The threshold value of the delayed cracking of M10B significantly changed along with relative humidity. When relative humidity increased from 60% to 80%, the threshold dropped sharply from 0.63 σb to 0.52 σb. We define 80% relative humidity as the ‘threshold humidity’ for M10B.Medium Mn steels have been considered as the next-generation materials for use in the automotive industry due to their excellent strength and ductility balance. To reduce the total weight and improve the safety of vehicles, medium Mn steels look forward to a highly promising future. However, hydrogen-induced delayed cracking is a concern for the use of high strength steels. This work is focused on the service characteristics of two kinds of medium Mn steels under different relative humidity conditions . Under normal relative humidity (about 40%) at 25 °C, the hydrogen concentration in steel is 0.4 ppm. When exposed to higher relative humidity, the hydrogen concentration in steel increases slowly and reaches a stable value, about 0.8 ppm. In slow strain rate tensile tests under different relative humidity conditions, the tensile strength changed, the hydrogen concentration increased and the elongation decreased as well, thereby increasing the hydrogen embrittlement sensitivity. In other words, the smaller the tensile rate applied, the greater the hydrogen embrittlement sensitivity. In constant load tests under different relative humidity conditions, the threshold value of the delayed cracking of M7B  steel maintains a steady value of 0.82 σ R. To date, several researchers have investigated the effects of initial martensite microstructure [With the rapid development of vehicles, the lightening of weight, energy saving and safety improvement are urgently required in the automobile industry. Over recent decades, automobile manufacturers have mainly considered the cost, formability and corrosion resistance of advanced high strength steels (AHSSs) with a good combination of tensile strength and ductility. In general, AHSSs are classified into categories of first-generation AHSSs, second-generation AHSSs and third-generation AHSSs (3rd-G AHSSs) . Of the tructure ,5,6, timtructure ,9,10,11,tructure  and cooltructure , and alltructure , Al [14,tructure , and Si tructure , on the Another phenomenon for this type of material is the strength decline due to the presence of hydrogen . Dutton As for corrosion behaviour in the service environment, many researches has studied the effect of dry–wet cycle, full immersion and simulated coastal-industrial atmosphere for different kinds of steels, such as carbon steel , and MnCRH values below 90% and above 20% are to be expected in almost any part of the world. In particular, early morning humidity of 80 to 100% is common in most coastal and low-lying areas. But only a few studies have focused on the atmospheric corrosion of MMSs and the effect of RH on service performance. In this work, we will systematically investigate the influence of hydrogen from different RH conditions on the hydrogen-induced delayed cracking of MMSs.MMS thin plate were supplied by Baoshan Iron&Steel Co., Ltd. In this study, the effect of the RH on hydrogen-induced delayed cracking of MMSs was investigated. The main differences between the two kinds of Mn steels are the Mn and V content. From the microstructure observation by our previous work , M7B shaThe heat treatment involved hot-rolled, intercritically batch-annealed, and cold-rolled processes, as follows. The slabs, after steelmaking, were heated to 1473 K for 2 h and hot-rolled at a temperature of 1153 K. The intercritically batch annealing was conducted at 893 K for 12 h. The thickness for hot-rolled sheets was 2.8 mm, consisting of austenite and ferrite. Then the sheets were pickled and cold-rolled to a thickness of 1.4 mm. The cold-rolled MMS sheets were heated to 893 K and held for 12 h. After heat treatment by manufactures, we received the thin plate medium Mn steels with a dual phase of austenite and ferrite . 4-7H2O + 45 g NiCl2-6H2O + 40 g H3BO4/L, with a current density of 10 mA/cm2 for 60 s. The hydrogen-charging solution on the charging side was 0.2 molL−1 NaOH + 0.22 gL−1 thiourea (H2NCSNH2) with 5 mAcm−2 charging current density at room temperature, approximately 25 °C, while the solution in the escaping side was 0.2 mol L−1 NaOH [The tensile test was prepared to obtain the mechanical properties and stress–strain curves of MMSs. X-ray diffraction  was performed to obtain the volume fraction of retained austenite. A study on the hydrogen permeability of specimens was conducted to obtain the effective hydrogen diffusion coefficient . The samL−1 NaOH .2SO4) solution was kept at 100% RH [Saturated aqueous solution of inorganic salts offers convenient secondary standards for the calibration of instruments for the measurement of RH . Saturat 100% RH . −1, and the measurement time is 20 min. Samples 1.3 mm thick  were exposed to different humidity conditions to observe the content of hydrogen entry. Samples were taken out to measure hydrogen content (diffusible hydrogen in wppm) after different exposing times: 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h. Due to the dispersion of experimental data for the hydrogen test, the average value of minimum three samples was used and scale bar was shown in −4 s−1 in air, 10−5 s−1 and 10−6 s−1 for M7B, 10−4 s−1 and 10−6 s−1 for M10B in wet air. Two parallel samples are used in CLT and SSRTT and the results showed great consistency. To ensure the data accuracy and repeatability, three specimens were prepared for hydrogen concentration