Humidity and temperature are fundamental for the balance in the life cycle of living beings and, consequently, for maintaining the well-being of the human population and reducing the prevalence of infectious diseases. Thus, in order to mitigate the impact of climate change, especially in the period when humidity is not the ideal, it is necessary to adopt some assistance measures. The present experimental study aims to elucidate what would be the recommended option to improve the quality of life of the human being and to clarify which resources  will be effective to improve the humidity of the air in times of drought and low moisture.The experimental study was carried out with INKBIRD hygrometers allowing the analysis of the variation of air humidity throughout the day. Three forms of treatment were established: humidifier, wet towel and bucket of water. In each room, two hygrometers were placed equidistant from the occupant of the room and their respective treatment that varied between 1m and 2m away from the headboard indoor each room. In addition, two environments were used as controls, one being an external environment and the other an internal closed environment, totaling five rooms for the study. The rooms were monitored between the end of July and the end of August 2019 in Goiania (GO).Although assistance measures are used to significantly improve air pollution in times of extreme drought, there was a significant difference between them. The humidifier and a wet towel had 7.50% and 5.71% more humidity in the external relation , respectively, more efficient. The volume of water, however, did not show significant difference (p>0.05) and, therefore, there was no variation.The humidifier and the towel are treatments considered more efficient, and that there was a significant effect of distance on humidity. Therefore, 1m of distance is more efficient in increasing and/or maintaining air humidity, inducing improvements in the populations’ health. However, urbanization has generated changes in climate profile and, consequently, it has aggravated or facilitated the emergence of diseases to the human population. To investigate the effects of climate change on health there is need to measure how these factors vary over time to allow the creation of measures that can mitigate health-related negative effects.   - 7 These effects range from the interaction between air humidity and temperature to air pollution, thermal sensation, and precipitation, which are factors responsible for regulating the metabolism of organisms. In the Center-West region of Brazil, the state of Goiás, on the months of July and August, the impact of climate change is already observed. Currently, these months present longer drought and low humidity that they used to be, and this change exacerbates health problems.   - 11 Humidity makes the atmosphere dense and prevents the dissipation of pollutants, bacteria, and viruses that easily survive in the period of low humidity. The excessive emission of pollutants has caused several direct damage to public health such as worsening of respiratory problems, respiratory, ocular and dermatological allergies, asthma, headaches, dryness of the upper airways that may lead to nosebleeds, dry and irritated throats, a feeling of sand in the eyes that become hyperemic, skin dryness, and fatigue. These health problems are worse when the air humidity is not at the ideal level recommended by the World Health Organization (WHO), which should be around 40% to 70%. Besides this ideal level of humidity, there is also a classification of the criticality states of low air humidity, according to a psychrometric scale: humidity between 21% and 30% is considered an attention state; between 12% and 20% an alert; and below 12% an emergency. While the humidity is below the ideal to adopt care measures whenever possible is necessary to minimize health- related problems. These measures involve the use of an air humidifier, bucket with water, and wet towel. However, little is known whether these resources have significant effectiveness in reducing the impacts caused by low air humidity, nor what would be the ideal location of these devices within rooms.Air humidity is a worldwide issue especially in times of drought. Low humidity influences the well-being of the population, affecting growth, development, and quality of life. These unfavorable relative humidity levels increase the prevalence of infectious diseases that may compromise the population and makes pathogens more contagious. This study aims to elucidate what would be the recommended resources and what should be the distance of them from the individual to enhance population welfare in times of drought and low humidity.To determine which resources are effective to improve the air humidity in dry seasons and indicate the best distance for these resources to be from the individual in order to obtain the appropriate level of air humidity, and improve the quality of life of human being.2 rooms at the same house using similar furniture accommodations at the indoor environment of a residential building. The INKBIRD hygrometers were attached to these accommodations to allow the analysis of the air humidity variation throughout the day. The humidifier used was the G-Tech ultrasonic humidifier, with a cloth wick system to take water from the reservoir, which made the air absorb humidity, and held 3L of water. The towel was 100% cotton and measured 70cmx140cm, manufactured by Karsten. The size of a bath towel and it was able to wet, on average, with 1L of water. The bucket used contained 5L of water.The study was carried out in the city of Goiania, state of Goiás, Brazil. To evaluate the most effective treatment to regulate air humidity, we analyzed air humidifier, bucket with water, and wet towel in three identical 15m In each room, two hygrometers were placed distant from the occupant of the room and its respective treatment, varying between 1m and 2m away from the head of the bed indoor each room. In addition, two rooms were used as controls, one outdoor (near the garage) and the other indoors, totalizing five rooms for the study. The rooms were monitored from the end of July to the end of August 2019.The data collected in percentages of humidity in each room were divided into four periods: dawn, morning, afternoon and evening. Subsequently, average humidity values were calculated for each period. The times established to perform the calculations were within a 2-hour interval, from 1:45 am until 11:45 pm. The times set for the calculations for the dawn period were 1:45 am, 3:45 am, and 5:45 am; for the morning 7:45 am, 9:45 am, and 11:45 am; for the afternoon 1:45 pm, 3:45 pm, and 5:45 pm; for the night 7:45 pm, 9:45 pm, and 11:45 pm.Repeated measures analysis of variance (ANOVA) was determined to evaluate the difference between the treatments used . In this way, we considered as similar the spatial and temporal dependence collected in the five rooms. However, the pre-analysis with this test indicated a collinearity between the monitored factors: period of the day  and treatment type .A linear mixed model was built, using humidity as the response variable, and treatment type and day period as predictor variables, which considered the fixed factors of the mixed model (see formula below). The room was considered as random factor of the analysis as having specific characteristics in each of the spaces, which could influence the observed humidity. In addition, the autocorrelation value was considered, since the experiment was repeated in the same rooms throughout the study.Finally, ANOVA was performed to assess whether there was an effect in terms of distance of the two most efficient treatments on air humidity quality. The treatments ranged from 1m to 2m distance from the bed within each room. The analyses were performed on R platform version 3.6.1 (2019), which is developed by R Development Core Team and freely available at http://www.r-project.org. This software functions as a language in which the codes used for the statistical analyses are open and the user can adapt them as needed.In general, we observed that efficacy of treatments and periods of the days ranged. The lowest value of humidity (27.40%) was observed in the outdoor in afternoon, whereas the highest value of humidity (82.60%)   . Measuri.e ., the predicted values, indicated a mean humidity value of 59.35% with a standard error of 1.44% , and the afternoon was the period that recorded the lowest average of humidity (about 21.32% less when compared with dawn).The outdoor area, where there was no treatment and, possibly the environment most exposed to evapotranspiration, was the one that differed most between the periods of the day   . The vaAs the humidifier and the towel were considered the most efficient treatments, the analysis of variance found no difference between treatments   , but th   The technical safety of several care measures is questioned because it is not known for sure which measure is really more effective to be exposed in headlines and magazines aimed at the general population in order to inform what really works.Hygrometers were chosen for the experimental study because they are able to provide an accurate monitoring of temperature and humidity conditions, keep in memory the records of the samples measured throughout the days even with a significant distance between the device and the cell phone that is connected to it via Bluetooth, therefore, guaranteeing high sample specificity. Air humidity is a variable that influences in a multifactorial way the balance of the ecosystem and the organism of all living beings, so its analysis would be more complex.   New studies, which evaluate which adaptive measures are really more effective and necessary, because the quality of life and the health of the population are two aspects of the inter-relationship between the city and the environment that are rarely addressed. The human being is the fundamental - but forgotten - point of the environmental issue in large metropolises. An effective feedback on low-cost methods to improve the living conditions of the population in times of drought is of major importance. The experiment, besides proving that the humidifier performs its function of increasing air humidity with mastery also demonstrated that the wet towel is an efficient treatment, and can bring these two statements back to the population, since there was no significant difference between these treatments, according to the analysis of variance. The bucket with water did not impact to the point of making the humidity more suitable for population welfare.  - 15 The ideal resources would be the humidifier or the wet towel. However, there are basic precautions to be taken regarding the humidifier. This is due to the accumulation of humidity and dust that occurs in air-conditioned environments, and therefore the proliferation of microbes and bacteria is much higher than in open environments. Suitable measures should be taken in relation to the humidifier, since it is not recommended to leave it on in the room all night long, because without sunlight the air humidity rises naturally, and the excess humidity promoted by the device becomes a problem, culminating in the appearance of mold and mildew. Among the main symptoms of people occupying these environments are infections, allergic and irritating reactions, headaches and joint pains, irritation in the eyes, nose and throat, dry cough, dermatitis, fatigue, drowsiness, difficulty to concentrate, sensitivity to odors, congestion, sinusitis, shortness of breath, allergic rhinitis, bronchial asthma, among others symptoms that are even more increased in dry season. The ideal would be to leave it on in the bedroom for 3 to 4 hours straight and turn it off before going to bed. However, one wonders how it would be possible for the small increase in humidity generated by the humidifier to cause these problems, since the humidity still remains at a level below that recommended by the WHO. Another observation regarding the use of the humidifier would be the need to wash it after each use, in order to prevent it from becoming a focus for fungi and other microorganisms that will be released into the air. The evident limitation of studies about which welfare measures are effective and their impact on the population’s life is notorious. However, through previous knowledge and the sum provided by data collection according to the hygrometers used, it was possible to analyze what really works and what is effective. We also identified two possible welfare measures that are very effective for the population, and can make it possible to better construct the strategy of environmental education and prevention methods for the population in times of drought and low humidity, therefore, reducing the impact of diseases.This study presented the results of a comparative evaluation of the performance of three support measures to promote humidification of the indoor environment of a residential building in a low humidity season between July and August 2019 in city of Goiânia, Goiás, Brazil. The humidifier and the towel were the treatments considered the best measures, and there was a significant effect of distance on humidity. The most efficient distance in increasing and/or maintaining air humidity was 1m. The use of wet towels is environmentally sustainable and economically feasible. This can improve sleep nights for lower to middle class population even in the low humidity and dry conditions in hot environments such as the Brazilian Midwest.   - 4 A umidade e a temperatura são fatores abióticos que compõem o clima e fundamentais para o equilíbrio no ciclo de vida dos seres vivos. Entretanto, a urbanização tem gerado mudanças no perfil climático e, consequentemente, agravado ou facilitado o surgimento de doenças na população humana. Para investigar os efeitos das mudanças climáticas sobre a saúde, é importante mensurar como esses fatores variam ao longo do tempo e, assim, permitir a criação de medidas que podem mitigar tais efeitos negativos.  - 7 Esses efeitos vão desde a interação entre a umidade do ar e a temperatura sobre a poluição atmosférica, a sensação térmica e a precipitação, as quais são responsáveis por regular o metabolismo dos organismos. Na Região Centro-Oeste do Brasil, estado de Goiás, nos meses de julho e agosto, já é possível perceber o impacto das alterações climáticas. Esse são meses de seca e baixa umidade, o que agrava os problemas na saúde.  - 11 A umidade torna a atmosfera densa, não deixando ocorrer a dissipação dos poluentes, das bactérias e dos vírus, que, no período de baixa umidade, sobrevivem com facilidade. A emissão excessiva de poluentes tem causado vários prejuízos diretos para a saúde pública, como piora dos problemas respiratórios, das alergias respiratórias, oculares e dermatológicas, asma, dores de cabeça e ressecamento de vias aéreas superiores, levando a sangramento nasal, garganta seca e irritada, sensação de areia nos olhos que ficam hiperemiados, ressecamentos de pele e fadiga. Esses problemas de saúde se agravam quando a umidade do ar não está no nível ideal recomendado pela Organização Mundial da Saúde (OMS), o qual deve ser em torno de 40% a 70%. Além desse nível ideal de umidade, também há uma classificação dos estados de criticidade da baixa umidade do ar, de acordo com uma escala psicrométrica: umidade entre 21% e 30% é considerada estado de atenção; entre 12% e 20%, alerta e abaixo de 12%, emergência.Enquanto a umidade está abaixo do ideal, é possível e necessária a adoção de medidas assistencialistas, com o objetivo de minimizar os agravos à saúde. Tais medidas envolvem o uso de umidificador de ar, balde com água e toalha molhada. No entanto, não sabemos qual desses recursos possui uma eficácia significante, a fim de reduzir os impactos causados pela baixa umidade do ar, e nem a localização ideal desses dispositivos dentro dos cômodos.Sabe-se que a umidade do ar é um problema de questão mundial, principalmente em épocas de seca. A baixa umidade causa efeito no bem-estar da população, afetando o crescimento, o desenvolvimento e a qualidade de vida. Além disso, esses níveis desfavoráveis de umidade relativa aumentam a prevalência de doenças infecciosas, comprometendo a população e tornando os patógenos mais contagiosos. Assim, este trabalho visa elucidar qual seria a opção recomendada e qual deve ser a distância do recurso ao indivíduo para melhorar o bem-estar populacional em épocas de seca e baixa umidade.Esclarecer quais recursos são eficazes para melhorar a umidade do ar em época de seca e indicar a melhor distância que esses recursos devem ficar do indivíduo, visando obter o nível da umidade do ar adequado para melhorar a qualidade de vida do ser humano.2na mesma casa com acomodações semelhantes dos móveis no ambiente interno de uma edificação residencial, no Bairro Jardins Milão. Os higrômetros INKBIRD foram fixados nessas acomodações, permitindo a análise da variação da umidade do ar ao longo do dia.O estudo foi realização no município de Goiânia, estado de Goiás, Brasil. Para avaliar qual o tratamento mais eficaz na regulação da umidade do ar, foram analisados três quartos idênticos de 15mForam estabelecidas três formas de tratamento: umidificador, toalha molhada e balde com água. O umidificador utilizado foi o da marca G-Tech ultrassônico, com sistema de pavio de pano para tirar água do reservatório, que fazia com que o ar absorvesse a umidade, e comportava 3L de água. A toalha era 100% de algodão, da marca Karsten. Media 70cmx140cm, correspondendo ao tamanho de uma toalha de banho, e era molhada, em média, com 1L de água. O balde continha 5L de água.Em cada quarto, foram colocados dois higrômetros equidistantes do ocupante do quarto e de seu respectivo tratamento, variando entre 1m e 2m de distância da cabeceira da cama dentro de cada cômodo. Além disso, dois ambientes foram utilizados como controle, sendo um externo (próximo à garagem) e outro fechado interno, totalizando cinco cômodos para o estudo. Os cômodos foram monitorados entre o final do mês de julho até final do mês de agosto de 2019.Os dados coletados em percentagem de umidade, em cada cômodo, foram divididos em quatro períodos: madrugada, manhã, tarde e noite. Foram, então, calculados valores médios de umidade para cada período. Os horários estabelecidos para realizar os cálculos foram com intervalo de 2 horas, a partir de 1h45 até 23h45. Os horários determinados para os cálculos no período da madrugada foram 1h45, 3h45 e 5h45; pela manhã foram: 7h45, 9h45 e 11h45; pela tarde: 13h45, 15h45 e 17h45, e 19h45, 21h45 e 23h45 pela noite.Foi determinada a realização da análise de variância (ANOVA) de medidas repetidas para avaliar a diferença entre os tratamentos utilizados . Dessa forma, assumiu-se a dependência espacial e temporal coletada nos cinco cômodos. Entretanto, a pré-análise com esse teste indicou a colinearidade entre os fatores monitorados: período do dia  e tipo tratamento .Foi construído um modelo linear misto, utilizando como variável resposta a umidade, e tipo de tratamento e período do dia como variáveis preditoras, sendo considerados os fatores fixos do modelo misto (ver fórmula a seguir). O cômodo foi considerado o fator aleatório da análise, por ter características particulares em cada um dos espaços, as quais poderiam influenciar na umidade observada. Além disso, foi considerado o valor de autocorrelação, já que o experimento foi repetido nos mesmos cômodos ao longo do estudo.R Development Core Team e disponível gratuitamente em http://www.r-project.org. Esse software funciona como uma linguagem, na qual os códigos utilizados para as análises estatísticas são abertos e o usuário pode adaptá-los conforme sua necessidade.Por último, foi realizada a ANOVA para avaliar se havia efeito da distância dos dois tratamentos mais eficientes na qualidade da umidade do ar. Os tratamentos variaram entre 1m e 2m de distância da cama dentro de cada cômodo. As análises foram realizadas em plataforma R versão 3.6.1 (2019), desenvolvida pelo De forma geral, houve variação dos tratamentos, bem como nos períodos do dia. O menor valor de umidade  foi observado na área externa no período da tarde, enquanto o maior valor  foi observado utilizando o umidificador no período da manhã   . Os perAs estimativas do modelo linear misto, ou seja, os valores preditos, indicaram um valor médio de umidade de 59,35%, com erro padrão de 1,44%   . Ao conTodos os períodos do dia se diferenciaram significativamente , sendo o período da tarde aquele que registrou menor média de umidade .A área externa, onde não houve nenhum tipo de tratamento e, possivelmente, o ambiente mais exposto a evapotranspiração, foi a que mais se diferenciou entre os períodos do dia   . Já a vSendo o umidificador e a toalha os tratamentos considerados mais eficientes, a análise de variância não constatou diferença entre os tratamentos   , porém bluetooth , assegurando, assim, alta especificidade da amostra. A umidade do ar é uma variável que influencia de maneira multifatorial no equilíbrio do ecossistema e do organismo de todos os seres vivos, logo sua análise seria mais complexa.  A segurança técnica de diversas medidas assistencialistas é questionada, pois não se sabe ao certo qual medida é realmente mais eficaz para ser exposta em manchetes e revistas voltadas para a população em geral, a fim de informar o que realmente funciona.A escolha dos higrômetros para o estudo experimental se deu por ele ser capaz de fornecer um monitoramento preciso das condições de temperatura e umidade, mantendo na memória os registros das amostras medidas ao longo dos dias, mesmo com distância significativa entre o aparelho e o celular que está conectado com ele via   Novos estudos, que avaliem quais medidas adaptativas são realmente mais eficazes, são necessários, pois a qualidade de vida e a saúde da população são dois aspectos da inter-relação entre cidade e meio ambiente raramente abordados, sendo o ser humano o ponto fundamental – mas esquecido da questão ambiental nas grandes metrópoles. É de extrema importância uma devolutiva eficaz sobre métodos de baixo custo para melhorar as condições de vida da população em épocas de secas.O experimento, além de comprovar que o umidificador exerce sua função de aumentar a umidade do ar com maestria, também demonstrou que a toalha molhada é um tratamento eficiente, podendo levar essas duas devolutivas para a população, visto que não houve diferença significativa entre esses tratamentos, de acordo com a análise de variância. O balde com água não impactou a ponto de tornar a umidade mais adequada para o bem-estar populacional.  - 15 Os recursos ideais seriam o umidificador ou a toalha molhada. No entanto, existem cuidados básicos a serem adotados em relação ao umidificador. Isso se deve ao acúmulo de umidade e poeira que ocorre em ambientes climatizados, e, portanto, a proliferação de micróbios e bactérias é muito maior do que em ambientes abertos. Medidas cabíveis devem ser adotadas em relação ao umidificador, uma vez que não é recomendado deixá-lo ligado no quarto durante toda a noite, pois, sem a iluminação solar, a umidade do ar já se eleva naturalmente, e o excesso de umidade promovida pelo aparelho torna-se um problema, culminando no aparecimento de mofo e bolor. Dentre os principais sintomas de pessoas ocupantes dos ditos ambientes, destacam-se as infecções, as reações alérgicas e irritantes, as dores de cabeça e articulares, a irritação nos olhos, no nariz e na garganta, a tosse seca, as dermatites, a fadiga, a sonolência, a dificuldade de concentração, a sensibilidade a odores, a congestão, a sinusite, a falta de ar, a rinite alérgica, a asma brônquica, entre outros que aumentam mais ainda nessa época de seca. O ideal seria deixá-lo ligado no quarto de 3 a 4 horas seguidas e desligá-lo antes de deitar. Porém, questiona-se como seria possível a pequena elevação na umidade gerada pelo umidificador causar esses problemas, pois a umidade ainda permanece em nível abaixo do recomendado pela OMS. Outra observação em relação ao uso do umidificador seria a necessidade de lavá-lo a cada uso, a fim de evitar que ele seja foco de fungos e outros microrganismos que serão jogados no ar.É notória a evidente limitação de estudo sobre quais medidas assistencialistas são realmente eficazes e seus impactos na vida da população. No entanto, por meio do conhecimento prévio e da somatória proporcionada pela coleta de dados de acordo com os higrômetros utilizados, foi possível analisar o que realmente funciona e é eficaz. Identificam-se ainda duas possíveis medidas assistencialistas de muita eficácia para população, que podem viabilizar um melhor direcionamento para a estratégia de educação ambiental e métodos de prevenção para população em épocas de seca e baixa umidade, diminuindo, portanto, o impacto de doenças.Este trabalho apresentou os resultados de uma avaliação comparativa do desempenho de três medidas assistencialistas para promover a umidificação do ambiente interno de uma edificação residencial em época de baixa umidade entre os meses de julho e agosto de 2019 em Goiânia, Goiás na Região Centro-Oeste. O umidificador e a toalha foram os tratamentos considerados mais eficientes, e houve efeito significativo da distância sobre a umidade. A distância mais eficiente no aumento e/ou manutenção da umidade do ar foi a de 1m. Assim, ambientalmente sustentável e economicamente viável, o uso de toalhas molhadas para uma população de classe média baixa pode induzir a melhores noites de sono, mesmo nas condições de baixa umidade e seca no Centro-Oeste brasileiro.


