HRI), and thermal conductivity were observed in 0.2 wt% nanocomposite samples compared with pure epoxy and other lower concentration GO nanocomposites. A high THRI and derivative of thermogravimetric analysis peak temperatures (Tm1 and Tm2) were exhibited by adding nano-fillers in the epoxy, which confirms higher thermal stability of nanocomposites than that of pristine epoxy.Thermomechanical modeling of epoxy/graphene oxide under quasi-static and dynamic loading requires thermo-mechanical properties such as Young’s modulus, Poisson’s ratio, thermal conductivity, and frequency-temperature dependent viscoelastic properties. In this study, the effects of different graphene oxide (GO) concentrations  within an epoxy matrix on several mechanical and thermal properties were investigated. The distribution of GO fillers in the epoxy was investigated using transmission electron microscopy (TEM). The digital image correlation (DIC) technique was employed during the tensile testing to determine Young’s modulus and Poisson’s ratio. Analytical models were used to predict Young’s modulus and thermal conductivity, with an error of less than 13% and 9%, respectively. Frequency–temperature dependent phenomenological models were proposed to predict the storage moduli and loss tangent, with a reasonable agreement with experimental data. A relatively high storage modulus, heat-resistance index (T Epoxy resin is one of the most commonly used thermosetting matrix systems in aircraft and automotive components, marine structures, and several coatings and adhesives due to its low shrinkage, high strength, and excellent physical and chemical stability . HoweverIn particular, graphene oxide (GO) and CNTs possess higher improvement in mechanical, thermal, and impact properties among the other types of fillers . Howeverg were also reported. Liu et al.  values and an increase in storage modulus values up to 0.5 wt% of GO content were observed, and then decreases with further addition of GO into the epoxy matrix. This confirms the drawback of incorporating higher concentrations of nano-fillers into the polymer matrix. Higher Tg values were also found in rGO samples compared with the GO-modified matrix, although not much variation was found in storage modulus and loss tangent. Tang et al.  at a filler content of 1.2 wt% were reported. In a recent study [Yu et al.  investignt study , it was nt study . Song etnt study  studied nt study  reportednt study ,34,35.Despite the availability of several experimental studies related to epoxy/GO nanocomposites, a limited number of studies are available for modeling the multifunctional properties such as the static and dynamic mechanical properties and thermal conductivity of epoxy/GO nanocomposites. Some researchers have attempted modeling the mechanical properties ,37 and tMoreover, to the best of our knowledge, there are no studies available on modeling the temperature–frequency-dependent viscoelastic properties of epoxy/GO nanocomposites. Therefore, our primary goal is not to enhance the properties by modifying the interaction between filler and matrix; instead, this study is aimed at modeling multifunctional properties of relatively low weight percentages (up to 0.2 wt%) of GO filler incorporated in the epoxy matrix. This work is novel in aspects related to the thermo-mechanical modeling and multifunctional properties analysis of epoxy/GO nanocomposites. In this study, we experimentally investigated the tensile properties based on DIC and performed thermomechanical analysis including DMA, DSC, TGA, thermal conductivity, and EMI shielding effectiveness of epoxy/GO nanocomposites. Besides, the theoretical models were used to predict the tensile modulus, thermal conductivity, and temperature–frequency-dependent viscoelastic properties. The DIC was used to report Poisson’s ratio of nanocomposites, which is a useful property to be used as an input parameter in multiscale modeling.Recently, epoxy/GO nanocomposites have found interest in several fiber-reinforced polymer composite applications. The materials used in this study were graphene oxide (GO) synthesized from graphite powder  through the modified Hummers method. Gurit’s Prime 20LV™ epoxy resin and hardener were used as the matrix material. The procedure for GO preparation was reported in our previous study . The ste2 F20 S-TWIN HR(S). The TEM images were captured at different magnifications, which are shown in Figure 3 in Initially, GO and epoxy resin was thoroughly mixed using a magnetic stirrer for 2 h, and then the hardener was added into the mixture. The resin to hardener ratio used was 100:28 by weight. After that, the samples were poured into a metallic casting mold and cured at room temperature for 24 h. Further, the samples were post-cured at 65 °C for 7 h before performing the experiments. The same procedure was used for preparing different weight concentrations  of epoxy/GO nanocomposite samples. The manufactured samples are shown in Figure 4. Before performing the experiments, the dispersion of GO nano-fillers in the epoxy matrix was studied using a high-resolution transmission electron microscopy  universal testing machine (UTM). These tests were performed at the displacement rate of 2 mm/min. The displacement was measured in situ by employing the hybrid non-contact displacement measurement techniques such as two-point and full-field (DIC) using the laser-based advanced video extensometer (AVE), as shown in The AVE camera equipped with Fujinon HF-35-HA-1B series lenses with the focal length ranging from 9 mm to 35 mm was used. A maximum number of 50 fps (frames per second) can be captured using this camera. A focal length of 35 mm was used in this study for capturing the images. The width and thickness of the samples in the gauge portion used were 13 mm and 3 mm, respectively. Finally, the captured images were post-processed using the DIC replay software to obtain the required displacement field and strain distribution in the region of interest. Further details about the DIC procedure employed and the advantages of non-contact strain measurement techniques over contact strain measurement techniques are given in our previous study ,42,43.The thermal tests under the cyclic load were also performed on different weight fractions of epoxy/GO nanocomposites using the dynamic mechanical analyzer  under three-point bending. The dimensions used for this test were 20 mm × 7 mm × ~3 mm . The tests were performed at different frequencies, namely, 0.1, 1, 5, 10, 15, and 30 Hz, over the temperature range from 23 °C to 150 °C at a heating rate of 2 °C/min.The thermal conductivity experiments were performed on pure epoxy and GO modified samples using the hot disk thermal constants analyzer (Model: TPS 2500 S). The specimen dimensions used for this test were 45 mm × 45 mm (length and width). The thickness of the samples used was approximately 3 mm.The heat flow experiments were performed on different weight percentages of GO reinforced epoxy samples using the TA instrument (Model: SDT Q600). These experiments were performed over the temperature range of 23 °C to 150 °C, at a heating rate of 10 °C/min.The percentage reduction in the mass of different weight percentages of GO-reinforced epoxy samples was studied through TGA analysis using the TA instrument (Model: SDT Q600). These experiments were performed over the temperature range of 23 °C to 900 °C, at a heating rate of 10 °C/min.EMI shielding effectiveness (SE) experiments were performed on pure epoxy and GO nanocomposites using a vector network analyzer . These tests were performed over a frequency range of 8.40 GHz to 12.20 GHz. The dimensions of the specimen used for this test were 22.5 mm and 12 mm (length and width). The thickness of the specimen used was approximately 3 mm.;;);); 3 and 2250 kg/m3, respectively [p and tp are the length and thickness of the GO filler, respectively. The values of lp and tp are approximately 0.85 μm and 2.696 nm, respectively [The tensile modulus of epoxy/GO nanocomposites  of pristine epoxy and epoxy/GO nanocomposites are discussed. The effect of frequency E′ f=Tg (E′) middle or Tmg corresponding to the inflection point of storage modulus, Tg (E″)max corresponding to the peak value of E″, and Tg (tan δ)max corresponding to peak values of tan δ. Tg (tan δ)max is the highest one and Tg (E′)middle is the lowest one among these three glass transition temperature values. Most researchers reported Tg as the temperature obtained from loss modulus and loss factor peak values [Tg values with the increase in frequency can be studied using the Arrhenius model via the activation energy expression and is given by [N are the applied frequency, activation energy, gas constant, and pre-exponential factor, respectively. Th strain line equation is (y = mx + c). y = ln (f) and x = The glass transition temperature of polymers and their nanocomposites can be classified as k values . The shigiven by (3)ln f=given by (4)Ea=−RIn this section, the theoretical models for predicting the effect of volume fraction on the storage modulus of epoxy/GO nanocomposites are discussed. The storage modulus of nanocomposites can be predicted using Einstein’s model ,49, whicly. Guth  modifiedHowever, in these models, the volume fraction of nanoparticles is the only controlling parameter. However, it is also important to use theoretical models that consider the temperature and frequency effects, as the viscoelastic properties of polymers and their nanocomposites rely on these parameters.T is the temperature. q are the intrinsic growth rate and the symmetry of the glass transition region, respectively. These parameters