after test. The specimen was about 1cm long, which was cut near the fracture. The prepared specimens were immediately washed with acetone and absolute ethanol, and then were blown dry with cold air [Constant load test (CLT) is a reliable method to obtain the threshold stress and evaluate the sensitivity of hydrogen-induced delayed cracking. Considering the experimental cycle and accuracy, the cut-time was set to no more than 200 h. The slow strain rate tensile test (SSRTT) is a classical method for exploring susceptibility to hydrogen embrittlement, which was determined by measuring the fracture strength drop, the reduction of the area and the elongation of the specimen under different humidity conditions in this study. The tensile rate was 10cold air . The aboTo facilitate clamping, a circular hole was machined on both sides of the sample, whose specific dimensions are designed according to ASTM E8. The gauge part of the sample was 20 mm long, 5 mm wide and approximately 1.5 mm thick. The schematic diagram of MMSs tensile specimen can be found in our previous work . Their sAccording to an early study, M7B and M10B are uniformly distributed with globular-shaped ferrite and retained austenite, with a grain size of about 1–3 μm. They consisted of a dual phase of austenite and ferrite, similar to other MMSs. XRD can be used to measure the volume fraction of retained austenite in MMSs. According to The nominal stress–strain curves of MMSs are shown in 0.63 was the corresponding time of Ia/I∞ = 0.63 using the Equation (1) [−7 cm2/s and 4.41 × 10−9 cm2/s, respectively.The different content of austenite contributed to effective different hydrogen diffusion coefficient for the MMSs. According to the result of hydrogen permeation test, as shown in tion (1) ,33,34,35After exposing to different humidity environments, samples were taken out to analyze their hydrogen content, which enters the material during the exposure process. 0: the length of gauge part before SSRT; l’: the length of the gauge part after SSRT; δ: the elongation of the specimen; δ0: the elongation of the specimen in air; δH: the elongation of the specimen under different relative humidity condition.−5 s−1 to 10−6 s−1, the material elongation decreases more obviously and the hydrogen embrittlement sensitivity increases significantly. The change of RH and strain rate have little effect on the fracture strength of M7B, but significant effect on the elongation and hydrogen embrittlement sensitivity. It can be seen that the increase in RH and the decrease in strain rate will lead to an increase of hydrogen concentration and hydrogen embrittlement sensitivity of the material, thus reducing its service performance.When the strain rate decreases from 10By comparing the tensile fracture of M7B under different conditions, SEM analysis was carried out on the tensile fracture in air and on the tensile specimen with a slow strain rate under different humidity conditions, shown in According to Cleavage/quasi-cleavage fracture is classified as a brittle transgranular fracture by separation across well-defined crystallographic planes, which occurs under high stress and related with dislocations . Quasi-c2 + M → H2M, H2M + M → 2HadM, HadM → MHab, MHab→M+H, where Had/Hab mean adsorbed/absorbed hydrogen.The area of brittle zone tends to increase with the increase of RH, which accelerated the entry of hydrogen into the sample by hydrogen adsorption and absorption. The detailed process is shown in The fracture surface shows fully ductile mode characterized with dimples in Akiyama et al. suggested SSRT under a wet condition using specimens with corrosion products formed under actual atmospheric environments or cyclic corrosion tests, to reproduce enhanced hydrogen entry influenced by the corrosion layer and to homogenize the hydrogen distribution in a specimen . Hydroge−4 s−1 and 10−6 s−1), while −4 s−1, with the increase of RH, the fracture strength of M10B steel reduced significantly. Given that the decrease of elongation causes the earlier fracture during the plastic deforamtion at a strain rate of 10−6 s−1, the fracture strength changes little but the hydrogen concentration and hydorgen embrittlement sensitivity gradually increases with the increase of RH. When the strain rate decreases from 10−4 s−1 to 10−6 s−1, the material elongation decreases more obviously and the hydrogen embrittlement sensitivity increases significantly. The increase of RH and decrease of strain rate accelerate the hydrogen degradation of material by reducing the elongation and increasing the hydrogen embrittlement sensitivity.By comparing the tensile fracture of M10B under different conditions, SEM analysis was carried out on the tensile fracture in air and on the tensile specimen with a slow strain rate under different humidity conditions; the fracture surface of M10B was shown in −4 s−1, shown in −4 s−1 to 10−6 s−1 at 60% RH, the fracture surface of M10B shows a total brittle mode characterized by transgranular (cleavage/quasi-cleavage) and intergranular fracture in As shown in b (the ratio of applied stress to tensile strength) and fracture time tF is shown in For the CLT, the test protocol followed was as discussed above. The results of the CLT test for M7B and M10B under three RH conditions are recorded and the relationship between applied stress σ/σThe corresponding stress threshold values under different RH conditions were obtained. Under the conditions of 60% RH, 80% RH and 100% RH, the threshold stress factors were 0.84, 0.8 and 0.79, respectively. It can be seen that for M7B steel, there was no obvious