Most importantly, the results demonstrated that the present silicon nanosheets revealed comparable and even larger NLO responses than graphene nanosheets. Undoubtedly, SiNSs could be strong competitors of graphene for applications in 2D-material-based photonics and optoelectronics.The present work reports on the transient nonlinear optical (NLO) responses of two different types of 2D silicon nanosheets (SiNSs), namely hydride-terminated silicon nanosheets (SiNS–H) and 1-dodecene-functionalized silicon nanosheets (SiNS–dodecene). The main motivation of this study was to extend the knowledge regarding the NLO properties of these Si–based materials, for which very few published studies exist so far. For that purpose, the NLO responses of SiNS–H and SiNS–dodecene were investigated experimentally in the nanosecond regime at 532 and 1064 nm using the Z-scan technique, while the obtained results were compared to those of certain recently studied graphene nanosheets. SiNS–dodecene was found to exhibit the largest third-order susceptibility χ CurreDMs repre2, WSe2) ,16,17,182, WSe2) , hexagon2, WSe2) , and 2D 2, WSe2) ,22.2–sp3 nature of the hexagonal Si lattice [2–sp3 hybridized network of silicene gives an excellent reactive ability, facilitating its chemical functionalization. Therefore, the latter can be used as a powerful strategy to improve the poor stability and widen the zero-bandgap of silicene, factors that otherwise restrict its practical applications, especially in optoelectronics and photonics [In particular, the success of graphene has triggered many researchers to study other graphene-like 2DMs made up of other IV group elements . Among them, the silicon-based counterpart of graphene, namely silicene, has gained great interest as it shares some of the outstanding properties envisaged for graphene, mainly due to the presence of Dirac fermions ,24. In a lattice ,24. It i lattice . In addihotonics ,28.2 (1 1 1) substrates [The main method for producing silicon nanosheets (SiNSs) involves epitaxial growth on various metallic substrates. To date, SiNSs have been successfully grown on Ag (1 1 1), Ir (1 1 1) and ZrBbstrates ,31,32,33bstrates , while iThe strong optical nonlinearity and ultrafast responses are some of the key properties of layered materials, leading to their high potential in optoelectronics and photonics as optical limiters, saturable absorbers, optical modulators, and wavelength converters ,36,37,382). After the deintercalation of calcium cations from the layered structure with aqueous HCl, the crystalline structure of CaSi2 collapsed, leading to the liberation of SiNS–H. The surface of the resulting hydride-terminating silicene was functionalized with 1-dodecene by utilizing hydrosilylation reactions to yield dodecyl–functionalized silicane (SiNS–dodecene). A schematic representation of the synthetic procedure used for SiNSs is presented in The synthesis and surface modification of the silicon nanosheets were performed as previously described in detail elsewhere . In brieSiNS–H and SiNS–dodecene were dispersed in different organic solvents; however, the toluene SiNSs dispersions showed greater uniformity. The prepared SiNSs toluene dispersions had a distinctive yellow color (turned to white or transparent upon oxidation) and were placed in 1-mm-thick quartz cells for the NLO measurements. Due to the strong oxophylicity of the Si atoms, the preparation of the SiNSs dispersions was performed inside a glove bag, while the cells containing the prepared samples were tightly sealed and were kept under argon atmosphere. Since the studied SiNSs precipitates relatively quickly in most of the common organic solvents, in turn forming agglomerates , the sam2 atmosphere until the solvent was completely evaporated and the mass of the sample remained stable. This mass was then weighed using a high-precision scale and the concentration of the sample was determined.To safely determine the concentrations of the dispersions used for the Z-scan measurements, the dispersed mass of the sample was weighed for a given sample volume. In more detail, several dispersions were prepared in vials and then left to rest for 1 day. After this, some of the supernatant was collected and transferred into the cells. For lower concentrations, a measured volume of solvent was added into the 1 mm quartz cells. Then, the dispersions in the cells were monitored through UV-Vis-NIR absorption spectra to ensure that no precipitation occurred, i.e., the concentrations were stable. From these dispersions, a measured volume was collected and the concentration was determined via a TGA Q50 V6.7 Build 203 . In more detail, the dispersion was heated in Nβ and nonlinear refractive index parameter γ′, respectively. The details of the Z-scan technique have been described in detail elsewhere [β > 0) or saturable absorption , respectively. Correspondingly, the presence of a transmission valley-peak or peak-valley configuration in the CA Z-scan indicates self-focusing or self-defocusing behavior, respectively, corresponding to γ′ > 0 or γ′ < 0. If the nonlinear absorption is negligible, the nonlinear refractive index parameter γ′ can be directly deduced for the CA Z-scan, while in the presence of nonlinear absorption the nonlinear refractive index parameter γ′ is deduced from the so-called “divided” Z-scan obtained from the division of the CA Z-scan trace by the corresponding OA one.The determination of the third-order nonlinear optical (NLO) properties of SiNS–H and SiNS–dodecene was performed using the conventional single-beam Z-scan technique . This telsewhere ,44, and β is calculated by fitting the experimental OA Z-scan data points with Equation (1), while the nonlinear refractive index parameter γ′ can be determined by fitting the corresponding “divided” Z-scan with Equation (2).x = z/z0, with z being the sample position and z0 the Rayleigh length; I0 is the on-axis peak irradiance; effL is the sample’s effective length; k is the excitation wavenumber. The nonlinear absorption coefficient The excitation laser sources employed for the Z-scan measurements were the fundamental and SHG outputs at 1064 nm and 532 nm, respectively, of a 4 ns Q-switched Nd–YAG laser system operating at a repetition rate between 1 and 10 Hz. However, during the experiments, the laser was operating at 1 Hz to prevent the manifestation of unwanted thermal or cumulative effects. For the experiments, the laser beam was focused into the sample by means of a 20 cm focal length quartz planoconvex lens. The beam spot radii at the focal plane were measured with a charge-coupled device (CCD) and was found to be 30 μm and 18 μm at 1064 nm and 532 nm, respectively. The laser beam energy was monitored by means of a calibrated joulemeter.Both SiNS samples were characterized by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) spectroscopy, and thermogravimetry analysis (TGA). A detailed description of the characterization results has been given elsewhere . BrieflyIn As mentioned previously, silicene not only possesses a graphene-like structure but also exhibits most of the remarkable properties of graphene, providing an alternative 2D material. Therefore, it is reasonably expected that silicon nanosheets may exhibit comparable NLO properties to those of graphene. As a matter of fact, in a recent study by our group reporting on the ultrafast third-order nonlinearities of SiNS–H and SiNS–dodecene ,47, it w(3)). To the best of our knowledge, the current work is the first experimental attempt to determine the effective NLO response of silicon nanosheets under 4 ns of visible (532 nm) and infrared (1064 nm) laser excitation and to compare the obtained findings with those of graphene nanosheets. For the accurate determination of the third-order susceptibility χ(3) and the related NLO parameters (nonlinear absorption coefficient β and nonlinear refractive index parameter γ′) of both SiNS dispersions and to gain a deeper insight into the physical mechanisms underlying their transient NLO response, several Z-scans of different dispersions with concentrations ranging from 0.05 to 0.4 mg/mL and for a wide range of incident laser intensities  were performed.It is noteworthy at this point to mention that the physical processes and mechanisms underlying the NLO response of an atomic or molecular system are strongly dependent on the laser excitation or pulse duration that interacts with the medium ,49. For z = 0) under both excitation wavelengths, corresponding to a saturable absorption  behavior. The SA behavior of SiNS–dodecene is opposite to the reverse saturable absorption  that can be observed for graphene under similar experimental conditions. This sign alternation of the nonlinear absorption coefficient β reflects the different operational physical processes underlying the NLO response of SiNSs and graphene under ns laser excitation. For example, the RSA behavior of graphene can be described in terms of two-photon absorption (TPA) or excited-state absorption (ESA) [In on (ESA) . On the on (ESA) . InteresFor SiNSs and graphene, electrons from the valence band can be promoted into the conduction band under sufficient laser intensity, while simultaneously empty states  are generated in the valence band a. As forγ′ > 0), similar to the nonlinear refractive response observed for graphene under both excitation wavelengths  of SiNS–H and SiNS–dodecene. The parameters summarized in Then, following the standard procedures used for the analysis of the Z-scan measurements , the valχ(3) of the 1-dodecene-modified SiNSs by factors of 2 and 1.5 for visible and infrared laser excitation, respectively. The possible mechanisms responsible for the enhancement of χ(3) are described below.The obtained experimental findings unambiguously confirm that chemical functionalization via hydrosilylation reaction can be used to tune the NLO response of silicane. H-terminated silicene showed a greatly improved NLO response after appropriate chemical modification, as expressed by the enhanced third-order susceptibility Moreover, it is interesting to note the absence of any measurable NLO response for SiNS–H. However, the functionalization of silicane with 1-dodecene switched on a strong saturable absorption behavior compared to SiNS–H under both excitation wavelengths. This macroscopic on–off-like behavior can be ascribed to the existence of point defects introduced by the localized orbitals, containing an unpaired electron, the so-called “dangling bond” orbitals. More precisely, reports about some Si–H films and amorphous Si–H have shown that the point defects can create mid-gap states and local potential fluctuations that can lead to spatially localized energy states at the edges of valence and conduction bands, the so-called Urbach band tails ,58,59. Sγ′ than crystalline Si [γ′ was ascribed to free carrier refraction, induced by two-step absorption due to the presence of defects states in amorphous Si–H [γ′ has the same sign as the Kerr effect, causing a refractive nonlinear response [−21 m2/W), can be useful and promising for designing all-optical switching devices [Concerning the nonlinear refractive response of both SiNSs, in some other studies it has been reported that amorphous Si–H possesses a larger nonlinear refractive index parameter lline Si ,62. The  devices .It is noteworthy that both silicon nanosheets exhibited ca. 1.5 (for SiNS–H) and 3 (for SiNS–dodecene) times larger NLO response under visible excitation than that of graphene. On the contrary, in the case of infrared excitation, graphene has been reported to exhibit a very similar NLO response to that of SiNS–dodecene and about double the NLO response of SiNS–H. Taking into account the negligible nonlinear absorptive response of SiNS–H, the larger third-order susceptibility of SiNS–H compared to graphene, especially under visible excitation, can be ascribed to the stronger Kerr effect. On the other hand, SiNS–dodecene exhibited an even stronger NLO response than graphene, most probably due to the presence of point defects. The present results indicate that both studied SiNSs can compete effectively with graphene, as they present comparable and even higher NLO responses than graphene, especially under visible excitation conditions. Concequently, SiNSs can eventually substitute graphene, being capable of serving better several photonic and optoelectronic applications.χ(3) among these 2D systems. These findings confirm that a new era of 2D materials such as graphene has already started in terms of advanced nanotechnology devices, since 2D silicon-based materials could eventually replace graphene nanosheets in several optoelectronic and photonic applications.Finally, the NLO properties of the present SiNSs will be compared to those of some graphene derivatives , nitrogen-doped GO (N-GO), boron–doped GO (B-GO) and fluorographene (CF)), which have been recently investigated under similar excitation conditions ,55,56. FSummarizing, the nonlinear absorption and refraction of two different silicon nanosheets, namely SiNS–H and SiNS–dodecene, were studied employing 532 and 1064 nm laser pulses for 4 ns. The present results revealed that the magnitude of the NLO response of silicon nanosheets is comparable and even higher than that of graphene nanosheets, especially under visible excitation. In addition, the functionalization of the hydride-terminated silicon nanosheets with 1-dodecene resulted in an important enhancement of its nonlinear refractive response and a noticeable “switch on” effect of the NLO absorptive response, most probably due to the presence of point defects. The present findings demonstrate the great potential of silicon nanosheets and that they can efficiently compete with graphene being very promising candidates for next-generation photonic and optoelectronic applications.