are determined by curve fitting degree of glass transition In this section, the theoretical models for predicting the effects of temperature and frequency on storage and loss moduli of pristine epoxy and epoxy/GO nanocomposites are discussed. The two-parameter viscoelastic model for polymers and polymer matrix composites is proposed by Feng et al. ,52 to prtion (9) :(9)φ=12qThe thermal conductivity of epoxy/GO nanocomposites can be determined through the Russell model using Equation (10), which is given by (10)λc=λterature .T) is the summation of EMI shield effectiveness of absorption (ASE) and reflection (RSE) phases [ASE and RSE) can be calculated using the scattering or S- parameters: S11, S12, S21, and S22. The first and second numbers in the subscript of S-parameters refer to the responding and incident ports, respectively. The EMI shielding effectiveness values of absorption and reflection can be calculated using Equations (11) and (12), respectively [In recent times, the EMI shielding effectiveness using GO films embedded in the polymer matrix composites has been investigated . Here, w) phases . The hig) phases ,57. Thesectively :(11)SEA=In this section, the tensile properties and the Poisson’s ratio of pristine epoxy (0 wt%) and epoxy/GO nanocomposites  are presented and discussed. It can be observed from p and q in Equations (7) and (8) were obtained using a curve fitting parameter for the T graph using Equation (9) in MATLAB. As can be seen in p and q decrease with the increase in frequency from 0.1 Hz to 30 Hz. Similar trends were reported in studies in the literature [The dynamic mechanical properties of pristine epoxy and GO samples for a temperature range of 23 °C to 150 °C at different frequencies  are presented here. The storage modulus and loss modulus are predicted for the corresponding temperature and frequency ranges using Equations (7) and (8), respectively. The tan δ values were determined from the predicted storage and loss moduli curves. The unknown parameters terature ,52,53.The evolution of storage and loss moduli of pristine epoxy and different GO samples is discussed in this section. The results were obtained for a temperature of 23 °C to 150 °C at different frequencies . The results of storage and loss moduli values for all frequencies are given in g (E″). This can be attributed to material softening at higher temperatures, as the mobility of molecules changes the state from immobile at room temperature to mobile.Initially, both the storage and loss moduli curves are slightly horizontal at room temperature, which is mainly because of the immobility of molecules in the polymer matrix. Room temperature (~23 °C) is not sufficient to cause adverse effects such as translational and rotational movements of molecular chains in the resin. Thus, as the temperature increases, adverse effects occur and gradually increase the molecular movements in the resin. A sudden drop in E′ value is seen in g. The enhanced molecular transport phenomena in the resin above Tg increases the rate of mobilization [In contrast to the E′ curve, the E″ curve increases up to Tlization . Therefomax for epoxy increases from 5.3 GPa at 0.1 Hz to 5.6 GPa at 30 Hz (4.6% increase), whereas the predicted (E′)max increases from 5.2 GPa at 1 Hz to 5.5 GPa at 30 Hz (5% increase). Similarly, for the same range of frequency variation, the (Tg)experimental increases from 69.7 °C to 72.8 °C (4.5% increase) and (Tg)predicted increases from 67.4 °C to 72.8 °C (8.1% increase). The experimental and predicted Tg values were noted from the respective peak values of the loss modulus curves. These observations confirm good correlation between experimental and predicted results. The time available for molecules to relax and rearrange within the epoxy matrix is less at high frequencies compared with low frequencies [g values are higher at the frequency of 30 Hz as compared with at 0.1 Hz. Similar trends in storage modulus and Tg values with the increase in frequency were also observed for 0.05 wt%, 0.1 wt%, and 0.2 wt% GO samples, as shown in It can be observed from quencies ,53,62,63The coefficient of frequency sensitivity (ζ) is obtained using Equation (2), which is a linear regression equation for estimating the effect of frequency on initial storage modulus of the pristine epoxy and different GO samples, as shown in g (tan δ)max values are shown in g values with the increase in GO content shows an improvement in self-healing properties owing to the shape memory effect of GO nano-fillers [g values with the increase in frequency is also observed in both experimental and predicted values, as shown in The loss tangent T-fillers . Similar-fillers ,24,28. Tg value corresponding to experimental (tan δ) max increases from 78.7 °C at 0.1 Hz to 90.9 °C at 30 Hz for pristine epoxy (15.5% increase). Similarly, the predicted Tg value corresponding to predicted (tan δ) max increases from 76.5 °C at 0.1 Hz to 88.7 °C at 30 Hz for pristine epoxy (16% increase), which confirms the excellent correlation between both approaches.It can be observed from max values for all frequencies compared with GO samples. This is because of an increase in interfacial bonding with the addition of GO content within the epoxy matrix [It can be observed from the figure that the pristine epoxy exhibits higher (tan δ) y matrix .g (tan δ) max values as the frequency (f) changes from 0.1 Hz to 30 Hz and the activation energy for all samples. These values are plotted using the Arrhenius model using Equation (3). The activation energy depends on the variation in Tg values as the frequency increases. The higher the change in Tg with the increase in frequency, the higher the resultant activation energy. It is worth noting from these figures that the change in Tg values are higher and almost the same in pristine epoxy and 0.05 wt% GO samples. As a result, the activation energy values were found to be higher in pristine epoxy and 0.05 wt% GO samples compared with other nanocomposites. The excellent agreement was assessed between the experimental values and curve fit from higher values (~>0.9) of the coefficient of determination (R2).g values are seen in the pristine epoxy compared with GO samples. These measurements follow the trend of Tg values measured using the DMA. g values measured through the DMA and DSC. Even though the average Tg values corresponding to loss modulus peak temperature and DSC correlate well at a lower amount of GO nanocomposites, higher deviation is obtained at nanocomposites of 0.2 wt% GO. However, the average Tg values corresponding to the loss tangent peak temperature value (71.9 °C) reasonably correlate with the Tg value of 68.8 °C measured using the DSC. The smaller deviation between the Tg values of DMA and DSC is due to the difference in heating rates. The heating rate used for DMA was 2 °C/min, whereas a heating rate of 10 °C/min was used for the DSC studies.m1 and Tm2) with the increase in filler content in the epoxy can be seen clearly in the high-temperature region derivative TGA curve. These peak values (Tm1 and Tm2) for pristine epoxy and different nanocomposites are given in m1 and Tm2) are obtained by adding nanofillers in the epoxy; as a result, the thermal stability of nanocomposites was higher than the pristine epoxy [HRIT) is calculated using Equation (14) [T5 and T30 correspond to the decomposition temperature of 5% and 30% weight loss, respectively, which are determined from ne epoxy ,69. The ion (14) ,32.(14)Tm1 and Tm2) of GO fillers added in epoxy samples are higher than the pristine epoxy sample. These observations lead to the important conclusion that the thermal stability increases by adding the GO fillers in the epoxy.From The present study aimed to investigate the effects of low concentrations of GO filler in the epoxy matrix on mechanical and thermal properties. Moreover, the theoretical models were used for determining the tensile modulus, Poisson’s ration, temperature–frequency-dependent viscoelastic properties, and thermal conductivity of different GO nanocomposites. The results are summarized below.The increase in tensile strength and modulus and decrease in failure strain (%) were observed with the increase in GO nanofiller. The deviation between the predicted and experimental tensile modulus values was less than 13%. Moreover, the Poisson’s ratio increased by 23.3, 26.7, and 6.7% with the increase in GO content.g values in all samples with the increase in frequency from 0.1 Hz to 30 Hz. A good correlation was obtained between the predicted values and experimental results. A good agreement was observed between the Tg values measured in all samples using the DMA and DSC.The dynamic mechanical analysis showed an increase in storage modulus and TThe increase in heat-resistance index and thermal conductivity values from 157.8 °C and 0.258 W/mK to 159.4 °C and 0.307 W/mK, respectively, was observed with the increase in GO content from 0 wt% to 0.2 wt% in the epoxy. The deviation between the predicted thermal conductivity values from the experimental results is lower than 9%.Modeling epoxy/GO nanocomposites using the finite element method (FEM)-based approach is difficult, in that several multi-functional properties are required as input values, which can be taken from the data presented in this study. Moreover, the experimental investigation and proposed models for predicting thermo-mechanical properties, such as Young’s modulus, Poisson’s effect, thermal conductivity, and temperature–frequency-dependent viscoelastic properties, can be used for thermomechanical modeling of epoxy/GO nanocomposites under quasi-static and dynamic loading.