change in the threshold stress value, along with the increase in RH. In a high humidity-containing service environment for M7B, the threshold stress does not significantly decrease with an increase in RH, and the change in hydrogen content is not obvious; thus, M7B has a good delayed cracking resistance.Similarly, the corresponding stress threshold values of M10B under different RH conditions were obtained. Under the conditions of 60% RH, 80% RH and 100% RH, the threshold stress factors were 0.63, 0.53 and 0.52, respectively. Under the same humidity environment, the threshold stress factor of M10B is less than that of M7B. It can be seen that, for M10B steel, the change in the threshold stress value decreased significantly with an increase in RH. At 80% RH, the threshold stress value decreases to a large extent, while hydrogen concentration increases synchronously, which can be maintained in a higher humidity range. 80% RH can be taken as the critical humidity of M10B. The service performance of materials in the environment fluctuates greatly at this level of humidity.−7 cm2/s and 4.41 × 10−9 cm2/s, respectively. The behavior of hydrogen diffusion is also influenced by different content of austenite, which hinders the diffusion of hydrogen.Different Mn contents will cause changes in the volume fraction of austenite. The content of austenite in M10B steel is much higher than that in M7B. The elongation of M10B is higher than M7B, because more austenite tends to deform during the tensile test. The effective hydrogen diffusion coefficients of M7B and M10B steel are 1.08 × 10When exposed to air, the hydrogen concentration in the M7B sample is 0.4 ppm. When M7B is exposed to 60% or higher RH, the hydrogen concentration increased to 0.8 ppm. For M7B, an increase of RH and a decrease of strain rate accelerate the hydrogen degradation with an increase of hydrogen concentration of the material, thus reducing its service performance.−4 s−1 with the increase of relative humidity and hydrogen concentration, resulting in final failure. Elongation decreases gradually under a strain rate of 10−6 s−1, with higher relative humidity increasing hydrogen content in M10B.Similar hydrogen degradation was also found in M10B, as fracture strength and elongation decrease gradually under strain rate of 10Due to the entry of hydrogen into samples in environments with high relative humidity, fracture morphology changes from ductile mode with obvious dimples to brittle mode characterization with intergranular fracture.For M7B, the threshold stress does not significantly decrease with an increase in RH, but shows a better delayed cracking resistance than M10B. Eighty percent RH can be accepted as the critical humidity for M10B.In this paper, the environmental corrosion sensitivity and hydrogen-induced delayed cracking behavior of M7B and M10B steels were studied by a combination of characterization methods, including X-Ray Diffraction analysis, the hydrogen permeation test, the humidity exposure test, the SSRTT and the CLT under humidity conditions. The conclusions are as follows:


A highly sensitive Au-graphene structure D-type fiber surface plasmon resonance biosensor is presented in this study to specifically detect biomolecules. The method of growing graphene is employed directly on the copper, and then a gold film of optimum thickness is sputtered, and the copper foil is etched to obtain the structure. This method makes the contact closer between the gold layer and the graphene layer to improve surface plasmon resonance performance. The performance of this type of surface plasmon resonance (SPR) sensor has been previously verified both theoretically and experimentally. With the proposed Au-graphene structure D-type fiber biosensor, the SPR behaviors are obtained and discussed. In the detection of ethanol solution, a red shift of 40 nm is found between the refractive index of 1.3330 and 1.3657. By calculation, the sensitivity of the sensor we designed is 1223 nm/RIU. Besides, the proposed sensor can detect the nucleotide bonding between the double-stranded DNA helix structures. Thus, our sensors can distinguish between mismatched DNA sequences.  Recently, surface plasmon resonance (SPR) technology has aroused huge attention because of its application in the detection of various physical, chemical and biological parameters ,2. The oCompared with the conventional prism-based sensors, the fiber-optic SPR sensor is particularly significant because of its integrated device, easy operation and excellent flexibility for remote detection and immune electromagnetic interference ,6. As a −1), which increases the area of the contact with analyte, allowing it to absorb more molecules [In recent years, two-dimensional (2D) nanomaterials have been widely studied because of their potential applications in transistors and opto-electrical converters ,17,18. Golecules . Moleculolecules ,23. Grapolecules . these aolecules . Subsequolecules . The graolecules ,28.Over the past decades, SPR based biosensors have aroused increasing attention because of their important role in real-time and level-free detection ,29. InduBesides, in order to investigate the practical application of our SPR sensor, we also studied the specificity of the sensor. In optical methods, label-free SPR is a convenient tool for DNA binding detection ,30. DNA 3 solution to protect the integrity of the graphene and gold layers, and this foil was allowed to stand for 4 h. After waiting for the copper foil to completely corrode, the resulting Au-graphene layer structure was transferred to the D-type