Trans-activation response DNA-binding protein of 43  kDa (TDP-43) regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Introduction of acetyl-lysine at the identified sites via amber suppression confirmed the results from site-directed mutagenesis. K84-acetylated TDP-43 showed cytoplasmic mislocalization, and the aggregation propensity of K136-acetylated TDP-43 was confirmed. We generated antibodies selective for TDP-43 acetylated at these lysines, and found that sirtuin-1 can potently deacetylate K136-acetylated TDP-43 and reduce its aggregation propensity. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting regulatory mechanisms for TDP-43 pathogenesis. TDP-43 is a nucleic acid binding protein, whose insoluble aggregates are neuropathological hallmarks of specific subsets of patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Post-translational modifications and acetylation of TDP-43 impact its interaction with RNA, its localization in the cells, and are linked to disease. Using antibodies generated against TDP-43 lysine acetylation sites, sirtuin-1 was found to potently deacetylate amber suppressed [acK136]TDP-43 and reduce its aggregation propensity. Thus, distinct lysine acetylations modulate nuclear import, RNA binding as well as phase separation and aggregation of TDP-43, suggesting regulatory mechanisms for TDP-43 pathogenesis. The TDP-43 polypeptide contains two RNA recognition motifs (RRM1 and RRM2), a nuclear localization signal (NLS) and a C-terminal glycine-rich low complexity domain  mRNA11. The C-terminal low complexity domain of TDP-43 promotes liquid-liquid phase separation (LLPS) as well as pathological protein aggregation12. Post-translational modifications (PTMs) can modulate TDP-43 functions in health and disease. Putative pathological PTMs of TDP-43 include C-terminal fragmentations and phosphorylations13, including the widely used marker pS409/41015. In addition, lysine modifications have been reported, including ubiquitinations at various residues16 and sumoylation and acetylation of the RRM118. The putative acetyl-mimic [K145Q]TDP-43 was distributed in a stippled manner in transfected cells, eventually recapitulating pathological phosphorylation and recruitment of ALS-related factors into TDP-43 aggregates19. In addition, [K145Q]TDP-43 had reduced CFTR splicing activity. Thus, lysine modifications may be important for physiology and pathological aggregation of TDP-43. However, the genesis of TDP-43 aggregates remains elusive.The trans-activation response DNA-binding protein of 43 kDa (TDP-43) regulates various RNA-processing stepssee Fig. . The NLS20. In the case of TDP-43, RNA binding prevents its phase separation in vitro and reduces droplet formation in vivo21. Wild-type (wt)TDP-43 is capable of going into liquid droplets but pathological mutants seem to form more resilient droplets. While the C-terminal glycine-rich domain of TDP-43 seems to play a crucial role in the formation of phase-separated droplets, the N-terminal domain may also contribute to the aggregation process24. Thus, the regulation of TDP-43 RNA binding could be pivotal in the pathophysiology of TDP-43. The molecular pathways regulating the disengagement from RNA are only beginning to be described.Research on LLPS is unveiling the molecular processes regulating the arrangement of membrane-less organelles. Heteronuclear ribonucleoprotein (hnRNP) LLPS is amply reported but whether and how this process is regulated in cells is poorly understoodIn the present study, we discovered that acetylation of K84 within the NLS reduced nuclear import whereas acetylation of K136 in the RNA recognition domain impaired TDP-43 RNA binding and splicing capabilities. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. To confirm the results from site-directed mutagenesis, we expanded the genetic code via amber suppression to introduce authentic acetyl-lysine at the identified sites. Indeed, [acK84]TDP-43 showed cytoplasmic mislocalization, and the increased aggregation tendency for [acK136]TDP-43 was confirmed. We generated antibodies selective for TDP-43 acetylated at K84 and K136. Using these tools, we found that the nuclear sirtuin SIRT1 can potently deacetylate [acK136]TDP-43. Moreover, SIRT1 reduced the aggregation propensity of [acK136]TDP-43. Thus, distinct lysine acetylations regulate nuclear import, RNA binding and phase separation of TDP-43 with possible relevance for TDP-43 pathogenesis.16, we became interested in how other PTMs could affect TDP-43 functionality and aggregation. Recently acetylation has been linked with TDP-43 pathology19, and lysine acetylation has the potential to disrupt ubiquitination by competing for the same residue. To explore this hypothesis more in depth, we re-examined the mass spectrometry data16 specifically for acetylated lysines. We found in transfected HEK293E cells that 6xHis-tagged wtTDP-43 was acetylated at lysine residues K79 and K84 around the NLS and K121 and K136 in RRM1 .After investigating the sites of TDP-43 lysine ubiquitinationsRM1 Fig. . We coul16.Each of the identified acetylated lysine residues was mutagenized to either arginine or glutamine to simulate the lack or the presence of acetylation, respectively. We first looked at the subcellular distribution of these N-terminally 6xHis-tagged TDP-43 mutants in transiently transfected HEK293E cells. Immunofluorescence (IF) staining of the 6xHis tag did not show visible deviation from the wtTDP-43 nuclear staining pattern for both acetyl-dead and acetyl-mimic mutations at residues K79 and K121 29 to minimize interference from the endogenous wtTDP-43. To assess protein solubility, RIPA-urea solubility fractionation assays were performed. While most of the 6xHis-tagged wtTDP-43 was RIPA soluble, a larger portion of both K136R and K136Q TDP-43 mutants shifted into the RIPA-insoluble fraction 129. While wtTDP-43 strongly bound to synthetic poly-(UG)12 but not the negative control poly-(UC)12 RNA, the acetyl-mimic [K136Q]TDP-43 showed a reduction in RNA binding 12 RNA retained on the nitrocellulose membrane  and four copies of tRNATAG30. Together the designed AcRS and the tRNATAG will incorporate acK added to the media at the introduced amber stop codon, directly incorporating an acetylated lysine site specifically during the protein translation.As K136 turned out to be a very delicate residue for site-directed mutagenesis, it was important to properly validate the pro-aggregative effects for [acK136]TDP-43. We succeeded to incorporate authentic acK at the apparently regulatory sites in TDP-43 with amber suppression methodology. TDP-43 mutants were generated with a C-terminal 6xHis tag and an amber stop codon (TAG) substituting the codon at position 84 and at position 136, respectively. Each of these plasmids was cotransfected into shCFTR minigene assay in shTDP-43-HEK293E cells. Indeed, [acK136]TDP-43 could only partially rescue CFTR exon 9 splicing TDP-43ΔNLS was uniformly distributed throughout the cytoplasm in most cells. However, there was a fraction of cells developing multiple cytoplasmic aggregates TDP-43ΔC showed a homogeneous nuclear distribution . HEK293E cells were transfected with either wtTDP-43-EGFP or [K136Q]TDP-43-EGFP. Then regions of interest (ROI) with bleached for half a second by 488 nm laser light irradiation and allowed to recover Fig. . The wtTted Fig. . Large TDP-43-EGFP. Transfected HEK293E cells were imaged for several hours and the distribution of TDP-43 was monitored. [K136Q]TDP-43 was recorded going into phase separation, forming droplets and these droplets were seen fusing with one another  1–8, representing class I and class II deacetylases. Each of these HDACs was cotransfected into amber suppressed [acK136]TDP-43 cells. None of the HDAC1-8 effectively reduced TDP-43 K136 acetylation,  as well as other sirtuins with similar level of expression (SIRT3 and SIRT6). When compared to cells without any sirtuin overexpression, SIRT1 and 2 caused a significant reduction in the number of cells with inclusions, even though some level of TDP-43 acetylation was detected 18. Consistent with this study by Maurel et al. (2020) using TDP-43 GFP fusion proteins, we found that 6xHis-tagged [K136R]TDP-43 formed intranuclear inclusions. The similarities in behaviour between K136R and K136Q TDP-43 mutants were puzzling, suggesting that instead of mimicking the presence or lack of acetylation, the mutants were introducing backbone changes in TDP-43. Point mutagenesis is a broadly used approach to study the cellular effects of protein lysine acetylation but in some cases can fail to rescue protein function42. In addition, glutamine and arginine contain delocalized electrons that are critical for the formation of phase-separated entities43. This provides an explanation for the stronger occurrence of phosphorylated aggregates in K136R/Q TDP-43. Amber suppression has its own disadvantages in terms of efficiency and availability of synthetic amino acids, in addition to the possibility of side effects. Nevertheless, transcriptomic analysis of stably amber-suppressed cells did not show upregulation of protein folding stress genes44. Another benefit of this approach is the possibility of removing the modification in vivo via deacetylases  and IF staining: rat anti-[acK84]TDP-43 , rat anti-[acK136]TDP-43 , rabbit pan-acetylated lysine , rabbit mAb mix pan-acetylated lysine , mouse anti-Flag HRP-coupled , mouse anti-GAPDH , rabbit anti-Hsp90 , rat anti-[pS409/410]TDP-43 , rabbit anti-TDP-43 , mouse anti-TDP-43 , mouse anti-hnRNPA1 ; Cell Signalling #5380), mouse anti-tubulin , mouse anti-ubiquitin , mouse anti-YY1 , mouse anti-β-actin ; rabbit anti-mouse HRP-coupled ; goat anti-rabbit HRP-coupled ; donkey anti-rat HRP-coupled  and secondary Alexa Fluor 488- , 555- , 647-  conjugated antibodies for IF produced in goat were from Invitrogen (1:1000).wtTDP-43 was cloned into pCMV 5′6His (Clontech) via SalI/NotI. Acetyl-mimic glutamine and acetyl-dead arginine substitutions were introduced by site-directed mutagenesis and cloned into pCMV 5′6His . The cloning and mutagenesis primers are listed in Supplementary Table 46. K136R and K136Q mutations were introduced via site-directed mutagenesis and cloned into pEGFP c1 via BamHI and HindIII.For the EGFP-tagged constructs, pEGFP c1 wtTDP-43 was used as a templatehttps://www.addgene.org/140008/; https://benchling.com/s/seq-ZKWKXmPrKBdp8jgpWEt5) and pPB_4xPylT_EF1_IPYE_chAcKRS_Puro (E451) (https://benchling.com/s/seq-6fpczrM3tMNE9fZ0P2nk). Constructs lacking the C-terminal part and with mutated NLS were in addition to K136TAG were cloned from previously described plasmids6 and cloned into pCMV 3′6His vector via SalI and Bsd120I. Cloning primers are listed in Supplementary Table For the C-terminally tagged amber suppression constructs, TDP-43 was cloned from the pCMV 5′ 6His construct previously mentioned into a pCMV 3’6His vector via SalI and Bsd120I. The amber stop codons from constructs K84TAG and K136TAG TDP-43 were introduced via 2-step point mutagenesis. For the N-terminally tagged amber suppression constructs, TDP-43 was cloned from the amber suppression C-terminally tagged constructs previously mentioned into the vector E400 via NheI and BamHI. The plasmids E400_pAS1_4x7SKPylT_EF1_In_IRES_Bsd (E400) , SIRT2 Flag , SIRT3 