Condition monitoring of rail transport systems has become a phenomenon of global interest over the past half a century. The approaches to condition monitoring of various rail transport systems—especially in the context of rail vehicle subsystem and track subsystem monitoring—have been evolving, and have become equally significant and challenging. The evolution of the approaches applied to rail systems’ condition monitoring has followed manual maintenance, through methods connected to the application of sensors, up to the currently discussed methods and techniques focused on the mutual use of automation, data processing, and exchange. The aim of this paper is to provide an essential overview of the academic research on the condition monitoring of rail transport systems. This paper reviews existing literature in order to present an up-to-date, content-based analysis based on a coupled methodology consisting of bibliometric performance analysis and systematic literature review. This combination of literature review approaches allows the authors to focus on the identification of the most influential contributors to the advances in research in the analyzed area of interest, and the most influential and prominent researchers, journals, and papers. These findings have led the authors to specify research trends related to the analyzed area, and additionally identify future research agendas in the investigation from engineering perspectives. Condition monitoring of certain devices is applied in a particular system in order to detect changes or analyze trends in controlling particular parameters, and most of all to prevent potential failures of a system. Early problem diagnosis ensures preliminary intervention for replacement of any equipment’s components before serious consequences. The term “condition monitoring” evolved—in the case of research connected to rail transport—with the development of instruments that allow the development of increasingly better measurement analyses. Such an evolution of terms, definitions, and approaches connected to condition monitoring in the case of the aforementioned mode of transport is presented in Recent decades have shown a certain evolution of approaches to the condition monitoring of various rail transport systems, especially in the context of rail vehicles and track subsystems. The approaches applied to rail infrastructure condition monitoring have evolved from manual maintenance, through methods connected to the application of sensors, up to the currently discussed techniques focused on the terms of Industry 4.0. The enormous increase in the quantities of generated data—namely, big data—requires diagnostic procedures to be automated. Such a challenge induced the need for up-to-date and improved methods of autonomous interpretation of condition monitoring data (mainly vibration methods). This has led the authors to the aim of this review paper, which is the consideration of several pieces of research focused on the scientific area of rail transport systems’ condition monitoring. The authors of this review paper took into consideration two distinct methods of scientific literature analysis: on the one hand, a bibliometric performance analysis  was carried out, and on the other hand, a systematic literature review was performed.condition monitoring techniques for rail vehicle dynamics ,applications and various soft computing methods used for condition monitoring in the context of intelligent systems ,description of onboard condition monitoring systems, methods, and devices—with a particular interest in microelectromechanical sensors, microprocessors, and transceivers—creating wireless sensor networks for freight rail transport ,methods, techniques, and applications connected to the condition monitoring of railroad switches and crossing systems ,condition monitoring systems and enabling automatic railroad track inspection ,railroad track condition monitoring with the use of inertial sensors  and GPS signals ,brief discussion on machine learning techniques in relation to rail track condition monitoring ,railway turnout condition monitoring ,53),various aspects connected to the industrial Internet of Things solutions related to condition monitoring ,various types of sensors in wired and wireless sensor networks , and wircondition monitoring associated with fault diagnosis of rolling bearings .According to the information contained in key scientific databases, nearly 20 review papers on the condition monitoring of railroad systems have been published in the last 4 decades. This is an insignificant number considering the essence of the subject matter; nevertheless, it is worth underlining that this number concerns some limited areas that will be defined later in the current article. By recalling the published review papers, it can be stated that their authors have taken under consideration topics such as :This briefly presented background allows us to state that no complex review papers connected to condition monitoring for rail transport were noted. As this can be treated as a research gap, the authors decided to consider an in-depth study consisting of a complex review of approaches to condition monitoring for railroad transport, under particular assumptions. This study is related both to passenger and freight railroad transport systems’ condition monitoring—in aspects connected to both rail vehicles and tracks.As was observed, there is a lack of review papers that treat condition monitoring of railway transport systems as complex systems composed of various facilities and subsystems, straightforwardly discussing both rail tracks and rail vehicles. This review paper’s authors have tried to fill such a research gap. Additionally, we decided to take into consideration the past few decades of condition monitoring considerations presented in numerous publications, which led us to formulate the main research aims of this publication. The research questions are as follows: RQ1: What are the current trends in condition-monitoring approaches, and how have these trends evolved over the last decades?RQ2: What are the future research directions and perspectives in the condition monitoring of rail transport systems?This paper is organized according to the following structure: The background resulted in research questions that need to be verified based on a certain methodology. The authors present the methodology of the current research in the next section. The two sections following the description of the methodology were selected based on methodological approaches, and are as follows: bibliometric performance analysis, and systematic literature review. Finally, in the last section, we discuss the main results of this study, and additionally present current research limitations and future research agendas.The type of research methodology and manner of data collection for further analysis depend on the research question being studied. Therefore, before we present our research methodology applied in the case of the current paper, we shall present potential well-known methods and techniques commonly used in the literature review context. Consequently, a proper compilation of methods and techniques is presented at the end of this section.This paper is based on coupled review methodologies—it combines a bibliometric performance analysis based on content-based analysis, and a systematic literature review as a result of the content-based analysis. The first of these methods is a form of quantitative analysis, while the second is qualitative. As a whole, this methodology adequately presents the full spectrum of publications connected to the condition monitoring of rail transport systems, both in quantitative and qualitative terms.Neuendorf  defined A bibliometric performance analysis based on content-based analysis is one aspect of the current paper, while the other is the systematic literature review. Literature review in general is characterized by certain typology, which was presented in detail in Marczewska and Kostrzewski  based onThe data searching process was limited to contributions published between 1980 and 2020 . The end date was based on the content of the scientific databases. The specific information connected to every stage of the research review is provided in the following subsections. The procedure of this research review is shown in The first stage of the search process was not limited to any period of time .“condition”* AND “monitoring” AND “railway*” AND “track” OR “vehicle”. This resulted in the return of 6115 records in total in the Scopus database . It should be mentioned that 23 publications in total were assigned to the year 2021, and these are included in this number, but excluded from the below statistics. The majority of the publications were released in English, and a significant number of them in Chinese—all of the languages of the publications are given in “(condition* AND monitoring) AND (railway*) AND (track*) AND )” allowed us to limit the number of searched publications to 5185 items . Once again, it should be mentioned that 23 publications were assigned to the year 2021, and these are included in this number, but not included in the below statistics.Since our interest was focused on the condition monitoring of railway vehicles and tracks, the following set of keywords was investigated: All of the papers in English were also filtered based on their type. As shown in i) in the mentioned research area per year (y) is presented in R2 = 0.964; this equation is noted as Equation (1). The end of the year 2020 was not excluded from this analysis; however, one should bear in mind that the number of publications will increase, considering that indexing in scientific databases is out of regulations; for example, if there was still a month of 2020 left until the end of the period between the completion of the analyzed data and the end of the year, not all of the released publications would have been indexed in the database yet.The number of publications in the research area of condition monitoring, released in English, grew annually. The number of publications , “railway”, “fault detection”, “railway track”, “vibration analysis”, “damage detection”, “failure analysis”, “railway infrastructure”, “fault diagnosis”, “vibration”, “monitoring system”, “railway accidents”, “surface defects”, “track irregularity”, “accident prevention”, “railroad engineering”, “condition monitoring system” , “track geometry”, “rail track”, “rail”, “railways”, etc. Although internal keywords such as “railroad”, “railroad transportation”, and “rails” describe more publications than “condition monitoring” terms, these are too general, and are characterized by a high degree of abstractness in connection to the particular subject matter. On the other hand, the remaining internal keywords are assigned to fewer publications than “condition monitoring”. Therefore, we assumed that the research publications of interest were hidden in a group focused on the internal keyword “condition monitoring”. Therefore, publications from the circle of this keyword are subjected to analysis in this article.A total of 617 publications assigned to the condition monitoring term were released in English between 1982 and the first half of 2020 . Most of the contributions were classified as articles and conference papers, reviews, and conference reviews; hence, these types of references were of interest to us. Unlike the sample before filtering via internal keywords, more publications in this sample were released as conference papers (54.9%) than articles (41.0%) .To satisfy the reader’s inquisitiveness, the full code applied in the Scopus database search engine is as follows:(condition*ANDmonitoring)AND (railway*)AND (track*)AND )AND )AND ).Notes and conference reviews were excluded from the research, since these are believed to be less influential in scientific terms and, thus, less relevant. For this reason, the studies to be assessed were selected from these areas, constituting a final research sample of 613 document results found with the following full code applied in the Scopus database search engine: (condition*ANDmonitoring)AND (railway*)AND (track*)AND )AND )AND )AND OR EXCLUDE .((TS=(condition* AND monitoring) AND TS=(railway*) AND TS=(track*)) OR (TI=(condition* AND monitoring) AND TI=(railway*) AND TI=(track*)) OR (AB=(condition* AND monitoring) AND AB=(railway*) AND AB=(track*)) OR (AK=(condition* AND monitoring) AND AK=(railway*) AND AK=(track*))) AND LANGUAGE: (English) AND LANGUAGE: (English) Refined by: DOCUMENT TYPES: (ARTICLE OR REVIEW OR BOOK CHAPTER OR PROCEEDINGS PAPER) Timespan: All years. Indexes: SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, BKCI-S, BKCI-SSH, ESCI, CCR-EXPANDED, IC . The search of the WoS database resulted in 473 records (checked on 8 January 2021). The majority of the WoS-indexed publications were also found on the Scopus database. Moreover, the Scopus database is characterized by a higher number of unique journals compared to WoS, as a result of which the total number of records in the Scopus database was greater than in the WoS database. A comparison of the records found in both databases is given in It is worth explaining the choice of the scientific database in these considerations. A couple of years ago, the authors of Baier-Fuentes et al.  mentioneR2 = 0.9147; this equation is noted as Equation (2).The increase in the number of publications from one year to another is different when the concept of publications is limited only to those for which the internal keyword “condition monitoring” was indicated. There is no exponential dependence as seen in Currently, the particular publication could be assigned to several subject areas, which means that publications assigned to the Engineering subject area can be assigned to several other areas as well. This is based less on the decision of a particular publication’s author(s), but more on an algorithm of the database. As can be observed, in the case of the “condition monitoring” internal keywords, most of the publications are indexed in the Engineering subject area, as is shown in Cooperation in publication and citation of the researchers cited in the case of the internal keywords “condition monitoring” is one of the most important analyses by which to present the level of researcher productivity. In order to conduct such an analysis, it was important to unify data imported from the database. The set of data connected to the published manuscripts were not given with the unique names or surnames of the selected researchers. Therefore, before analysis, the following unifications were performed: every time “Goodall R.M.” appeared in the data file it was changed to “Goodall R.”; “Weston P.F.” to “Weston P.”; and “García Márquez F.P.” to “Márquez F.P.G.”, etc. After this kind of correction, it was possible to obtain a network of cooperation in publication and citation of researchers cited in the case of the internal keywords “condition monitoring”; This network is presented in For the total number of 57 unique researchers, the total strength of co-authorship links with other authors was calculated , which were not unified, as can be observed in It is worth mentioning, despite perhaps seeming obvious, that different keywords characterize a given publication. Therefore, it is not possible to group all publications characterized by identical keywords in the text within—for example—a single paragraph; this is the purpose of As mentioned at the end of the previous section, we initially present an analysis of selected papers connected to various types of sensors used in different solutions connected to condition monitoring in railway transport The list of review papers’ specific keywords starts with “acceleration sensors” and “accelerometers”. These keywords were ascribed by the authors in both singular and plural form. In Liu et al. , two meaAcceleration sensors were also applied by Känsälä et al. he biggest challenge for condition monitoring is ensuring selection of the right technologies”, Brant and Liang .