region (60 °C for 30 min) of the fabricated D-type fiber using a transfer method to obtain the sensor we wanted to manufacture. The entire sensor production flow chart is shown in We took a piece of copper foil of 0.5 cm × 1.5 cm size. First, to remove the stain, the foil was soaked three times with acetone for 15 min each time. Subsequently, the copper foil was cleaned three times with alcohol for 15 min each time. Finally, the foil was ultrasonically cleaned three times with deionized water for 10 min each time. The single layer of graphene was grown on clean copper by a chemical vapor deposition system, and the obtained optimum thickness of the Au film was calculated by sputtering on the graphene using a magnetron sputtering system. The copper foil with graphene and Au film was suspended in a suitably configured FeClAs shown in Scanning electron microscopy (SEM) was used to characterize the surface morphology of the D-zone of the sensor. The Raman spectrometer was used to collect the Raman spectra in the D-zone of the sensor to determine the presence of graphene and biomolecules. The laser wavelength used was 532 nm, the integration time was 4 s, and the effective power of the laser source was maintained at 50 mW.To detect the specificity of the sensor, the previously prepared sensor was used as a sample, and the probe DNA was fixed to the D-type region of the sensor by 1-butyric acid succinimide ester (PBASE). Through the real-time detection of biosensors, we obtained the hybridization kinetics of tDNA and probe DNA. Probe DNA and tDNA were purchased from Sangon Biotech Inc. . PBASE, dimethyl sulfoxide (DMSO), and phosphate buffered saline (PBS) were obtained from Sigma-Aldrich . The sequence of the probe DNA was 5′-AGT ACA TCA CAA CTA-3′, while the sequence of the complementary tDNA was 5′-TAG TTG TGA TGT ACT-3′, and the mis DNA was 5′-CCT CCA CAG CTC GAG-3′.Graphene’s properties give it a significant advantage in fixing DNA molecules . The strThe following is the specificity detection of the D-type fiber biosensor for DNA hybridization. We used PBASE as an adhesive to attach the probe DNA to the surface of graphene so that we could fix the probe DNA without creating defects . First, To assess the performance of the sensors developed here and to verify that SPR is better represented, the normalized transmission spectra of the sensors were collected in an ethanol solution with an RI of 1.3330 to 1.3657, as shown in In addition to sensitivity, stability and repeatability are vital parameters for estimating sensing performance. Under the same conditions, the D-type fiber sensor developed here is studied in the normalized transmission spectrum of the 1.355 RI alcohol solution for six cycles, and the reproducibility of the sensor is achieved under the same RI. As shown in To further evaluate the performance of the sensor in DNA detection, we used this sensor for real-time detection of FC DNA with 100 nm concentration and recorded the normalized projection spectra. We can see in To sum up, a highly sensitive Au-graphene structure D-type fiber SPR biosensor is presented here. The Au-graphene structure obtained here makes the contact more closely between the gold layer and the graphene layer. Graphene makes the designed sensor higher in sensitivity. The proposed sensor exhibits excellent stability and repeatability. The designed sensor can detect nucleotide bonding between the dsDNA helix structures, and therefore mismatched DNA sequences can be detected.


Sarcoidosis is a complex disease that belongs to the vast group of autoinflammatory disorders, but the etiological mechanisms of which are not known. At the crosstalk of environmental, infectious, and genetic factors, sarcoidosis is a multifactorial disease that requires a multidisciplinary approach for which genetic research, in particular, next generation sequencing (NGS) tools, has made it possible to identify new pathways and propose mechanistic hypotheses. Codified treatments for the disease cannot always respond to the most progressive forms and the identification of new genetic and metabolic tracks is a challenge for the future management of the most severe patients. Here, we review the current knowledge regarding the genes identified by both genome wide association studies (GWAS) and whole exome sequencing (WES), as well the connection of these pathways with the current research on sarcoidosis immune-related disorders. Sarcoidosis belongs to the group of multifactorial diseases and implicates various etiological triggers, including environmental , infectious, and genetic factors ,2. This Genetic loci linked to specific phenotypic traits have largely been determined by genome-wide association studies (GWASs) in many multifactorial common and rare diseases . The priBTNL-2 (Butyrophilin-like 2 protein), a cofactor of CD80/CD86 acting as a negative regulator of T-cell activation during the presentation of antigens by APCs (antigen presenting cells) [BTNL-2 SNP related to sarcoidosis, rs2076530, induces the activation of a cryptic splice site located 4 bp upstream of the exon five-intron wild-type donor splice-site site and a premature a premature stop in the spliced mRNA [BTNL-2 protein and disturbs the regulation of T-cell activation. This is probably one of the significant success stories of GWAS in sarcoidosis allowed by the analysis of 947 independent cases of familial and sporadic sarcoidosis. Nevertheless, the BTNL-2 rs2076530 (G16071A) variant is commonly observed in the baseline population, with a mean minor allele frequency (MAF) of 0.4194 (41.94%), varying