Flag , SIRT4 Flag , SIRT5 Flag , SIRT6 Flag , and SIRT7 Flag . All sirtuin plasmids were characterized by North et al.http://n2t.net/addgene:36829; RRID:Addgene_36829), HDAC3 Flag , HDAC4 Flag , HDAC5 Flag , and HDAC7 . HDAC2 plasmid was described by Reyon et al.48, HDAC3 plasmid was characterized by Emiliani et al.49 and HDAC4, 5, 7 and 8 were characterized by Fischle et al.50, HDAC1 and HDAC6 plasmids were generated by Fiesel et al.29.Plasmids containing HDACs 2–5, 7 and 8 were ordered from Addgene. The plasmids ordered were HDAC2 Flag  cells were generated and characterized previously29. Cells were plated at different concentrations corresponding to the surface of the plate for different experiments. Transfection of plasmids was done 24 h after plating using FuGENE6 (Promega) at a DNA/FuGENE ratio of 1:4.5 according to the manufacturer’s instructions.HEK293E (ATCC) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% foetal bovine serum (FBS), at 37 °C in 5% COl-lysine . Cells were lysed or fixed after being 24 h in the presence of acetylated lysine. When mentioned, Ex527  was dissolved in DMSO and added to the cultured cells 24 h before lysis to a final concentration of 1 or 10 µM.HEK293E cells were plated and cultured in DMEM with 10% FBS and transfected with FuGENE6 (Promega) 24 h later. Twenty four or 48 h after transfection the media was substituted with DMEM with 10%FBS and 5 mM Nε-Acetyl-TDP-43 HEK293E cell pellets from confluent 10 cm plates were washed twice in ice-cold PBS and then resuspended in 500 µl of hypotonic buffer A  and incubated on ice for 5 min. Cell membranes were ruptured with 20 strokes in an ice-cold Dounce homogenisator and resulting samples were centrifuged at 500 × g for 5 min at 4 °C. Supernatants (cytosolic fraction) were mixed with 5× RIPA buffer, incubated for 10 min on ice and cleared by spinning the samples at 5000 × g for 15 min at 4 °C. Pellets containing nuclei and residual cytoplasmic proteins were washed twice with hypotonic buffer A. After resuspension in 500 µl S1 buffer (250 mM sucrose and MgCl2), samples were layered carefully over 500 µl S3 buffer (880 mM sucrose and MgCl2) and centrifuged at 3000 × g, for 10 min at 4 °C. The supernatant was discarded, and the remaining pellet was lysed with urea buffer. DNA was sheared by passing lysates 30 times through a 23-gauge needle. The cleared supernatants in loading buffer were analyzed via Western blotting.For the nuclear-cytoplasmic fractionation shown in Supplementary Fig. For the nuclear-cytoplasmic fractionations of HEK293E cells shown in Supplementary Fig. 2PO4, 8 M urea). For the sequential extraction cells were first lysed with RIPA buffer  with proteinase inhibitor cocktail (Roche). Samples were centrifuged at 5000 × g for 15 min, leaving the RIPA-soluble proteins in the supernatant. The remaining pellet was then lysed in urea buffer, and insoluble material was removed with a centrifugation step at 5000 × g for 15 min. DNA was sheared with a 23-gauge needle. Protein concentration in lysates in urea buffer was quantified with a Bradford Protein assay kit (Biorad) and lysates in RIPA buffer with BCA protein assay kit (Pierce). 3x Laemmli loading buffer with 100 mM DTT was added to the samples and afterwards they were boiled at 95° for 5 min. The denatured samples were then subjected to western blot analysis. Denatured samples were loaded and run in polyacrylamide gels. The gels were then blotted using the wet-transfer Trans-Blot® Cell system from Bio-Rad. Proteins were transferred to Hybond-P polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% non-fat milk/TBS-T for an hour at room temperature (RT). Primary antibody incubation took place overnight at 4 °C. The membranes were washed and incubated with HRP-coupled secondary antibody for 1–2 h at room temperature. Membranes were washed and proteins were detected with Immobilon Western chemiluminescent HRP substrate (Millipore) using the ChemiDoc XRS+ Imaging System.Cells were collected with a cell scraper and lysed in urea lysis buffer  with 2 ovalbumin-coupled synthetic peptides at the same time: one with amino acids corresponding to residues 79-89 of TDP-43, with K84 acetylated (C-KDNKR(Ac)KMDETD); and the second one corresponding to residues 131-141, with K136 acetylated (C-LMVQV(Ac)KKDLKT). Animals were sacrificed and splenocytes were fused with mouse myeloma cells. Hybridoma supernatants were tested for binding to acetylated and non-acetylated peptides by ELISA. Those supernatants that were positive for the acetylated peptides and negative for the non-acetylated peptides were further validated on lysates of cells expressing either wt or amber-suppressed TDP-43 by Western blot analysis (dilution 1:10) and IF staining (dilution 1:2). Hybridoma clones TDACA 9G9 (IgG2c/κ) against [acK84]TDP-43, TDACB 23B3 (IgG1/κ)  to coverslips in a 24-well plate coated with poly-D-lysine (PDL) and collagen. Cells then grew for 24–72 h before fixation with 4% PFA/PBS for 20 min at RT depending on the experiment. Permeabilization of the cell membrane was done with 1% Triton-X-100/PBS for 5 min at RT. Blocking was done with 10% Normal Goat Serum in PBS, for 1 h at RT. The primary antibody incubations were done at the already mentioned concentrations, in 1%BSA/PBS for 2 h at RT. The secondary antibody incubation was done in 1% BSA/PBS for 1 h. Mounting was done using 40 µl of Dako mounting media per slide, following a 4° incubation overnight. Immunofluorescence images were acquired using a Zeiss fluorescence microscope with Apotome attachment (Axio imager z1 stand).HEK293E cells were plated on glass bottom chambered sides  coated with PDL and collagen and transfected with C-terminally EGFP-tagged wt or K136Q TDP-43 after 24 h. Imaging and photobleaching was done with a Zeiss LSM510 META confocal microscope, using a x63/1.4 oil objective. ROIs were bleached for 1 s using a 405 nm and a 488 nm laser at maximum power output. Cells were continuously imaged for 3 min after bleaching. Reference ROIs were measured from unbleached cells to correct for photobleaching due to fluorescence imaging.2. Imaging was done with an Axio observer Z1.HEK293E cells were plated on glass-bottom chambered slides coated with PDL. They were transfected with C-terminally EGFP-tagged wt or K136Q TDP-43 after 24 h. While imaging cells were kept in a temperature-controlled chamber at 37° with 5% CO12-3′ and 5′-(UC)12-3′ were ordered from Sigma. The oligonucleotides were biotinylated using the Pierce RNA 3′ End Desthiobiotinylation kit (Cat no 20163) with small variations. For each 1nmol of biotinylated cytidine bisphosphate 100 pmol of RNA were added to the biotinylation reaction. Biotinylated RNA oligos were purified using chloroform:isoamyl alcohol. For the RNA-protein pulldown the Pierce Magnetic RNA-Protein Pulldown kit (Cat no 20164) was used with small variations. Cells were lysed in NP-40 buffer  and 400 µg of protein was used for each RNA-protein binding reaction. After 1 h incubation proteins were eluted in 25 µl Laemmli buffer.High-performance liquid chromatography purified RNA oligonucleotides 5′-(UG)TDP-43 cells were plated in 10 cm plates. After 24 h, cells were transfected with the respective plasmids. After 48 h, cells were lysed in Native NiNTA lysis buffer  and protein concentration was measured via Bradford assay (Bio-Rad). 500-800 µg of protein were incubated with NiNTA beads at 4 °C overnight. Beads were washed with Native NiNTA lysis buffer with 20 mM imidazole. After washing, proteins were eluted in Binding buffer  containing 300 mM imidazole, which was then quantified measuring the absorbance at 280 nm using a Nanodrop 2000 (Thermo Fisher Scientific). The purified protein was incubated at various concentrations with 2 nM of biotinylated poly-(UG)12 RNA for 30 min at room temperature. The resulting complex was run through a Bio-Dot® SF Microfiltration apparatus  loaded with a nitrocellulose and a positively charged nylon membrane. Membranes were then cross-linked at 120 J/m2 and then labelled RNA was detected using the Chemiluminescent Nucleic Acid Detection module kit  according to the manufacturer’s instructions. In addition, for quantification purposes pulldown fractions were analyzed via SDS-PAGE electrophoresis and further Western blot. Band intensity corresponding to wtTDP-43 and K136R TDP-43 was quantified using ImageJ 1.52a and the resulting data was used to normalized the Dot Blot densitometry.HEK293E shCells were plated in 10 cm plates for 24 h before transfection. Seventy-two hours after transfection cells were lysed in urea lysis buffer containing 10 mM imidazole. DNA was sheared with a 23-gauge needle. After quantifying the amount of protein in the lysates with a Bradford Protein assay kit (Biorad), 500–800 µg of protein were incubated with NiNTA agarose beads (Qiagen) at 4 °C overnight. Beads were washed with Urea wash buffer  containing 20 mM imidazole. Afterwards proteins were eluted in 3x Laemmli buffer at 95° for 10 min. Eluted proteins were analyzed in Western blot.TDP-43 cells were plated in 6-well plates and after 24 h were double-transfected with the corresponding TDP-43 construct and a CFTR minigene construct11 at a ratio of 2:1. After 72 h RNA was isolated using the RNeasy Mini Kit (Qiagen) following the indications from the provider. From the isolated RNA cDNA was obtained using the Transcriptor High Fidelity cDNA Synthesis Kit (Sigma) following the protocol of the provider. The cDNA of interest was then amplified via a standard PCR and the product was run in a 2% agarose gel. DNA was visualized using Midori Green from Biozym and the ChemiDoc XRS + Imaging System.HEK293E shP values below 0.01 were considered significant. Unpaired t-tests were performed to compare two variables and assess significance. Chi-squared test was used to assess significance in the IF experiments. n values detailed in each figure legend. Graphical representations of TDP-43 mutants and acetylated forms was made with Pymol (version 2.4.0). Post translational modifications were modulated using the PyTMs plugin by Warnecke et al.51.Cellprofiler 4.1.3 was used for quantification of IF pictures. Statistical significance was calculated using Microsoft Excel 2016 and Prism 9.3.0 (Graphpad). Western blot images were captured with ImageLab 5.0. Densitometry of electrophoresis gels was performed using Imagej 1.52a (NIH). Further information on research design is available in the Supplementary InformationPeer Review FileSupplementary video 1Supplementary video 2Supplementary video 3Reporting Summary