Prmt5 deletion results in a progressive loss of SSCs and male infertility. The proliferation of Prmt5-deficient SSCs cultured in vitro exhibited abnormal proliferation, cell cycle arrest in G0/G1 phase and a significant increase in apoptosis. Furthermore, PLZF expression was dramatically reduced in Prmt5-deficient SSCs, and the levels of H3K9me2 and H3K27me2 were increased in the proximal promoter region of the Plzf gene in Prmt5-deficient SSCs. Further study revealed that the expression of lysine demethylases  was significantly reduced in Prmt5-deficient SSCs and that the level of permissive arginine methylation H3R2me2s was significantly decreased at the upstream promoter region of these genes in Prmt5-deficient SSCs. Our results demonstrate that PRMT5 regulates spermatogonial stem cell development by modulating histone H3 lysine modifications.Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of mono- or symmetric dimethylarginine residues on histones and non-histone substrates and has been demonstrated to play important roles in many biological processes. In the present study, we observed that PRMT5 is abundantly expressed in spermatogonial stem cells (SSCs) and that  Plzf, Gfra1, Pou5f1, Lin28A, and Nanos3 are expressed in undifferentiated spermatogonia, and c-Kit is considered a marker gene of differentiating spermatogonia. Several Sertoli cell-produced growth factors, such as glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF2), and insulin-like growth factor (IGF), are also crucial for the maintenance and proliferation of spermatogonial stem cells (SSCs)  . Homozygs mutant .Plzf  belongs to the Kruppel family and is expressed in hematopoietic stem/progenitor cells  causes male and female sterility. PRMT5 represses the activation of LINEs and IAP transposons via symmetric dimethylation of arginine 3 on histone H2A and H4 (H2A/H4R3me2s)  and male infertility. Prmt5-deficient SSCs cultured in vitro exhibited abnormal proliferation, and the cell cycle was arrested in G0/G1 phase. Further study revealed that the expression of PLZF was dramatically reduced in Prmt5-deficient SSCs. We also observed that inactivation of Prmt5 resulted in enrichment of H3K9me2 and H3K27me2 in the promoter region of the Plzf gene, which in turn caused downregulation of Plzf expression and defects in SSC development.PRMT5, a type II protein arginine methyltransferase, catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM or AdoMet) to histones and non-histone substrates to form mono- or symmetric dimethylarginine  . PRMT5 p4R3me2s) . PRMT5 i4R3me2s) . The resertility , suggestin vitro by both immunofluorescence  and B-type cyclins  were all significantly reduced in Prmt5-deficient SSCs. In contrast, the expression of cyclin-D, cyclin-E1, and cyclin-G was not altered after Prmt5 inactivation. It is worth noting that p21 and p53 expression dramatically increased after inactivation Prmt5 at both the mRNA and protein levels. These results indicate that PRMT5 is important for stemness maintenance and the proliferation of spermatogonial stem cells.The differentially expressed genes associated with stemness maintenance, proliferation and the cell cycle in both control and stemness . As showent SSCs , whereasent SSCs . The expBlimp1  of the Plzf gene. The ChIP-qPCR results showed that H4R3me2s, H3R2me2s, and H2AR3me2s levels remained unchanged (data not shown), whereas those of H3K27me2 and H3K9me2 were significantly increased at the proximal promoter region (Site 2: −175∼−347 bp) of Plzf in Prmt5-deficient SSCs  remained unchanged. The mRNA levels of lysine methylases for H3K27me2  were not increased in Prmt5-deficient SSCs, while the expression of demethylase for H3K27me2 (KDM6B) was significantly deceased  of JMJD1A, JMJD1B, JMJD1C, and KDM6B. The ChIP-qPCR results showed that inactivation of Prmt5 led to a significant increase in the repressive histone modification H4R3me2s at the promoter region (−417 ∼−546 bp) of JMJD1C  and H3K27me2 (KDM6B) is caused by changes in histone arginine methylation in f JMJD1C , whereasf JMJD1C . Moreoveent SSCs . The levent SSCs . These rPrmt5 in male germ cells using Stra8-Cre led to defects in meiosis and male infertility  via recruitment of Polycomb protein in a Pax2/Grg4-dependent manner  was significantly reduced at both the protein and mRNA levels, indicating that the increase in H3K9me2 and H3K27me2 was most likely due to the downregulation of histone H3 lysine demethylase expression. These results suggested that the expression of target genes regulated by PRMT5 is cell context dependent.As a protein arginine methyltransferase, PRMT5 catalyzes MMA or sDMA in histones and non-histone substrates . Interest manner . In hemahylation . In the ent SSCs . Moreoveent SSCs , the expMvh-Cre leads to defects in SSC maintenance  results in aberrant meiotic progression. However, the expression of meiosis-associated genes, such as Stra8, Sycp3, Dmc1, and γH2AX, was not affected Wtsi) Wtsi) , Prmt5+/ail tips .+/Δ;Mvh-CrePrmt5 and control mice immediately after euthanasia, fixed in 4% paraformaldehyde for up to 24 h, stored in 70% ethanol, and embedded in paraffin. Five-micrometer-thick sections were cut and mounted on glass slides. Then, the tissue sections were processed for immunohistochemistry and immunofluorescence.Testes were dissected from IHC and IF procedures were performed as described previously . Antibodflox/flox;Cre-ERTMPrmt5 mice at 5–7 days postpartum (dpp) were digested with collagenase IV and DNase I for 5 min into small fragments and then centrifuged at 400 rpm for 2 min. The seminiferous fragments were suspended in mouse embryo fibroblast (MEF) medium containing 10% FBS . Twenty-four hours later, the SSCs were collected and then transferred to mitomycin C-treated MEF feeder cells. The specific medium for SSCs contained GDNF and FGF2 for later culture. flox/flox;Cre-ERTMPrmt5 SSCs treated with ethanol or 1 μM tamoxifen were harvested for western blot, real-time PCR or MTT assay.Establishment and maintenance of SSCs were performed as previously described . In brieWestern blotting procedures were performed as described previously . TissuesCT–ΔΔ. Gapdh was used as an internal control for quantification. The primers used were listed in For Real-Time PCR, RNA was isolated from cultured SSCs using EASYspin Plus Tissue/Cells RNA Rapid Extraction kit following manufacturer’s instructions. The relative expression level was calculated using the formula 22 incubator. Then, the medium containing MTT was removed, and 750 μl DMSO was added. After an incubation for 10 min on a shaking table at 75 rpm/min, the OD value at 490 nm was measured.The relative number and viability of SSCs were evaluated by MTT assays. In brief, SSCs seeded in 24-well plates were washed with PBS twice and incubated with MTT solution (0.5 mg/ml) for at least 4 h at 37°C in a CO® Plus Sonication Chromatin IP Kit (# 56383) following the manufacturer’s instructions. For ChIP-qPCR, the input genomic DNA or immunoprecipitated DNA was used as a template. Quantitative real-time PCR was performed according to the manufacturer’s instructions. The input DNA was used as a normalization control. The primers used are listed in ChIP assays were conducted using a SimpleChIPP-values based on three to six independent experiments in PCR reactions or western blotting assay. Multiple t-test-one per row was used to compare the differences between two groups. P < 0.05 or P < 0.01 was considered statistically significant.Experiments were repeated at least three times. GraphPad Prism7 software was used for analysis of The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Zoology, CAS (AEI-09-02-2014).FD and MC (co-first author): formal analysis and investigation. FG: funding acquisition. FD, MC (co-first author), MC (third author), LJ, ZS, LM, CH, and XG: methodology. FG: project administration. MC (co-first author) and LM: resources. FD: writing—original draft. FD and FG: writing—review and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N = 1,058) and measured trauma exposure, perpetration, mental health problems , perceived stigma , experienced stigma, and skepticism toward reintegration with ex‐combatants. Male ex‐combatants  had more past trauma and violence perpetration than other community members and a greater number of recent conflicts (including both victimization and perpetration) within the community and with strangers/organized violence. They reported more experienced stigma, more severe PTSD symptoms but were less skeptical about reintegration. Ex‐combatants’ ongoing violence was predicted by an interplay of the community's skepticism toward reintegration and ex‐combatants’ perceived and recently experienced stigma (often attributed to the armed group history) and mental health problems, in addition to lifetime traumatization. These findings promote the need for combined interventions that address individual mental health problems including aggression and collective discriminatory attitudes and behaviors.Reintegration of ex‐combatants involves multiple challenges. In addition to the trauma‐related psychological sequelae, social obstacles in the community can aggravate psychopathological aggressive tendencies and lead to the continuation of violence in civilian life. However, the association between others’ negative attitudes and ex‐combatants’ ongoing perpetration of violence remains largely unexplored. Between September 2018 and May 2019, we assessed a representative community sample of adults in Eastern DR Congo ( This article showed that stigmatization presents as additional burden for ex‐combatants in Eastern DRC beyond posttraumatic experiences. Rejective attitudes toward ex‐combatants form the basis of stigmatization and by predicting both mental health problems and aggression present as social drivers of the cycles of violence. Our study demonstrates the importance for intervention programming that combines individual treatment with interventions including ex‐combatants' social environment. Rather, it can be understood as adaptation to a highly violent environment in which aggression and violence increase social status  differences between ex‐combatants and other community members in trauma exposure, perpetration, mental health problems, perceived and experienced stigma, and skepticism toward reintegration of ex‐combatants; (2) the prevalence rates for the community's skeptical attitudes toward reintegration; and (3) the interplay of the community's skepticism toward reintegration with ex‐combatants’ perceived and experienced stigma, mental health problems, and ongoing violence controlled for trauma exposure. We hypothesize that the community's skepticism toward reintegration in combination with ex‐combatants’ perceived and recently experienced stigma predicts their mental health problems and ongoing violence in addition to trauma exposure.22.1Between September 2018 and May 2019, a representative sample from six communities with 687 households and 1946 adult inhabitants were interviewed in the Kivu regions, Eastern DRC. This survey represents the baseline of a longitudinal randomized controlled trial with three follow‐up timepoints  measuring the effectiveness of a community intervention to reduce stigmatizing attitudes toward trauma survivors including ex‐combatants and increase outreach of mental health services after trauma. Hereafter, only baseline data were analyzed. However, information about participants who disclosed armed group involvement not at baseline but at a later timepoint was used to determine the group of ex‐combatants. For each community, we calculated the sample size by controlling for sex and age distributions  with a 5% margin of error and 95% CI. Diagnostic interviewers selected participants in door‐to‐door visits by blind and random drawing of folded papers labeled with the respective sex and age category. If selected participants were absent, interviewers organized later appointments and if multiple inhabitants of one household were eligible (within the chosen category), the person who chose a paper marked with a black‐cross out of a subset of empty papers was interviewed. In isolated cases, residents denied participation due to lack of time (because of work or school responsibilities) a further paper was drawn. After visiting all households, a subsample of community members was invited for interviews nonrandomly, in order to obtain a representative sample size. This recruitment was facilitated by individuals with respected local authority. After randomization and indication of availability, individuals were informed about the study procedure. None of these individuals declined participation. Twenty‐one trained local psychologists conducted clinical interviews (1.5 to 2.5 hr) under supervision by the authors on site. Questionnaires were translated from English to Kiswahili and back by two native Congolese interpreters and discrepancies discussed with two of the authors. Participants received light refreshment and small financial compensation . Interviewers double rated forty‐two assessments for the calculation of interrater reliability. The study was approved by the ethic commission of the University of Konstanz (31/2016) and the Social Fund of the DRC.2.2n = 137) indicated a history of armed group involvement, whereby only a minority of them were female . As female and male recruits present with essential characteristic disparities, this paper focuses on male ex‐combatants only. The final sample contains 1,058 participants with 129 (12%) ex‐combatants. Participants were almost equally distributed in regard to sex , on average 36 years old , mostly in a partnership , had on average four children  and indicated on average five years of education  and low financial wealth . All were Congolese nationals. The most common native languages were Kiswahili  and Hunde . One fifth  spoke more than one language. Almost everyone was religious . About half of the participant migrated into the communities , for example, due to security issues , marriage , stigmatization in their last communities , or other reasons . Ex‐combatants were younger than other community members (t(216) = 2.9, p = .004), more often in a partnership (χ2(1) = 7.9, p = .005) and more educated  = −7.0, p < .001). The groups did not differ in the number of children, immigration status (born in village versus immigrated), and wealth (p > .05).A total of 1,066 community members participated in the study. 