from 0.3304 (33.04%) to 0.4596 (45.96%) in African and European populations, respectively [BTNL-2 variant could not explain the autosomal dominant predisposition to sarcoidosis in affected families [BTNL-2 remains an interesting candidate gene that may explain a basal predisposition to the disease, but requiring additional genetic factors until reaching a threshold of predisposition, a prerequisite for inducing the abnormal accumulation of granuloma.As introduced previously, variations in susceptibility to sarcoidosis in different ethnic groups, familial clustering, and increased concordance in monozygotic twins are the three main observations suggesting that a genetic background may act synergistically to environmental factors for triggering the disease . Genome-g cells) ,18,19. Tectively ,22. Thisfamilies . HoweverBTNL-2 history is a critical step for understanding that sarcoidosis, as many multifactorial and common diseases, has a complex genetic architecture and, as a result, DNA analysis to predict disease will implicate a panel of genes and the establishment of a polygenic risk score to identify patients who could benefit from the knowledge of their susceptibility to the disease, but also those with particular and/or severe forms of the disease. In sarcoidosis, specific class HLA class II antigens have been associated with an increased risk of disease occurrence and some of them with certain sarcoidosis phenotypes [An extensive description of the enotypes ,25. The enotypes . Regardienotypes ,28. An aenotypes ,30. As denotypes . Variousenotypes ,33. Takeenotypes ,34. An aenotypes . Interesenotypes . Even inenotypes .Behind the complexity of the HLA genes allelic combinations and the relative risks involved, interesting observations link sarcoidosis to infectious agents and autoimmunity. The immune response to mycobacterial antigens has been shown to be dependent on DRB1 haplotypes, with a more significant association to DRB1*0301, the one associated with Lofgren syndrome and resolving disease . The proBTNL-2 was the most extensively analyzed in sarcoidosis. Other genes, such as NOTCH4 (neurogenic locus notch homolog 4), TAP2 , TNFα (tumor necrosis factor α), LTA (lymphotoxin α), HSPA1L (heat shock 70 kDa protein 1L), and several open reading frames have been putatively associated with the immune process involved in the disease [NOTCH4 (rs715299) has been shown to be associated with sarcoidosis in African Americans and European patients independently from others in the MHC region in the same sample [NOTCH4 protein, but Notch family proteins are involved in regulation of T-cell immune response and are related to other autoimmune diseases such as multiple sclerosis and lupus [TAP2  moves protein fragments from foreign invaders into the endoplasmic reticulum in close connection with major histocompatibility complex (MHC) class I proteins and TAP2 polymorphisms have been highlighted in European cases of sarcoidosis [Within the region of the major histocompatibility complex and between the regions of class I and class II genes, several genes have been described that appear to be involved in both global and specific inflammatory responses. Immune-related functions are characteristic to molecules belonging to major histocompatibility complex (MHC) class III as genes for some complement proteins, cytokines, and heat shock proteins lie in the region . Among t disease ,41. A spe sample . To datend lupus . TAP2  probably generate a background of genetic predisposition for the development of sarcoidosis, but other GWAS studies performed identify a set of genes and loci that may contribute synergistically to the genesis of the disease. We may imagine a complex polygenic model in which the progressive accumulation of mutations in various genes increases the relative risk of the onset of sarcoidosis . VariousANXA11 (Annexin A11), a gene located on chromosome 10q22-23 and implicated in several biological pathways, including apoptosis and proliferation [2+-dependent processes and mainly apoptosis [ANXA11 R230C variant might be owing to a defective apoptosis and/or autophagy inside granuloma, thus inducing a persistent inflammatory reaction and a higher risk of onset of sarcoidosis and poorer outcome [One of the most relevant associations targets feration ,55. GWASferation ,57. A siferation . A non-cferation . Annexinferation . This inpoptosis . Interespoptosis ,62. S100poptosis . Taken t outcome .CCDC88B (coiled-coil domain-containing protein 88B) located on chromosome 11q13, which encodes a positive regulator of T-cell maturation and inflammatory function involved in inflammatory bowel diseases [Another interesting gene identified by GWAS was diseases ,66. Togediseases .IL23R gene, encoding the receptor of interleukin 23, which is involved in differentiation of the differentiated Th17 T cell population, and the development of autoimmune and inflammatory disorders has been identified as susceptibility variants for sarcoidosis [Three SNPs  are located in the coidosis . Two of coidosis ,70; and coidosis . Interescoidosis . IL-23 scoidosis . In a lacoidosis . IL-12 dcoidosis .Various SNPs located near the nuclear factor kappa B subunit 1 (NFKB1) and MANBA (beta-D-mannoside mannohydrolase) genes on chromosome 4q24 showed a significant association with sarcoidosis, but no functional relationship has been established despite the well-known role of NF-kappa-B, a pleiotropic transcription factor in a vast array of stimuli related to inflammation, immunity, differentiation, cell growth, and apoptosis ,21. NFKBA very common variant (rs3184504) of