While attention has consistently been shown to be biased toward threatening objects in experimental settings, our understanding of how attention is modulated when the observer is in an anxious or aroused state and how this ultimately affects behavior is limited. In real-world environments, automobile drivers can sometimes carry negative perceptions toward bicyclists that share the road. It is unclear whether bicyclist encounters on a roadway lead to physiological changes and attentional biases that ultimately influence driving behavior. Here, we examined whether participants in a high-fidelity driving simulator exhibited an arousal response in the presence of a bicyclist and how this modulated eye movements and driving behavior. We hypothesized that bicyclists would evoke a robust arousal and orienting response, the strength of which would be associated with safer driving behavior. The results revealed that encountering a bicyclist evoked negative arousal by both self-report and physiological measures. Physiological and eye-tracking measures were themselves unrelated, however, being independently associated with safer driving behavior. Our findings offer a real-world demonstration of how arousal and attentional prioritization can lead to adaptive behavior. The influence of negative arousal on attentional biases and behavior has been consistently observed in laboratory tasks, but the functional consequences of such biases in real-world situations are largely unexplored. Drivers often view bicyclists as unpredictable and carry negative perceptions toward bicyclists, with potential consequences for driving behavior, offering a real-world context within which to examine this issue. In this study, using a high-fidelity driving stimulator, we examined the relationship between self-reported anxiety and arousal evoked by bicyclists, visual attention to bicyclists, and behaviors related to safe driving. Physiological and eye-tracking measures suggested that negative arousal and attentional bias toward bicyclists are independently associated with safer driving behaviors and can be functionally adaptive.The world is filled with vast amounts of information in a dynamically changing environment. Attention is the cognitive process that selectively filters incoming sensory information to determine which stimuli are ultimately represented in the brain . First, we evaluated the degree to which bicyclists evoked arousal by measuring changes in physiological responses when the bicyclist appeared in the driver’s field of view, and the degree to which the driver’s attention was biased toward the bicyclist by measuring eye movements. If the bicyclist does evoke an arousal response, it is unclear whether the magnitude of this response would be associated with closer encounters with bicyclists and more inconsistent control of the vehicle or instead facilitate greater distance between the driver and bicyclist and more consistent lane position, which are related to bicyclist safety. Likewise, greater attention to bicyclists could benefit driving performance (more careful tracking and avoidance) or hinder it (insufficient attention to lane position and other roadway events and/or a tendency to drift toward the point of fixation when looking at the bicyclist). Although measured physiological arousal is necessarily ambiguous with respect to valence, we interpret bicyclist-evoked arousal in our experiment as negative given the prior literature concerning bicyclist–driver interactions  were recruited from the Texas A&M University community. All participants were English-speaking, reported normal or corrected-to-normal visual acuity and normal color vision, held a valid driver’s license, reported driving experience of at least 1.5 years, and were not on any medications that may have affected the operation of a moving vehicle. All procedures were approved by the Texas A&M Institutional Review Board (IRB2018-0659D) and were conducted in accordance with the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from each participant. Participants received monetary compensation of $10/h. No participants withdrew early from the study due to simulator sickness, and all participants completed the entire experiment.One hundred and five participants  whose ages ranged from 18 to 54 years , and a speaker system to provide ambient roadway noise . The sensor was connected to a chest strap that was worn underneath the subject’s clothing to maintain skin contact. The BioHarness 3.0 can detect heart rate in the range of 25–240 bpm and breathing rate in the range of 4–70 bpm. In addition, EDA was measured using the Shimmer3 GSR sensor  from the palm. The Shimmer3 sensor can measure EDA in the range of 10–4700 kΩ.Eye movements were captured in the experimental drives using a 60 Hz single-camera FOVIO system . The FOVIO system was interfaced with the Eyeworks Data Record software , and movie files of a moving dot (denoting where the participant was looking) were saved using a script from EyeWorks Design (Eyetracking Inc.). Participants were calibrated to the FOVIO system using a four-point calibration screen and were instructed to look at the exterior edges of the panoramic display while maintaining a directly forward field of view.After completing the consent forms and demographic surveys, participants were connected to the EDA and BioHarness monitors. The participants were then seated on the driving simulator and monitored for stable connectivity with live readings from both physiological monitors. Lastly, participants were calibrated to the FOVIO eye-tracking camera. All participants completed three virtual drives: one practice and two experimental drives. The goals of the practice drive were to familiarize the participants with the simulator controls and screen participants who experienced severe simulator sickness. The two experiment drives differed in the posted speed limit: 40 mph (64.37 kph) in the first drive and 25 mph (40.23 kph) in the second drive. Before the start of each drive, the experimenter told the participant the speed limit of each drive, and the virtual world contained speed limit signs before and after every programmed vehicle–bicyclist interaction (described below). Since interactions were triggered based on distance from the bicyclist, the experimenter verbally instructed the participant to monitor their speed if they exceeded the speed limit. The total length of the experiment took approximately 1 h.The simulated driving environment and scenarios are described in detail in Goddard et al. . In brieFollowing the two experiment drives, participants were disconnected from the EDA and BioHarness sensors. Lastly, participants were relocated to a separate room where completed a short survey before completing the experiment.Participants completed a self-report survey probing explicit attitudes toward bicyclists, which was previously described in Goddard et al. . Here, wAs a part of a different study focused on relating driver attitudes to driver–bicyclist interactions, participants completed an implicit association test (IAT), which was previously developed to assess subconscious attitudes concerning drivers and bicyclists Goddard, . We did The movie files exported from the eye-tracking software were used to quantify saccades made to, and the fixation duration on, three categories of interest: the road and other vehicles, the bicyclist, and the side/rear-view mirrors . Two different individuals manually coded the videos by counting the saccades to and the fixation duration on (using the elapsed time in the movie files) these three categories of objects during each interaction. We defined the start of each interaction as when the bicyclist first becomes visible on-screen and the end of each interaction as after the driver passes the bicyclist and it is no longer in the participant’s field of view. For the overtake, right turn, and left turn events, the bicyclist was defined as visible when the participant was driving toward the bicyclist and was 225 m away, while the end of the event was defined as when the driver had passed the bicyclist and was 25 m away. For the intersection event, the bicyclist became visible to the participant when the driver was 70 m away from the bicycle and the end of the event was defined as when the bicyclist had gone through the intersection and was 15 m away from the driver.rs > 0.71 .To verify the consistency of the data produced by the two coders, we calculated the inter-rater reliability by correlating their scores for each measure. The raw number of saccades to and fixation duration on each measure was z-scored for each coder. In addition, to account for human error, a third individual acted as an “arbitrator” when there was substantive disagreement among the coders. We calculated the difference in z-scores between the two coders for each measure and determined that the third individual would code measures that differed by more than 2 z-score units. For these instances, the two data points that were closest in proximity  were chosen as the final data points. The inter-rater reliability between the two coders for all measures was To account for individual differences in overall eye movements made between participants , the saccades to and fixation duration on the bicyclist were analyzed as a proportion of all valid gaze data, as a measure of attentional bias toward the bicyclist. Lastly, we calculated the time to fixate the bicyclist on the first overtake interaction during the first experimental drive as a proxy measure for orienting time to the bicyclist when it is first introduced to the driver .For all physiological measures , we focused on two time periods of interest: baseline and when visually encountering the bicyclist. Baseline time periods were defined as the 12 s time period of free driving in the simulator world before seeing the bicyclist. The “encountering bicyclist” time period was defined as the 12 s time period after the bicyclist became visible. Given the inherent variability in the temporal unfolding of the driver–bicyclist interaction and potential variability in the resulting time course of physiological responses, we established a standardized time window of 12 s a priori to allow for ample time for capturing physiological changes and to not overlap with other events in the drive. In addition, we focused analyses on the peak response to better account for variability in the total duration of the bicyclist interaction and individual differences in time to notice the bicyclist. All EDA responses were preprocessed using the AcqKnowledge 5.0 software . To analyze event-related skin conductance responses (ER-SCR) elicited when seeing the bicyclist, phasic EDA responses were derived by delineating the presence of SCRs within the tonic signal. All preprocessing steps used the default AcqKnowledge 5.0 software parameters and guidelines recommended in Braithwaite and Watson .overall pattern of relationships among our variables and whether that pattern is more consistent with our hypothesized interrelationships among variables than would be expected by chance, providing a wholistic picture that avoids the multiple comparisons problem. This analysis accounts for both the direction and magnitude of individual correlations, and whether the sum total of the correspondence between these correlations and the hypothesized relationships is greater than would be expected under the null hypotheses that our variables were not related to each other.We calculated a correlation matrix consisting of the physiological, eye-tracking, driving, and self-report measures and conducted a randomization test to quantify the evidence for or against our aforementioned hypothesis linking arousal (and self-reported emotion), eye movements, and driving behavior. For the randomization test, correlations that were predicted to be negative were sign-flipped such that the strength and direction of each correlation corresponded to the strength and direction of evidence for or against our hypothesis. Then, the sign