13% , mental health problems, and skepticism toward reintegration of ex‐combatants.2.3.1The frequency of different traumatic event types, experienced stigma, and perpetration was assessed with the 41‐item version of the Threats to Human Life Scale . Participants indicate the experience (yes/no) of recent (last three months) and lifetime (prior to the last three months) threat types to physical  and social integrity  and types of perpetration . For each event, the scale asks for the aggressor  and ex‐combatants additionally indicated if they believed to have experienced social threats because of their armed group history. For perpetrated events, participants were asked against whom the violence was directed . The subscale sum scores are based on event occurrence  and context .2.3.2 to 4 (6 or more times a week/severe) in relation to an index trauma. A sum score indicated PTSD symptom severity (range 0–80) and diagnosis was calculated following the manual. Prior research demonstrated the scale's applicability in African countries  to 3 (Nearly every day). A sum score (range 0–27) indicated depression symptom severity and diagnosis of Major Depression was ascertained according to DSM‐5  to 4 (Agree), whereby the sum score indicates a stronger desire for violence (range 0–60). The scale has been used in DRC among male and female ex‐combatants  to 4 (Completely/I felt this very strongly) for a time in the last four months when they felt the most shame or worst about themselves. A sum score was calculated (after recoding two inverse items) indicating feelings of shame (range 0–56). The scale was successfully applied in a sample with Africans  to 3 (I completely agree) on five items with reference to a traumatic event after which they needed social support. The sum score indicated the perceived lack of general social acknowledgement (range 0–15). Reliability measures were satisfying .We administered the “general disapproval” subscale of the 2.3.4Social ReconstructionScale  to 4 (Agree). To our knowledge, the scale has not been used in a comparable setting yet. The sum score indicates skeptical attitudes toward reintegration of ex‐combatants (range 0–76). Internal consistency and interrater reliability were satisfying .Skepticism toward reintegration of ex‐combatants was assessed with the 2.4SPSS 26 IBM,  was used33.1SD = 6.5, range: 0–30, note that some ex‐combatants are born and raised within armed groups). The majority were part of military missions  mostly with combat experience . At baseline, 61  ex‐combatants indicated that their armed group history was socially disclosed: In 16 (27%) of the cases, confidents were the only persons who were informed, whereas 44 (73%) stated that neighbors or the whole community also knew about their past. Reluctance to disclose was likewise reflected in this study as 57 (44%) ex‐combatants had not disclosed their armed group history to interviewers at baseline but at a later timepoint of the research trial. The main reasons reported to interviewers were initial skepticism about confidentiality and fear of disclosure to and persecution by the community or even prosecution by the police.Ex‐combatants enrolled in armed groups at an average age of 18 years (3.2n = 301) fulfilled the diagnostic criteria of PTSD with higher prevalence rates among ex‐combatants  than other community members . Ninety‐two (9%) presented with a diagnosis of major depression, whereby ex‐combatants  and other community members  showed comparable prevalence rates. Almost all ex‐combatants approved at least one item of appetitive aggressive .Table 3.3X2  = 28.8, p < .001, whereby one community in particular presented with less beliefs in reintegration than others in post hoc tests. The percentage of ex‐combatants in this community  was higher than in the others  = 7.1, p = .008).Prevalence rates for the community's skeptical attitudes toward reintegration of ex‐combatants are presented in Table 3.4ß = 0.46, SE = 0.12, p < .001 and recently experienced stigma, ß = 0.43, SE = 0.15, p = .004. Mental health problems were predicted by perceived stigma, ß = 0.13, SE = 0.06, p = .031, recently experienced stigma, ß = 0.29, SE = 0.10, p = .006, and trauma exposure, ß = 0.45, SE = 0.10, p < .001. Ex‐combatants’ ongoing violence was predicted by high recently experienced stigma, ß = 0.28, SE = 0.11, p = .009, but neither by low or moderate experienced stigma , and by mental health problems, ß = 0.16, BootSE = 0.07, 95% BootCI = . Conditional analyses revealed no indirect effects of the community's skepticism in reintegration on ex‐combatants’ ongoing violence via perceived stigma and mental health problems (p > .05).The relation between the community's skepticism toward reintegration and ex‐combatants’ perceived and recently experienced stigma, mental health problems, and ongoing violence controlled for trauma exposure is presented in Figure 4This study showed that ongoing violence perpetrated by male ex‐combatants in Eastern DRC is not only enhanced by mental health problems but also by the social environment. Specifically, implicit beliefs and overt interaction with the community, specifically their skepticism in reintegration of the demobilized and stigmatization against them as well as ex‐combatants’ feelings of being stigmatized. Although all community members presented with a high level of trauma exposure, ex‐combatants constitute a group with a particularly destructive mixture of lifetime trauma and perpetration. As consequence to their war trauma history, appetitive aggression is highly prevalent and PTSD symptoms were more severe than the average in the community.4.1Twelve percent of participants who admitted an armed group history also presented with significantly more perpetration and lifetime trauma  including aggressive tendencies (e.g., Narrative Exposure Therapy for Forensic Offender Rehabilitation, FORNET, original paper Elbert et al., 4.5Limitations include the constrain on explicit measures, which may imply a bias of social desirability. All data were collected in rural areas where insecurity was somehow calculable. Moreover, female ex‐combatants who make up to 48% of armed groups in Eastern DRC (Johnson et al., 5Ex‐combatants in Eastern DRC face multiple obstacles to reintegration after returning from the battlefield and present with the risk of fueling the cycles of violence in the community. This study demonstrated that, in addition to lifetime trauma, social adversities account for ex‐combatants’ mental health problems and continuation of violence in civilian life. Comprehensive reintegration programs should address trauma‐ and combat‐related mental health and behavior problems both at the individual and the community level.The authors have no conflict of interest to declare.Sabine Schmitt involved in conceptualization, methodology, formal analysis, investigation, data curation, writing–original draft, review and editing, visualization, and project coordination. Katy Robjant involved in investigation, writing–review and editing, project management, and funding acquisition. Anke Koebach involved in conceptualization, methodology, supervision, writing–review and editing, and funding acquisition.https://publons.com/publon/10.1002/brb3.2156.The peer review history for this article is available at


Systemically, circulating CX3CR1+ monocytes reversibly increase in individuals with renal impairment and correlate with their cardiovascular risk. In this review, we discuss role and regulatory mechanisms of the CX3CL1-CX3CR1 axis in both localized and systemic effects of renal inflammation.Attraction, retention, and differentiation of leukocytes to and within the kidney are governed by chemokines. The chemokine CX3CL1  and its receptor CX3CR1 are exemplary in this regard as they are highly expressed and further upregulated in a range of kidney diseases. CX3CL1 is chiefly produced by renal endothelium and tubular epithelium, where it promotes leukocyte attraction. Recent data suggest that in addition to established soluble mediators, cellular interactions may enhance CX3CL1 expression. The receptor CX3CR1 is essential in myeloid phagocyte homing to the kidney at homeostasis, after acute cell depletion and in inflammation. CX3CR1 and its ligand are highly regulated in human kidney diseases such as IgA nephritis, systemic lupus erythematosus, and inflammatory conditions such as transplant rejection. A mechanistic role of CX3CR1 has been established in experimental models of nephrotoxic nephritis and renal candidiasis. It is debated in fibrosis. Recent publications demonstrate a role for CX3CR1 The chemokine (C-X3-C motif) Ligand 1  , reporting both deleterious (Li et al. + cells already in the absence of injury (Emal et al. + cells was unaffected. Cell depletion was reversible after fecal transplantation. After ischemia reperfusion injury, renal damage was significantly reduced in these mice. This proposes to investigate a possible impact of a plastic CX3CR1+ cell phenotype remains in this condition.An interesting aspect to non-renal modulators of the severity of renal IRI was published in 2017: depletion of gut microbiota by broad-spectrum antibiotic therapy decreased abundance of renal CX3CR1During acute kidney injury in polymicrobial sepsis, a combination of murine and human studies demonstrated worse outcome in mice lacking CX3CR1 (Chousterman et al. Escherichia coli, at least 1 day after infection (Hochheiser et al. The CX3CR1-CX3CL1 axis plays a differential role in renal infection. The lack of CX3CR1 was irrelevant for host-response in pyelonephritis caused by + phagocytes were central for contrast agent uptake, which occurred within minutes. For ensuing damage, the NLRP3 inflammasome was required, since IL-1β blockade was protective. The impact of the myeloid cells, but not necessarily monocytes, was demonstrated by decreased damage after anti-Ly6G treatment and, more specifically for phagocyte impact, by a beneficial effect of liposomal clodronate, which depletes these cells. It was not tested whether CX3CR1 was mechanistically involved, but such a role appears likely given its major role in renal homing.Another recent study addresses myeloid cells in kidney injury induced by radio-contrast agents (Lau et al. There are conflicting data on a mechanistic impact of CX3CR1 in renal fibrosis. While some studies suggest profibrotic action after ischemia reperfusion injury (Furuichi et al. Newer data from a different organ system, namely, the lung, may contribute to the understanding of the CX3CL1-CX3CR1 system in fibrosis. After demonstrating local and systemic CX3CL1 upregulation in human pulmonary fibrosis, the authors continued with functional studies of human monocytes from patients and controls. Here, they found differential effects of endothelial CX3CL1 with a pro-migratory effect on patient, but not control cells (Greiffo et al. + cells are also abundant in tissues lining the intestine. In their absence, the integrity of the intestinal barrier was compromised, resulting in an altered microbiome, endotoxemia in the portal blood, and aggravation of inflammatory conditions of the gut and liver (Schneider et al. CX3CR1+ cells in induction of tertiary lymphoid structures and IgA response in salmonella colitis and determined a mechanistic requirement of the CX3CR1HIGH expressing subtype (Koscsó et al. + cells in the intestine and possibly their renal homing, or the homing of T cells activated by them, may be tested as a mechanistic link between both diseases.A recent publication addresses the role of CX3CR1Another recently described systemic effect of CX3CR1 is dead cell disposal. An experiment addressing muscle cell death induced by influenza virus infection demonstrates that CX3CR1+ cells are required for phagocytic disposal (Runyan et al. The CX3CL1-CX3CR1 ligand-receptor pair plays a complex role in kidney disease and continues to raise interest in the treatment of vascular complications and in patients with kidney disease (Low et al. In the future, emerging technologies such as single-cell mRNA sequencing may allow better understanding of expression and gene regulation of the CX3CL-CX3CR1. As mRNA does not always correlate with functional receptor surface expression in terminally differentiated migratory cells, novel technological platforms for spatially resolved, multiplexed immunofluorescence may further our understanding by providing high-dimensional proteomic information on the single-cell level. Such information may close the gaps in our understanding of the complex biology of the CX3CL-CX3CR1 axis and its unique role in renal disease, thereby opening new avenues for more specific therapies of kidney disease.


