional templates may have a functional value in visual search (Bravo & Farid, While not c et al.  asked obc et al. . Resultsc et al.  and its c et al. . Thus, fc et al.  showed tc et al. , observec et al.  was usedc et al. , the allc et al.  suggestsresource hypothesis of visual search comprises a set of three main principles to conceptualize the complex relationships between WM resources and attentional templates. Here, we expose each of the three principles that constitute this hypothesis and discuss their relevance in relation to existing models of template-guided visual search.Based on resource models of WM (Franconeri et al., First, the allocation of resources to an attentional template has an effect on visual search. While appearing trivial, this first principle received considerable amount of support from studies that manipulated the number of features specifying attentional templates and demonstrated clear causal effects on visual search (Bravo & Farid, Second, as laid out above, the allocation of the largest amount of resources to a representation in WM is not sufficient to give this representation the status of attentional template and thus, the ability to guide visual search (Dube & Al-Aidroos, Third, representations that obtain the status of attentional template, whether at encoding or during maintenance, receive an amount of WM resources proportional to their relevance for visual search. Therefore, a single attentional template receives the largest amount of WM resources because it is the only relevant representation (Kerzel & Witzel, So far, we have shown that WM representations receiving the largest amount of resources do not necessarily act as attentional templates. However, single attentional templates inevitably receive the largest amount of resources, which makes them more precise than any other representations in WM. While the increase in resources appears to improve visual search, the exact processes involved are still to be determined. Here, we present three proposals about the role of WM resources in template-guided visual search that may not be mutually exclusive see Fig. . For easAs introduced earlier, attentional templates contribute to two distinct processes in visual search. First, attentional templates allow for the selection of objects with template-matching attributes by converting display-wide enhancement of relevant features into spatially specific enhancement, thus guiding visual attention Eimer, . Second,Recently, Berggren et al.  investigWhile no previous study investigated protection from interference in the context of template-guided visual search, this topic has been particularly fruitful in the WM literature. First, studies investigating individual differences have repeatedly demonstrated that interference impaired memory performance in individuals with low WM capacity, but not in those with high WM capacity Engle, . As inteBehavioral evidence from continuous delayed-estimation tasks indicates that single attentional templates receive the largest amount of WM resources as their recall precision is higher than the recall precision of other WM representations. However, these measures are usually collected after visual search is performed, allowing intervening stimuli to contaminate them. For instance, orienting visual attention toward a distractor disrupts information already stored in WM (Hamblin-Frohman & Becker, Recently, WM has been conceptualized as a limited resource, distributed flexibly and strategically between stored representations. As attentional templates are thought to be represented in WM, we reviewed empirical evidence that the allocation of WM resources has consequences not only on memory, but also on visual search. We have argued that three main principles govern the relationships between WM resources and template-guided visual search. First, the allocation of resources to an attentional template has an effect on visual search, as it may improve the guidance of visual attention, facilitate target recognition, and/or protect attentional templates against interference. Second, the allocation of the largest amount of resources to a representation in WM is not sufficient to give this representation the status of attentional template and thus, the ability to guide visual search. Third, the representation that obtains the status of attentional template, whether at encoding or during maintenance, receives an amount of WM resources proportional to its relevance for visual search. Thus formalized, the resource hypothesis of visual search describes how the internal representation of the target is maintained in WM and how it affects the exploration of visual environments. Moreover, the concept of WM resources gives new insights on previous debates and complements existing models of template-guided visual search.