the SH2B3 (SH2B adaptor protein 3) on chromosome 12q24.12 has been considered as a susceptibility factor for sarcoidosis . DespiteThe cytoskeleton, which includes microtubules and actin filaments, is crucial for many cellular processes, such as proliferation and differentiation. An interesting association has been shown in a German study between the RAB23 gene on chromosome 6p21 and sarcoidosis, focusing on a non-synonymous SNP variant, rs10484410 (G207S) . RAB23 pTAB1/TAB2, defined as TAK1 (TGFβ-activated kinase activators) binding proteins 1 (TAB1) and 2 (TAB2), results in macrophage death [NOD2 germline mutations are associated with Crohn disease and early onset sarcoidosis or Blau syndrome, a rare systemic inflammatory disease characterized by early onset granulomatous polyarthritis, uveitis, and dermatitis [NOD2 in sarcoidosis, sarcoid-related uveitis, and orofacial granulomatosis, including those associated with Blau syndrome, suggesting that NOD2-related pathways could play an important role in basal predisposition to the risk of occurrence of uncommon forms of sarcoidosis [Among the studies using the GWAS technique, paradoxical results have been obtained on the role of NOD2 (nucleotide-binding oligomerization domain-containing protein 2) in sarcoidosis . NOD2 prge death . NOD2 germatitis ,86. Studcoidosis ,88,89. TZNF592 were found to be associated with neurosarcoidosis in African American and European American patients [Other genes have been suggested as susceptibility factors for sarcoidosis from single, unreplicated association studies. Among these, the XAF1 gene (X-linked inhibitor of apoptosis or XIAP associated factor 1) located on chromosome 17 shares an interesting intronic variant (rs6502976) in an African American cohort of patients . Functiopatients . Homozygpatients .Polymorphisms of CCR5 (C-C chemokine receptor type 5), predominantly expressed on T cells, macrophages, and dendritic cells, have been identified in a Czech series of patients, and further confirmed as being more specifically associated with lung disease progression ,96. CCR5Taken together, GWAS studies were able to identify minor and/or major genetic factors of susceptibility to sarcoidosis, but were unable to identify strong genetic triggers, which may explain the occurrence of familial forms of the disease. As shown in the Swedish study including 23,880 participants with sarcoidosis, the relative risk associated with having at least one first-degree family member with sarcoidosis was estimated at 3.73, and increased to 4.69 if at least two first-degree family members were identified as having sarcoidosis . The FreGenetic screening of a complex disease by WES may be firstly done for the search of de novo mutations by analyzing a classical familial situation, the so-called trio, in which a child, despite healthy parents, develops the disease at an early age. Subtraction of parental genotypes from the affected child allows the identification of new mutations putatively linked to the disease. This protocol has been applied in three trios identified by the French GSF and RespiRare networks and allowed a focus on a series of genes sharing de novo and recessive mutations in affected children . This sthttps://string-db.org/) protein network, mapped to pathway gene sets curated in the Wikipathway 2016 database and submitted to an additional gene set over-representation pathway analysis using ConsensusPathDB, a meta-database that integrates different types of functional interactions from multiple data resources [MTOR, RICTOR, and MLST8) to be the top three protein complexes that encode the variant-affected transcripts. While this familial analysis confirms the genetic heterogeneity commonly reported in sarcoidosis, it allowed to stratify heritable pathogenic variants in mTOR and Rac1 signalling pathways, as well as in autophagy and vesicular transport processes. These data were highlighted by one of the critical mouse models of granuloma induced by the deletion of Tsc2 (tuberous sclerosis 2), a negative regulator of the metabolic checkpoint kinase mTORC1 belonging to the mTOR complex [This first study was followed by a WES screening of five families sharing at least two first-degree affected relatives and comparing the genotypes of patients to intrafamilial healthy controls . Analysiesources . From th complex . Deletio complex ,111,112. complex . Similar complex ,105,113.A recent WES analysis focused a Finnish series of 72 patients selected based on disease activity, half resolved and half persistent, in order to identify prognosis genetic markers . This woIL17 gene within a complex containing RORγt (retinoic acid-related orphan receptor gamma), the master transcription factor of Th17 cells promoting Th17 cell differentiation [All the genetic data confirm the very strong genetic heterogeneity in sarcoidosis. Our first impression is that we must probably distinguish the familial and sporadic forms and, in each of these groups, the particular clinical expression of the disease. The challenge of genetic studies is to identify biological markers for early diagnosis of the disease and, more reasonably for sarcoidosis, to define the genetic profiles that may be associated with severe and persistent disease, and more specifically with multi-organ lesions. We recently hypothesized that genetic mutations observed in familial sarcoidosis mostly occur on autophagy-related genes and two regulatory hubs, mTOR and Rac1, which can hamper the clearance of pathogens (opportunistic viruses or bacteria) or non-organic particles and alter both macrophages and T