V1.1 and β-catenin interact in various ways to control NMJ development. They differentially regulate nerve branching and presynaptic innervation patterns during the initial phase of NMJ formation. Conversely, they cooperate in regulating postsynaptic AChR clustering, synapse formation, and the proper organization of muscle fibers in mouse diaphragm. CaV1.1 does not directly regulate β-catenin expression but differentially controls the activity of its transcriptional co-regulators TCF/Lef and YAP. These findings suggest a crosstalk between CaV1.1 and β-catenin in the activity-dependent transcriptional regulation of genes involved in specific pre- and postsynaptic aspects of NMJ formation.Activity-dependent calcium signals in developing muscle play a crucial role in neuromuscular junction (NMJ) formation. However, its downstream effectors and interactions with other regulators of pre- and postsynaptic differentiation are poorly understood. Here, we demonstrate that the skeletal muscle calcium channel Ca  •Neuromuscular junction formation requires either muscle calcium or β-catenin signaling•V1.1 and β-catenin control presynaptic innervation patternsComplementary actions of Ca•V1.1 and β-catenin are crucial for postsynaptic AChR clusteringParallel actions of Ca•V1.1 differentially regulates activity of β-catenin targets TCF/Lef and YAPLoss of Ca Biological sciences; Developmental neuroscience; Cell biology; Developmental biology V1.1 , is a member of the L-type calcium channel family, whose expression is specific to skeletal muscle  antagonistically controlling presynaptic motor nerve branching and (b) cooperatively controlling postsynaptic AChR clustering and (c) that parallel acting CaV1.1 and β-catenin signaling pathways converge at the level of transcriptional regulators.The striking parallels in the presynaptic phenotypes in Cachanisms . Here, wV1.1 coordinates several aspects of the presynaptic motor nerve differentiation by initiating activity-driven muscle calcium signaling , then the concurrent loss of CaV1.1 and β-catenin is expected to alleviate the presynaptic defects. In order to examine this possibility, we cross-bred HSA-cre, β-catflox/flox, and CaV1.1+/− mice to generate muscle-specific β-catenin knockout mice in the CaV1.1−/− background  mice with CaV1.1+/−; TCF/Lef:H2B-eGFP (GFP positive) mice to ensure that GFP-expressing mice are hemizygous containing only one allele of TCF/Lef:H2B-eGFP transgene. If in mouse diaphragm β-catenin acted through TCF/Lef, we would expect an increased reporter gene activity in CaV1.1−/−; TCF/Lef:H2B-eGFP mice at E18.5.In the canonical Wnt signaling pathway, β-catenin primarily regulates TCF/Lef (T-cell factor/lymphoid enhancer factor)-dependent transcription. TCF/Lef transcription factors then bind to Wnt response elements to regulate Wnt target genes . To examith eGFP . BecauseV1.1−/− mice, average GFP fluorescent intensity of nuclei in CaV1.1−/−; TCF/Lef:H2B-eGFP was significantly reduced compared to that in CaV1.1+/?; TCF/Lef:H2B-eGFP controls at both E14.5 and E18.5. At both time points, the cumulative GFP intensity plots of nuclei in CaV1.1−/−; TCF/Lef:H2B-eGFP mice showed a left-shift indicating an overall decline in TCF/Lef activity in the absence of CaV1.1 function  and total YAP antibodies. In E14.5 CaV1.1−/− diaphragms, Western blot analysis showed no difference in expression of either pYAPS112 or total YAP. However, at E18.5, we observed a significant increase in both pYAPS112 and total YAP protein expression  (Birc5 and Bcl2l1)  and β-catenin-independent (TEAD) pathways, suggesting that YAP itself is the main target of CaV1.1.In order to test the β-catenin-dependent and -independent transcriptional activity of YAP, we performed qRT-PCR analysis of TEAD/YAP target genes (and Axl)  and of β Bcl2l1)  in CaV1.−/− mice C and 5D.V1.1 and β-catenin work together in various ways in the regulation of distinct features of neuromuscular synaptogenesis and that CaV1.1 differentially impacts the activity of transcriptional co-activators of β-catenin. Therefore, these findings reveal a crosstalk between two of the key regulatory mechanisms of NMJ formation and identify novel transcriptional targets of CaV1.1, likely involved in activity-dependent calcium signaling in muscle , the growth cones will keep growing, allowing sufficient time to cover the entire diaphragm.Interestingly, the active time window for their complementary presynaptic action appears to be limited to early stages of NMJ formation. Later in development, β-catenin knockout no longer is capable of rescuing the hyper-innervation defects caused by CaV1.1 and β-catenin in pre- and postsynaptic development. Whereas presynaptically CaV1.1 and β-catenin exert opposing effects on nerve branching, postsynaptically they show synergistic effects on AChR clustering. Both individual knockouts cause widening of the endplate band and a reduction in AChR cluster size. In the double knockouts, this phenotype is exaggerated in that there is an extreme reduction of AChR cluster number and size, with no recognizable restriction of the remaining clusters within the diaphragm muscle. This demonstrates that each signaling mechanism, CaV1.1-dependent and β-catenin-dependent, can maintain a certain degree of AChR clustering and patterning in the absence of the respective other. Thus, the two signaling mechanisms possess partially redundant actions on the formation and organization of AChR clusters. Previous cell culture studies showed the importance of CaV1.1 and β-catenin for agrin-induced AChR clustering   , little clusters  and 3 .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Mehmet M. Kaplan  and Cav1.1+/− mice. In parallel, HSA–Cre mice were crossed with β-catfloxed/floxed mice to generate HSA–β-catfloxed/+ mice, which were then crossed with Cav1.1+/− mice to obtain HSA–β-catfloxed/+; Cav1.1+/− mice. β-catfloxed/floxed; Cav1.1+/− mice were crossed with HSA–β-catfloxed/+; Cav1.1+/− mice to obtain HSA–β-catfloxed/floxed; Cav1.1+/−  embryos. At both E14.5 and E18.5, the observed number of HSA–β-cat−/−, Cav1.1−/− and HSA–β-cat−/−; Cav1.1−/− embryos collected, genotyped and analyzed during the course of this study corresponded to the expected ratio of Mendelian inheritance. Embryos heterozygous for HSA–β-cat and/or Cav1.1 did not show any phenotype. Control embryos analyzed were carrying at least one Cav1.1 and one β-catenin expressing allele. Cav1.1+/− mice were crossed with TCF/Lef:H2B-eGFP mice (purchased from Jaxson lab) to generate Cav1.1+/−; TCF/Lef:H2B-eGFP mice, which were then crossed with Cav1.1+/− mice to obtain Cav1.1−/−; TCF/Lef:H2B-eGFP embryos. Either Cav1.1+/+; TCF/Lef:H2B-eGFP or Cav1.1+/−; TCF/Lef:H2B-eGFP mice were used as controls. During mating periods sperm plugs were checked daily at 8:00 a.m. The day on which a sperm plug was detected was counted as embryonic day E0.5. Embryos were collected at the indicated days of pregnancy by cesarean section of sacrificed pregnant mice.All animal protocols conformed to the guidelines of the European Community (86/609/EEC) and were approved by the Austrian Ministry of Science (GZ: 2020-0.073.957 and GZ: 2020-0.073.961). Cav1.1-/- , HSA–β-c-/-  for 1 h at room temperature. Diaphragms were dissected in PBS and incubated in 0.1 M glycine in PBS for 1 h at room temperature, permeabilized and blocked in PBS containing 1% BSA, 5% normal goat serum (NGS) and 0.2% Triton X-100 overnight at 4 °C. Primary antibodies for rbSynapsin , rbNeurofilament  or rbMuSK  were applied at 4°C overnight. Then muscle samples were washed three times at 1 h intervals and incubated with anti-rabbit Alexa594 , anti-rabbit Alexa488 , anti-mouse Alexa594  according to the primary antibodies applied and/or α-BTX conjugated with Alexa488 or Alexa594  for 2 h at room temperature. After extensive washing, diaphragms were mounted in Vectashield. Images were captured on a Leica microsystems SP5 laser scanning confocal microscopy using LasAF acquisition software (Leica microsystems). Fluorescence was excited using the 488 nm and 561 nm laser lines and recorded at a bandwidth of 493-556 nm (green channel) and 566-752 nm (red channel), respectively. 8-bit images with 1024x1024 pixels were acquired at 400 Hz scan speed. Maximum projections of acquired z-serial images were assembled and analyzed with Metamorph software. Micrographs in t values of Hprt and gene of interest was used to calculate relative expression. Taqman gene expression assays were purchased from Applied Biosystems. Total RNAs were extracted from diaphragm muscles at E14.5 or E18.5 using RNeasy Fibrous Tissue kit (Qiagen). cDNAs were synthesized by Superscripts II Reverse Transcriptase (Invirtogen). We performed RT-PCR by using TaqMan gene expression assays in master mix with QuantStudio 6 Pro RT-PCR system . We measured duplicates for each sample and used Hprt (Hypoxanthine guanine phosphoribosyl transferase) as a reference gene. For relative RT-PCR, the difference between C7), mouse YAP , rabbit pYAP127 , or α-tubulin  overnight at 4°C and then with secondary antibodies conjugated with HRP for 1 hour at room temperature. ECL Supersignal West Pico was used to detect the chemiluminescence, which was visualized with ImageQuant LAS4000. Image Studio Lite was used to quantify Western Blot bands. Values were first normalized to tubulin and then to the average of control mice for each litter.E14.5 or E18.5 diaphragms were dissected in ice-cold PBS and scraped in RIPA buffer containing 50 mM TRIS-HCl (pH:8), 50 mM NaCl, 10 mM NaF, 0.5 mM EDTA, 10%SDS, 10% glycerol, 1%igepal, 1x protease inhibitor complete cocktail (Roch). Lysates were centrifuged at 13,100 g for 15 minutes at 4°C. A BCA assay (Thermo Scientific) was used to determine protein concentrations. We separated 10 μg of diaphragm lysates by 4–12% SDS-PAGE at 80V for 30 minutes followed by 160V for 90 minutes, transferred the protein to a PVDF membrane at 25V, 100 mA for 2,5-3 hours at 4°C using a semi-dry blot . The membrane was stained with mouse β-catenin , mouse active β-catenin  was used to present all data. The two-tailed student t-test was applied to assess statistical differences between two genotypes. Ordinary one-way ANOVA was applied to assess statistical significance for the differences between more than two genotypes. A p < 0.05 ANOVA value was considered as significant, as indicated in the figure legends. ANOVA was followed by a Tukey’s multiple comparisons post hoc test to reveal statistically significant differences among experimental groups.