of each correlation was randomly flipped through 100,000 iterations, and a distribution of the sum of the correlation coefficients was computed, against which the observed sum of the actual correlations was compared. Such a test allows assessment of the We included all three of our physiological measures , all three of our eye-tracking measures , all driving measures related to safe driving behavior , and self-report measures that could influence perceiving the bicyclist as a threat and self-evaluated driving skill . When including the distance from bicyclist measure in our correlation matrix, we separated the measure into the overtake interaction and the average distance over the other three interactions  because the overtaking maneuver is unique with respect to the other types of maneuvers concerning intersections, particularly in that it necessitates a close encounter . Thus, driving data were analyzed from a total of one hundred and one participants. To account for movement artifacts in the physiological data due to the effect of poor fitting to the sensors and changes in posture during the experimental drives, we removed data from epochs that featured at least one value outside the valid range of measurement from both physiological sensors. Thus, heart rate and breathing rate data were analyzed from a total of 92 participants and EDA was analyzed from a total of 88 participants. Lastly, eye-movement data from six participants were unable to be collected, due to major movements of the head that the tracking software could not tolerate that resulted in an inability to maintain stable eye position. Thus, eye-movement data from a total of 95 participants were acquired and analyzed. For each individual correlation computed, all retained data points were used, with different measures having a slightly different number of data points.M = 78.7, SE = 0.52; encounter bicyclist: M = 79.7, SE = 0.52), t(91) = 4.08, p < 0.001, dz = 0.46, and EDA , t(87) = 4.12, p < 0.001, dz = 0.44, when the bicyclist appeared in their field of view. In contrast, participants showed significant decreases in peak breathing rate when encountering the bicyclist , t(91) = 3.09, p = 0.003, dz = 0.29, potentially to some degree holding their breath during the interaction.We first evaluated whether the bicyclist evoked negative arousal by measuring physiological changes upon viewing the bicyclist compared to baseline periods of the task Fig. . Particip = 0.005); that is, the hypothesized relationship among our dependent measures was collectively stronger than would be expected by chance.Next, using the randomization test see , we examGiven that the collection of measures reliably supports our hypothesis, we further explored the individual relationships between measures. That is, we broke down individual correlations with respect to the aspect of our hypothesis that they speak to. This is tantamount to performing post hoc contrasts to probe a significant main effect in an analysis of variance.  and self-reported driver skill . No other pertinent correlations were significant. Given these individual relationships and the overall pattern evident from our randomization test, our data are consistent with the idea that greater bicyclist-evoked arousal is associated with safer driving.Consistent with our hypothesis, heart rate was correlated with distance between the driver and the bicyclist during the overtake maneuver , and also with the decision to pass in front of the bicyclist . No other pertinent correlations were significant. Given these individual relationships and the overall pattern evident from our randomization test, our data are consistent with the idea that greater attention to the bicyclist is associated with safer driving.Consistent with our hypothesis, proportion of saccades toward the bicyclist and fixation duration was correlated with distance from the bicyclist during the non-overtake interactions . Counter to our hypothesis, self-reported nervousness near bicyclists was positively correlated with variability (standard deviation) in lane position  and self-reported driver skill . These contradicting relationships offer no clear conclusions on our hypotheses.Consistent with our hypotheses, self-reported hold-up pressure was correlated with both the proportion of saccades toward the bicyclist and fixation duration on the bicyclist , predictable given that they are non-independent measures. However, we also investigated whether increased attentional biases as indicated by saccades and fixation duration were associated with faster first fixation time on the bicyclist. Time to first fixate the bicyclist was correlated with both the proportion of saccades toward the bicyclist and the fixation duration on the bicyclist , demonstrating a correspondence between initial orienting and sustained attentional priority. Several strong relationships were identified among driving measures, particularly those that were non-independent . Comparing self-report measures of anxiety concerning bicyclists and physiological indicators of arousal, self-reported nervousness near bicyclists was correlated with EDA . In general, there was no clear relationship between the self-report measures evaluating real-life bicyclist interactions and the magnitude of physiological arousal evoked by bicyclists in our simulated driving task. We include these significant relationships within categories of measures in Fig. We investigated whether variables within a specific category had significant relationships to provide more context to our group measures. We first evaluated whether our physiological measures were in-sync with each other. However, no significant correlations were identified, suggesting that these three measures capture different components of arousal in our task. Within the eye-movement measures, proportion of saccades toward the bicyclist was highly correlated with proportion of fixation duration on the bicyclist . It is impossible to know to what degree such attention was strategic or involuntary. A more purely involuntary measure of attention to the bicyclist might be more closely linked to physiological arousal and can only be assessed in an experiment in which a suitable comparison stimulus can be used as a basis for comparison to the bicyclist. Such an experiment would likely need to include portions of the drive in which bicyclists are in view but legitimately irrelevant to the task, precluding a measure of safe driving during driver–bicyclist interaction (which necessarily renders the bicyclist task-relevant) which was the focus of the present study.Overtaking is a complex set of behaviors with multiple phases, and work remains to be done to truly understand just what are safe interaction behaviors and what are the best proxies for safe interactions. Caution should be taken in interpreting whether the observed variation in driving behavior that included maintaining a greater distance from the bicyclist and being less likely to pass ahead of a bicyclist is in fact a reflection of In the present study, we measured heart rate, breathing rate, and EDA as indicators of bicyclist-evoked arousal. Prior research involving this sample of participants indicated self-reported feelings of nervous and being startled by a bicyclist’s presence  are each associated with safer driving behavior, consistent with the facilitation of goal-directed behavior. Counter to our hypothesis, however, attention and arousal were themselves unrelated; it does not seem to be the case that physiological arousal was responsible for variation in attention in our task. We found evidence that self-reported anxiety concerning driver–bicyclist interactions encountered in everyday life was positively correlated with attention to bicyclists in our task, although greater self-reported anxiety was not itself associated with safer driving. Interestingly, the speed of initial orienting was predictive of the sustained attention measures (fixation duration and number of saccades to the bicyclist) which were themselves predictive of safe driving; this highlights the potential value of quickly recognizing and attending to bicyclists.One implication of our findings is that efforts to reduce driver anxiety concerning bicyclists should be taken with caution; although at certain levels, bicyclist-evoked anxiety and arousal may become maladaptive (e.g., Etkin & Schatzberg,


The use of genetically modified (GM) mesenchymal stromal cells (MSCs) and preconditioned MSCs (pMSCs) may provide further opportunities to improve the outcome of core decompression (CD) for the treatment of early-stage osteonecrosis of the femoral head (ONFH). GM interleukin-4 (IL4) over-expressing MSCs (IL4-MSCs), platelet-derived growth factor (PDGF)-BB over-expressing MSCs (PDGF-BB-MSCs), and IL4-PDGF-BB co-over-expressing MSCs (IL4-PDGF-BB-MSCs) and their respective pMSCs were used in this in vitro study and compared with respect to cell proliferation and osteogenic differentiation. IL4-MSCs, PDGF-BB-MSCs, IL4-PDGF-BB-MSCs, and each pMSC treatment significantly increased cell proliferation compared to the MSC group alone. The percentage of Alizarin red-stained area in the IL4-MSC and IL4-pMSC groups was significantly lower than in the MSC group. However, the percentage of Alizarin red-stained area in the PDGF-BB-MSC group was significantly higher than in the MSC and PDGF-BB-pMSC groups. The percentage of Alizarin red-stained area in the IL4-PDGF-BB-pMSC was significantly higher than in the IL4-PDGF-BB-MSC group. There were no significant differences in the percentage of Alizarin red-stained area between the MSC and IL4-PDGF-BB-pMSC groups. The use of PDGF-BB-MSCs or IL4-PDGF-BB-pMSCs increased cell proliferation. Furthermore, PDGF-BB-MSCs promoted osteogenic differentiation. The addition of GM MSCs may provide a useful supplementary cell-based therapy to CD for treatment of ONFH. Osteonecrosis of the femoral head (ONFH) is a debilitating disease in which the femoral head may collapse, subsequently causing osteoarthritis of the hip . This diThe role of mesenchymal stromal cells (MSCs) in the pathogenesis of osteonecrosis has only recently received attention; it has been reported that ONFH inhibits the differentiation of osteoblasts from mesenchymal progenitor cells . The capThe MSC secretome, which contains a variety of molecules such as cytokines and growth factors, has attracted much attention in recent years because the secretome has been reported to improve tissue repair . The MSCBased on the interaction of MSCs with cells of the immune system, regeneration occurs in damaged mesenchymal tissues, including bone. MSCs and other progenitors are ultimately involved in the regeneration of bone and other tissues, and macrophages have been shown to play an essential role in modulating the mobilization and differentiation of these cells. MSCs and macrophages coordinate their functions with each other to facilitate the physiological transition from acute inflammation to tissue regeneration . We consBased on these findings, we investigated the efficacy of CD in combination with MSCs, preconditioned MSCs (pMSCs), and IL4-MSCs with injectable hydrogels as a cell-based therapy for the treatment of steroid-associated ONFH in rabbits. Although the injection of pMSCs into the