cell responses . Rac1, antiation . As for DDIT4 (DNA damage inducible transcript 4 gene), also called Rtp801, encoding a factor that turns off the metabolic activity triggered by mTOR by stabilizing the TSC1–TSC2 inhibitory complex [DDIT4 is an essential mediator of cigarette smoke-induced pulmonary injury and emphysema and promotes NFκB activation, alveolar inflammation, oxidative stress, and apoptosis of alveolar septal cells [Variations in the direct regulation of mTOR probably have major consequences on autophagy. In two families with a severe form of sarcoidosis, we demonstrated deleterious mutations in  complex . Interesal cells . As suggal cells . In sarcal cells ,124. Theal cells . Similaral cells .The analysis of the transcriptome makes it possible to evaluate the over expression or, on the contrary, the loss of expression at the level of the messenger RNA of certain genes by comparing the samples of patients with those of control subjects. This method is complementary to genomic analysis techniques, GWAS and WES, but will not necessarily target the same genes, because a mutation in the structure of a gene is not necessarily correlated with an abnormality in its expression. The transcriptome will thus provide information on the pathways involved in the genesis of sarcoidosis, and will have to be performed on different types of biological samples, as well as in different clinical situations. An excellent review of the literature was published in 2017 on transcriptome data published since 2006 using either extracts of pathological tissue from sarcoidosis or preparations from BAL and, in some studies, the peripheral blood of patients . All of The compilation of the data obtained by GWAS and, more recently, by whole exome sequencing shows how high the locus heterogeneity is. This does not facilitate a priori the screening of a particular gene for predicting the disease, for example, in familial forms. Undoubtedly, many more studies will be needed using either NGS gene panels, or more likely large-scale sequencing of the exome, possibly the genome. Nevertheless, a careful reading of the scientific literature on the function of the genes identified in genetic studies on sarcoidosis leads us to think that genetics will have a major utility (1) to identify specific pathways that may lead to innovative therapies for corticoid-resistant sarcoidosis and (2) to allow the characterization of genetic markers associated with particularly severe forms of the disease, either owing to persistence and progression to pulmonary fibrosis, or by lesions in many organs, or by the occurrence of opportunistic  infections suggesting an immune weakness or autophagic of genetic origin. As described previously, these assumptions make sense when we analyzed the functional target  of the treatments currently used in advanced forms of sarcoidosis. This relationship between therapeutic and pathway can also be considered realistic for less specific treatments such as corticosteroids and leflunomide, which impact S6 kinase, a critical effector of mTORC1 ,140. Rec


Studies on control of and preventive measures against asphalt pavement moisture damage have important economic and social significance due to the multiple damage and repair of pavements, the reasons for which include the poor interfacial adhesive ability between acidic aggregates and asphalts. Anti-stripping agent is used in order to improve the poor adhesion, and decomposition temperature is regarded as being important for lots of anti-stripping products, because they always decompose and lose their abilities under the high temperature in the mixing plant before application to the pavement. A novel anti-stripping composite, montmorillonoid/Polyamide (OMMT/PAR), which possesses excellent thermal stability performance and is effective in preventing moisture damage, especially for acidic aggregates, was prepared. Moreover, the modification mechanisms and pavement properties were also investigated with reference to the composites. The results show that OMMT/PAR was prepared successfully, improving the interfacial adhesion between the acidic aggregate and the modified asphalt. Due to the nanostructure of OMMT/PAR, the thermal stability was enhanced dramatically and the interfacial adhesion properties were also improved. Furthermore, asphalts modified with OMMT/PAR and their mixtures showed excellent properties. Finally, the moisture damage process and the mechanisms by which OMMT/PAR improves the interfacial adhesion properties are explained through adhesion mechanism analyses. The rapid development of the highway has received much attention in the past few years because of its perfect pavement properties, operational safety and convenience ,2,3. AspThe most important reason for the occurrence of moisture damage is the poor interfacial adhesive property between the asphalt and the aggregate . CurrentGao et al.  studied As a result, the interfacial adhesion properties between acidic aggregates and asphalt have not yet been thoroughly reported, and the anti-stripping products that are available on the market always exhibit weaker thermal stability, leading to unsatisfactory construction. Therefore, in this study, in order to improve thermal stability and for its effective application between acidic aggregates and asphalt, a novel anti-stripping composite was prepared, and the thermal, adhesive, and pavement performances are discussed in comparison with other ASA products. To study the interface adhesive performance between asphalt and aggregate, two brands of matrix asphalts KL-90# and SK-90# were