Bmal1 and Per2 exhibited circadian expression in ALCs. Circadian disruption mice showed reduced amelogenin (AMELX) expression and enamel matrix secretion and downregulated expression of BMAL1, PER2, PPARγ, phosphorylated AKT1 and β-catenin, cytokeratin-14 and F-actin in ameloblasts. According to previous findings and our study, BMAL1 positively regulated PER2. Therefore, the present study focused on PER2-mediated ameloblast differentiation and enamel formation. Per2 knockdown decreased the expression of AMELX, PPARγ, phosphorylated AKT1 and β-catenin, promoted nuclear β-catenin accumulation, inhibited mineralization and altered the subcellular localization of E-cadherin in ALCs. Overexpression of PPARγ partially reversed the above results in Per2-knockdown ALCs. Furthermore, in in vivo experiments, the length of incisor eruption was significantly decreased in the circadian disturbance group compared to that in the control group, which was rescued by using a PPARγ agonist in circadian disturbance mice. In conclusion, through regulation of the PPARγ/AKT1/β-catenin signalling axis, PER2 played roles in amelogenin expression, cell junctions and arrangement, enamel matrix secretion and mineralization during ameloblast differentiation, which exert effects on enamel formation.Circadian rhythm is involved in the development and diseases of many tissues. However, as an essential environmental regulating factor, its effect on amelogenesis has not been fully elucidated. The present study aims to investigate the correlation between circadian rhythm and ameloblast differentiation and to explore the mechanism by which circadian genes regulate ameloblast differentiation. Circadian disruption models were constructed in mice for in vivo experiments. An ameloblast-lineage cell (ALC) line was used for in vitro studies. As essential molecules of the circadian system,  It is important to determine the correlation and regulatory mechanism between circadian rhythm and ameloblast differentiation as well as enamel formation.Circadian rhythm plays roles in physiological and behavioural processes in organisms. However, the association between circadian rhythm and tooth development, especially enamel development, has not been fully elucidated. As the hardest tissue in the human body, dental enamel forms to protect the tooth from external trauma as well as physical and chemical stimuli.Pparγ) in mice abolished or dampened circadian rhythm at both behavioural and cellular levels without affecting locomotor activity under regular LD conditions.17 The circadian gene PER2 might regulate the timing and steps of cell lineage and cell fate.18 PER2 interacts with PPARγ and regulates the role of PPARγ in the transcriptional activity of target genes.19 Researchers have found that PPARγ agonists regulate AKT activity, while the effect of PPARγ on AKT is still controversial. Activation of PPARγ by agonists such as rosiglitazone and pioglitazone induced AKT phosphorylation, thus activating AKT.21 In contrast, another agonist, troglitazone, inhibited the phosphorylation of AKT.22 Moreover, AKT could phosphorylate β-catenin directly at Ser552, a site that differs from the regular phosphorylation target of GSK3β in the WNT upstream signalling pathway, and influence the subcellular localization of β-catenin, which is independent of WNT upstream signalling.25 Our previous study showed that translocation of β-catenin into the nucleus could inhibit mineralization in ameloblasts.26 Whether PER2, as an essential molecule of the circadian system, is involved in the ameloblast differentiation process via the above series of molecular signalling pathways needs to be clarified. Therefore, the present study focused on PER2-mediated regulation of ameloblast differentiation and enamel development.Deletion of peroxisome proliferator-activated receptor gamma  mice , Bmal1-knockdown ALC cells  and control cells  were successfully constructed and verified  identified the top 20 enriched pathways, which included the Pi3k-Akt signalling, Wnt signalling and Pparγ signalling pathways  and presented immunofluorescence staining similar to that in ALC-Con-pEnCMV cells ,15 suggesting a correlation between circadian rhythm and ameloblast differentiation. In the present study, consistent with the above findings, we found that Per2 mRNA followed circadian rhythms in ALC cells. Notably, Per2, Pparγ and Amelx mRNAs followed similar fluctuating expression patterns, which indicated that PPARγ might be involved in the process of ameloblast differentiation. In addition, previous studies demonstrated that PER2 is dampened when the circadian rhythm is disrupted.32 Here, in circadian disruption mice, we observed decreased expression of PER2, PPARγ, AKT1-Ser473, β-catenin-Ser552 and AMELX in ameloblasts, reduced enamel matrix secretion and decreased incisor eruption, showing the possible association between these molecules and circadian disturbed ameloblast differentiation.34 Moreover, PPARγ is expressed in type II alveolar epithelial cells and is necessary for lung development,35 which indicates the involvement of PPARγ in epithelial cell differentiation. However, the role of PPARγ in ameloblasts, the most important epithelium-derived cell in enamel formation, remains unclear. Previous studies suggested that PER2 could directly regulate the transcriptional activity of PPARγ to target genes.19 Recent studies showed that AKT was related to ameloblast differentiation37 and could be regulated by PPARγ.22 In accordance with previous studies, we found that the knockdown of Per2 reduced the expression of PPARγ and AKT1-S473 and that overexpression of PPARγ increased the phosphorylation of AKT1. In addition, in neonatal mice with circadian disruption, the expression of PER2, PPARγ and AKT1-Ser473 was decreased, accompanied by inhibited β-catenin-Ser552 expression. AKT was reported to phosphorylate β-catenin directly at Ser552, thus influencing its relocalization.25 Moreover, our previous studies have demonstrated that activated WNT/β-catenin signalling and accumulation of β-catenin in the nucleus could inhibit mineralization in ameloblasts and cementoblasts.28 In the present study, we found increased translocation of β-catenin into the nucleus and reduced differentiation activity in ALC-Per2-sh cells. Meanwhile, overexpression of PPARγ partially rescued the inhibited differentiation activity in ALC-Per2-sh cells, which was characterized by enhanced AKT1 and β-catenin phosphorylation levels, decreased expression of β-catenin in the nucleus and increased ALP staining and ALP activity in the ALC-Per2-sh-pEnCMV-Pparγ group. In addition, a PPARγ agonist reversed the inhibitory effect of circadian disturbance on incisor eruption. Based on these findings, it was suggested that a novel signalling network could regulate β-catenin subcellular localization and thus influence ameloblast differentiation through the PER2/PPARγ/AKT1/β-catenin signalling axis. In addition, as shown in our study, overexpression of PPARγ simply partially rescued the impeded ameloblast differentiation resulting from Per2 knockdown, which implied that other signalling pathways were involved in the PER2-mediated ameloblast differentiation process. Therefore, further studies are required to determine the additional mechanism between PER2 and ameloblast differentiation.PPARγ, a transcription factor of the ligand-activated nuclear receptor superfamily, plays an essential role in pulp cell survival, pulp homeostasis and dentin mineralization.39 In the present study, obvious voids and decreased expression of cytoskeletal proteins CK14 and F-actin in ameloblasts of neonatal mice with circadian disruption indicated a disturbance of the ameloblast arrangement, which might be explained by the dysregulation of β-catenin. The change in E-cadherin subcellular localization in Per2-knockdown ALC cells also implied an effect on the cell adherent junctions. Establishing and maintaining cell polarity is critical for cell migration, differentiation and transportation of the secretory matrix.39 On the