bone tunnel significantly enhanced osteogenesis of the femoral head in vivo compared to MSCs, there was no significant difference in the number of empty lacunae, a histological marker of bone cell death. The injection of IL4-MSCs significantly decreased the number of empty lacunae compared to MSCs but resulted in less osteogenesis .Platelet-derived growth factor (PDGF)-BB promotes both angiogenesis and osteogenesis . PDGF-BBThis in vitro study investigated the efficacy of genetically modified over-expressing MSCs, i.e., IL4-MSCs, PDGF-BB-MSCs, and IL4-PDGF-BB-MSCs and pMSCs for use as adjunctive cell-based therapy for CD.We used rabbit bone marrow-derived MSCs purchased from Cyagen Biosciences  in all experiments. MSCs were expanded with MSC growth medium  until passage 5.The vectors containing elements of rabbit IL-4 (rIL-4) or human PDGF-BB (hPDGF-BB) were generated according to our previous report . BrieflyThe lentivirus vector pCDH-CMV-copGFP for control, the lentiviral vector pCDH-CMV-rIL4-copGFP for rabbit IL4 secretion, or the lentiviral vector pCDH-PGK-hPDGF-BB-copGFP for human PDGF-BB secretion were produced in human embryonic kidney 293 T cells  by co-transfecting with the control, rabbit IL4 over-expressing, or human PDGF-BB over-expressing transfer plasmid, packaged plasmid (psPAX2), and enveloped plasmid (pMD2G VSVG) using a calcium phosphate transfection kit  with 25 mmol/L chloroquine. The virus was mixed in a serum-free medium with 6 μg/mL of polybrene  and incubated at the multiplicity of infection (MOI) of 80 for 6 h to infect into rabbit MSCs. The infected cells were confirmed to be GFP-positive cells by fluorescence microscope on day 4 post-infection. These established MSCs were used from passages 8–10. IL4-MSCs or PDGF-BB-MSCs were also used after testing the IL4 or PDGF-BB secreting level with enzyme-linked immunosorbent assay (ELISA).The lentiviral vector pCDH-PGK-hPDGF-BB-mRFP for human PDGF-BB secretion was produced in human embryonic kidney 293 T cells  by co-transfecting with the human PDGF-BB over-expressing transfer plasmid, packaged plasmid (psPAX2), and enveloped plasmid (pMD2G VSVG) using a calcium phosphate transfection kit  with 25 mmol/L chloroquine. The virus was mixed in a serum-free medium with 6 μg/mL of polybrene  and incubated at the MOI of 80 for 6 h to infect into previously established rabbit IL4-MSCs. The infected cells were confirmed to be GFP- or RFP-positive cells by fluorescence microscope on day 4 post-infection. IL4-PDGF-BB-MSCs from passages 8–10 were used after testing the IL4 and PDGF-BB secreting levels with ELISA. Thus, four kinds of MSCs were established in this study .GFP-MSCs, IL4-MSCs, PDGF-BB-MSCs, or IL4-PDGF-BB-MSCs were seeded into T75 flasks and cultured in an MSC growth medium for 1 day.In the GFP-MSC, IL4-MSC, PDGF-BB-MSC, and IL4-PDGF-BB-MSC groups, the medium was replaced with a fresh MSC growth medium, and the cells were cultured for 3 more days. The cells from these four groups were washed three times with Dulbecco’s phosphate-buffered saline (DPBS), then the cells were trypsinized and seeded for each experiment.Preconditioning must always be performed immediately before use. To precondition the GFP-MSC, IL4-MSC, PDGF-BB-MSC, and IL4-PDGF-BB-MSC groups, the cells 1 day after seeding were cultured in the MSC pre-conditioning medium (MSC growth medium containing TNFα (20 ng/mL) and LPS (20 μg/mL)) for 3 days. The resulting GFP-pMSCs, IL4-pMSCs, PDGF-BB-pMSCs, and IL4-PDGF-BB-pMSCs were washed three times with DPBS, then the cells were trypsinized and seeded for each experiment. 4 cells/well) using the MSC growth medium. The cells were used on days 1, 3, 5, and 7. For the day 7 cells, the medium was replaced with fresh medium on day 3. Cell proliferation was analyzed using the Quant-iT PicoGreen dsDNA Assay Kit . After washing the cells seeded in a 6-well plate three times with DPBS, 1 mL of DNase/RNase-free water was added to each well. The freeze/thaw cycle was repeated three times to release cellular DNA, which was stained with PicoGreen according to the manufacturer’s protocol. The amount of dsDNA was calculated by measuring the fluorescence at the wavelength of 480/520 nm using a plate reader .The cells from all eight groups  were washed three times with DPBS, and then the cells were trypsinized and seeded in a 6-well plate (2 × 104 cells/well) using the MSC growth medium. The supernatants on day 3 of the IL4-MSC, IL4-pMSC, PDGF-BB-MSC, PDGF-BB-pMSC, IL4-PDGF-BB-MSC, and IL4-PDGF-BB-pMSC groups were used. The expression levels of IL4 and PDGF-BB on day 3 were measured using the rabbit IL4 ELISA Kit  and the human PDGF-BB ELISA kit , respectively, according to the manufacturer’s protocols. The optical absorbance at 450 nm was measured using a plate reader . The IL4 expression level-dsDNA ratio or PDGF-BB expression level-dsDNA ratio were also calculated using the absolute value of IL4 or PDGF-BB expressions divided by the amount of dsDNA, respectively.The cells from all eight groups  were washed three times with DPBS, and then the cells were trypsinized and seeded in a 6-well plate  staining and with 2 × 104 cells/well for Alizarin red-staining. Cells were cultured in the osteogenic medium . The medium was changed to a fresh medium twice a week.The cells from all eight groups were washed three times with DPBS, and then the cells were trypsinized and seeded in 24-well plates with 4 × 10ALP staining was performed 2 weeks after seeding. Cells were fixed in 4% paraformaldehyde (PFA) for 30 min, washed three times with DPBS, and stained with ALP substrate solution  at 37 °C overnight. After washing three times with DPBS and drying, photographs of the entire wells were taken using a BZ-X 810 digital microscope . Nine 20x micrographs were taken and then stitched together to create each photograph of the entire well. The percentage of ALP-stained area per well was analyzed using ImageJ 1.53e (NIH).Alizarin red-staining was performed 4 weeks after seeding. Cells were fixed in 2% PFA for 10 min, washed three times with DPBS, and stained with 40 mM pH 4.1–4.3 Alizarin Red S  for 15 min. After washing three times with DPBS, photographs of the entire wells were taken using a BZ-X 810 digital microscope . Nine 20x micrographs were taken and then stitched together to create each photograph of the entire well. The percentage of Alizarin red-stained area per well was analyzed using ImageJ 1.53e (NIH).p < 0.05.All data are shown as mean ± standard error. All the in vitro experiments were performed in triplicate. A one-way ANOVA with post-hoc Tukey test was performed with IBM SPSS statistical software . Statistical significance was set at The fold changes in the amount of dsDNA on days 3, 5, and 7 compared to day 1 are shown in The IL4 expression level on day 3 in the IL4-MSC group was significantly higher than the IL4-pMSC group. The PDGF-BB expression level on day 3 in the PDGF-BB-MSC group was higher than the PDGF-BB-pMSC group. On the other hand, the IL4 expression level on day 3 in the IL4-PDGF-BB-pMSC group was higher than the IL4-PDGF-BB-MSC group. Furthermore, the PDGF-BB expression level on day 3 in the IL4-PDGF-BB-pMSC group was significantly higher than the IL4-PDGF-BB-MSC group A. The same tendency as each absolute value of IL4 and PDGF-BB expression levels was observed in the IL4 and PDGF-BB expression level-dsDNA ratio among IL4-MSC, PDGF-BB-MSC, and IL4-PDGF-BB-MSC groups B.The percentage of stained area in the IL4-MSC and IL4-pMSC groups was significantly lower than the GFP-MSC group. However, the percentage of stained area in the PDGF-BB-MSC and PDGF-BB-pMSC groups was significantly higher than the GFP-MSC group. There were no significant differences in the percentage of stained area among the GFP-MSC, IL4-PDGF-BB-MSC, and IL4-PDGF-BB-pMSC groups. The percentage of stained area in the PDGF-BB-MSC group was also significantly higher than the IL4-PDGF-BB-pMSC group .The percentage of stained area in the IL4-MSC and IL4-pMSC groups was significantly lower than the GFP-MSC group. However, the percentages of stained area in the PDGF-BB-MSC and PDGF-BB-pMSC groups were significantly higher than the GFP-MSC group. The percentage of stained area in the IL4-PDGF-BB-pMSC was significantly higher than in the IL4-PDGF-BB-MSC group. There was no significant difference in the percentage of the stained area between the GFP-MSC and IL4-PDGF-BB-pMSC groups. The percentage of stained area in the PDGF-BB-MSC group was higher than the IL4-PDGF-BB-pMSC with no statistical difference .The summary of all the above results is shown in Various methods have been developed to increase the survival of transplanted cells and promote tissue regeneration at the transplant site . One exaThe purpose of gene modification of MSCs is to facilitate MSC survival, affect their capacity for differentiation, regulate the expression of specific genes and alter the secretion of specific proteins ,34,35. ORegarding osteogenic differentiation, the percentage of ALP and Alizarin red-stained area in the IL4-MSC and IL4-pMSC groups was lower compared to the GFP-MSC group. IL4 expression levels on day 3 in the IL4-MSC and IL4-pMSC groups were 77.7 ± 4.4 ng/mL and 34.6 ± 5.7 ng/mL, respectively. We have previously reported in vitro experiments in which the concentration of IL4 necessary for macrophage polarization and immunomodulation was greater than 20 ng/mL . It is iPDGF-BB stimulates MSCs, promoting angiogenesis and osteogenesis. PDGF-BB secreted by preosteoclasts may determine the spatial and temporal vascularization required for support of osteogenesis . We inveThe IL4 expression level in the IL4-MSC group and the PDGF-BB expression level in the PDGF-BB-MSC group was higher than in the IL4-pMSC and PDGF-BB-pMSC groups, respectively. These phenomena may be attributed to the inferior cell proliferative ability of IL4-pMSCs and PDGF-BB-pMSCs compared to IL4-MSCs and PDGF-BB-MSCs, respectively. On the other hand, the IL4 and PDGF-BB expression levels in the IL4-PDGF-BB-pMSC group were higher than the IL4-PDGF-BB-MSC group. This phenomenon may be due to some synergistic effect of preconditioning MSCs with co-over-expression of IL4 and PDGF-BB, but this is a subject for future research.There is one other report on the co-over-expression of genetically modified MSCs with two different agents using the lentiviral vector. The added genes were vascular endothelial growth factor and B-cell lymphoma-2; the co-over-expressing MSCs showed significantly higher expression of each agent than singly modified MSC . HoweverThis in vitro study investigated the efficacy of genetically modified MSCs  and corresponding preconditioning of these MSCs as a potential adjunctive cell-based therapy for CD. The use of PDGF-BB-MSCs or IL4-PDGF-BB-pMSCs increased cell proliferation compared to MSCs alone. Furthermore, PDGF-BB-MSCs promoted osteogenic differentiation compared to MSCs alone. Although osteogenic differentiation for MSCs alone and IL4-PDGF-BB-pMSCs showed no difference, IL4-PDGF-BB-pMSCs co-expressed sufficient amounts to exert a biological effect IL4 and PDGF-BB and could be expected to have significant osteogenic differentiation potential in vivo. Thus, these genetically modified MSCs may provide a useful supplementary cell-based therapy for CD.