one hand, β-catenin in the cytoplasm is involved in normal cell adherent junctions and polarity arrangements, which ensure the secretion and transport of ameloblasts.40 On the other hand, β-catenin translocating to the nucleus activates Wnt signalling, which functions to regulate enamel mineralization.In addition, β-catenin functions in cell adhesion associated with E-cadherin, which is relevant to cell polarity and migration, and functions as a trigger in regulating WNT/β-catenin signalling.Overall, the study presented findings showing that circadian disruption in mice resulted in reduced enamel matrix formation and molecular dysregulation during amelogenesis. Moreover, it was further revealed that PER2, an essential molecule of the circadian system, exerted pivotal effects on the function and differentiation of ameloblasts, including amelogenin expression, cell junctions, enamel matrix secretion and mineralization, which were regulated by the PPARγ/AKT1/β-catenin signalling axis. However, the regulatory mechanism of circadian rhythm during tooth development has not been completely elucidated and needs further exploration.In the limitations of the present study, the results highlighted that PER2 played an essential role in ameloblast differentiation through the PPARγ/AKT1/β-catenin signalling axis. The findings not only provide insights to understand the association and mechanisms between circadian rhythm and amelogenesis but also propose a new perspective for the prevention of developmental defects of enamel.The reagents used for cell treatment were Lipofectamine 3000 , ascorbic acid , sodium β-glycerophosphate , and dexamethasone . Primary antibodies included PER2 , BMAL1 , PPARγ , AKT1-Ser473 , β-catenin-Ser552 , AKT1 , AMELX , β-catenin , LAMIN B1 , β-tubulin , GAPDH , CK14 , F-actin , and E-cadherin .n = 6 per group) used in different experiments are specified in Supplemental Table Kunming mice were purchased from the Provincial Centre of Disease Control  and housed at 22–24 °C and 55%–60% humidity under specific pathogen-free (SPF) conditions. Mice were maintained on a 12 h light/12 h dark cycle for at least 2 weeks before the study. Eight-week-old female mice were used in the experiments to estimate the incisor eruption rate: small dimples were created on the labial enamel surface of the alveolar bone crest, and the amount of lower incisor eruption was estimated after 1 week. The neonatal offspring of 10- to 12-week-old pregnant mice were used in the experiments to detect the development of tooth germs and ameloblasts. Mice had free access to a standard rodent chow diet and water and were randomly allocated into different groups. Normal conditions of a 12 h light/12 h dark cycle were disturbed by controlling lighting from 20:00 at night to 8:00 in the morning. The number and each group of mice  overnight, demineralized if necessary with 10% EDTA, dehydrated in a graded ethanol series, embedded in paraffin, and serially sectioned at 5 μm. The sections were stained with haematoxylin and eosin (HE) for histology and processed for immunohistochemistry and immunofluorescence using the antibodies described below.41 (donation from Professor Souichi Koyota) was maintained in DMEM/F12 (HyClone) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (HyClone) for regular culture.The ameloblast-lineage cell (ALC) lineFor the synchronization study, ALC cells were exposed for 2 h to serum-free medium to induce cell synchronization. After that, serum-free medium was changed to regular culture medium, and ZT0  was initiated after the medium change. The synchronized cells were cultured for 48 h and harvested every 6 h. Total RNA was extracted and analyzed by qRT-PCR.Per2 knockdown, the lentivirus expression vectors Per2-sh and control (empty vector) were purchased from GeneChem . ALC cells were transfected with the lentivirus particles and named ALC-Per2-sh and ALC-Con. After 72 h of transfection, puromycin (Invitrogen) was used to select and maintain the stably transfected cell lines for further experiments. The protocol for Bmal1 knockdown was the same as that for Per2 knockdown.For Pparg-m-3×FLAG and pEnCMV-MCS-3×FLAG (control vector) were purchased from Miaolingbio . ALC-Con and ALC-Per2-sh were transfected with plasmids diluted using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol.For PPARγ overexpression, the plasmids pEnCMV-−1 ascorbic acid (Sigma-Aldrich), 10 mmol·L−1 sodium β-glycerophosphate (Sigma-Aldrich), and 10 nmol·L−1 dexamethasone (Sigma-Aldrich) for the differentiation assay. The cell samples were collected for qRT-PCR, western blot, ALP staining and ALP activity  at days 3, 7, 14, and 21.When cells grew to near 60% confluence, regular culture medium was changed to differentiation medium containing 50 mg·LFor ALP staining, a BCIP/NBT Alkaline Phosphatase Colour Development Kit  was used according to the manufacturer’s protocols. Briefly, the cultured cells were fixed with 4% PFA for 15 min, rinsed with PBS, and incubated in BCIP/NBT staining solution for 15 min followed by rinsing with PBS again.ALP activity was quantified using the Alkaline Phosphatase Assay Kit . The optical density was read spectrophotometrically at 405 nm. Relative ALP activity was normalized to the total protein concentration.Each group of cells used in different experiments is specified in Supplemental Table The sections mentioned above “tissue preparation” were used in this part. After deparaffinization and rehydration, the slices were treated by autoclaving in citric acid buffer (pH 6.0) for 5 min to expose antigens. Immunohistochemistry was performed with a streptavidin-peroxidase kit . Samples were incubated overnight at 4 °C with the following primary antibodies: PER2 (1:200), BMAL1 (1:500), PPARγ (1:400), AKT1-Ser473 (1:300), and β-catenin-Ser552 (1:400). After incubation, the samples were managed according to the manufacturer’s protocols. The colour reaction was performed using diaminobenzidine . The sections were then counterstained with haematoxylin.42The immunohistochemistry paraffin sections were subjected to microscopic analysis. Light yellow to brown staining was recorded as positive immunostaining. The mean optical density of proteins was measured by Image-Pro Plus 6.0 .For CK14 and F-actin immunofluorescence staining, paraffin sections were treated with anti-CK14 mouse monoclonal antibody (1:500) followed by goat anti-mouse IgG secondary antibody Alexa Fluor® 488  and anti-F-actin mouse monoclonal antibody (1:50) followed by FITC-conjugated goat anti-mouse IgM .For ALC cell immunofluorescence, cells were fixed with 4% PFA for 20 min, rinsed with PBS, and blocked in serum in a 37 °C incubator for 1 h. Anti-E-cadherin mouse monoclonal antibody (1:50) and anti-β-catenin (1:100) rabbit antibody were used overnight incubation at 4 °C. Cells were then incubated with goat anti-mouse and goat anti-rabbit IgG secondary antibody Alexa Fluor® 488  for 1 h at room temperature followed by staining with DAPI.The immunofluorescence paraffin sections and cells were analyzed with a confocal laser scanning microscope .Total proteins of tooth germs and ALC cells were extracted using RIPA buffer supplemented with 1 mM PMSF. NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) were used to extract cytoplasmic and nuclear proteins according to the manufacturer’s protocol. The concentration of all protein samples was measured by a BCA protein assay kit . Equal quantities of proteins were subjected to SDS-PAGE and then electrophoretically transferred onto PVDF membranes. Specific primary antibodies included PER2 (1:1 000), BMAL1 (1:1 000), PPARγ (1:1 000), AKT1-Ser473 (1:500), AKT1 (1:500), AMELX (1:1 000), β-catenin (1:1 000), β-catenin-Ser552 (1:1 000), LAMIN B1 (1:500), β-tubulin (1:1 000) and GAPDH (1:5 000). The immunoreactive proteins were detected by an ECL system (Thermo Fisher Scientific). GAPDH was used as the normalized control for total protein lysis buffer. LAMIN B1 was set as the normalized control for nuclear protein lysis buffer, while β-tubulin was set as the normalized control for cytoplasmic protein lysis buffer.Per2-sh cells was extracted using TRIzol reagent  and then used for mRNA sequencing . The criterion for differential gene expression was set as |fold change| > 1 and false discovery rate less than 0.05.Total RNA of ALC-Con and ALC-−Δ(ΔCt), where ΔCt = Ct (mRNA) − Ct (β-actin). PCR primers for each gene are listed in Supplemental Table Total RNA of tooth germs and ALC cells was extracted using TRIzol reagent. One microgram of total RNA was reverse-transcribed into cDNA using Hieff® 1st Strand cDNA Synthesis SuperMix for qPCR . qRT-PCR was performed using Hieff® qPCR SYBR Green Master Mix . The relative gene expression was calculated using the equation 2t-test. Multiple comparison tests were performed using one-way ANOVA with Bonferroni’s test for more than two groups. For all tests, a P value < 0.05 was considered statistically significant.All quantitative experiments were performed in triplicate. GraphPad Prism 7.0 software was used for data analyses and graphing. All quantitative data are presented as the mean ± s.e.m. (standard error of mean). Statistical comparisons between two experimental groups were analyzed by unpaired, two-tailed Student’s supplemental materials