employed, and their physical properties are shown in 2 and gradually heated to 280 °C for 4 h, with OMMT/eleostearic dimer acid nanocomposites (OMMT/Di-EA) being obtained. Then the OMMT/Di-EA were gradually added into the diethylenetriamine (EDTA) system, which was heated to 120 °C under protection by N2; the system was kept under 200 °C for 2 h before EDTA was distilled under vacuum, and the resulting liquid was a polyamide nanocomposite solution. Moreover, diluent and compatilizer were added under mechanical stirring to achieve a uniform mixture when the temperature was reduced to 80 °C, and the anti-stripping layered silicate nanocomposites of OMMT/Polyamide (OMMT/PAR) were finally achieved. For the purpose of easily injecting it into the mix plant, OMMT/PAR was also diluted by glycerol in a ratio of 5:1 (OMMT/PAR: glycerol), referred to as modification anti-stripping material (MAS). PAR with no OMMT was also prepared in this work.Anti-stripping composites were prepared according to Matrix asphalts were modified by 0.3 wt % OMMT/PAR, ASA-1, and ASA-2, respectively, where the KL-90# modified by OMMT/PAR, ASA-1 and ASA-2 was named as KL-90-OMMT/PAR, KL-90-ASA-1, and KL-90-ASA-1, respectively. Similarly, the names SK-90-OMMT/PAR, SK-90-ASA-1 and SK-90-ASA-2 were used when the asphalt was replaced by SK-90#. Matrix asphalt should always be assumed to be KL-90 unless otherwise stated.Asphalt concrete AC 16 with a maximum grain size of 16 mm was prepared as the asphalt mixture in this work due to the particle reinforced composites and the common application on highly trafficked road pavements according to the Technical Specification for Construction of Highway Asphalt Pavements . First of all, the modified asphalts and the acidic aggregates were heated to 150 °C and 160 °C, respectively. The hot modified asphalts and aggregates were mixed at 150 °C for 60 s, and then impacted for the revolving compaction sample with dimensions of 100 ± 2 mm in diameter and 150 ± 2.5 mm in height, and for the Marshall sample, with dimensions of 101.6 ± 0.25 mm in diameter and 63.5 ± 1.3 mm in height, according to the Standard Test Methods of Bitumen and Bituminous Mixture for Highway Engineering .By using the TG-DTA thermal analyzer , the thermal performances were characterized. The test conditions were as follows: 10 mg samples were placed in an aluminum container and tested from room temperature to 800 °C at a rate of 10 °C/min.According to the industrial standard STMBM, the adhesion properties between asphalts and aggregates are tested by utilizing the poaching method according to the rule (T0616) of STMBM. The aggregates with sizes bigger than 13.2 mm were washed and immersed into a hot asphalt sample after being heated to 105 °C and immersed for 45 s, and were then lifted out. The aggregates with the asphalt were boiled in water for 3 min after cooling in room temperature for 15 min, and the adhesion level can be marked up as having a grade of 1–5 according to the movement of hydration on the aggregate rock surface and the desquamation of the asphalt, for which grade 1 is the lowest grade and grade 5 is the highest one. To confirm the durability, adhesive tests between acidic aggregates and the modified asphalts were also carried out after aging.The freeze–thaw splitting tests divided the Marshall samples into two groups: A and B. Firstly, the A group samples were immersed in water for 15 min in a vacuum and 30 min under normal pressure; then, they were frozen at −18 °C for 16 h, and immersed in 60 °C water for 24 h. Finally, the A and B group samples were all immersed in 25 °C water for 2 h and the splitting strengths were tested, respectively. The splitting strengths of the B group were defined as the splitting strengths of these samples. Moreover, the ratio of splitting strengths of the A and B groups were defined as the splitting strength ratios (TSR) of these samples according to STMBM. The dynamic elasticity moduli of the asphalt mixtures were also evaluated according to STMBM under a temperature of 20 °C and a frequency of 10 Hz.The thermal stability of anti-stripping composites is considered to be one of the most significant factors, due to the high temperature in the mix plants used by building contractors and asphalt suppliers. Generally speaking, the anti-stripping composite is injected into the asphalt several hours or even days before use. However, modified asphalt needs to be stored at a temperature higher than 130 °C (it may be much higher in some areas), sometimes for a number of weeks, and thus it is essential for the anti-stripping composite to hold a better thermal stability, which can help keep them effective throughout the whole mixing and compaction processes.TG curves of the three kinds of anti-stripping materials are shown in −1, and the peak of the bending vibration of C-ONH in PAR is also shown at 1250 cm−1, which can be considered to be typical functional groups of PAR compared with that of OMMT. Therefore, it can be concluded that the layered silicate nanocomposites of montmorillonoid were prepared successfully, based on the peak of 1250 cm−1 and 3350 cm−1 according the curve of OMMT/PAR.The results of the adhesion properties between the acidic aggregates and asphalts modified by anti-stripping composites are shown in In brief, all the asphalts modified by the anti-stripping products give a temporary upgrade when they have not undergone aging, but their adhesion properties are so different after RTFOT and PAV aging. Also, other acidic aggregates of sandstone and quartzite have shown similar results. That is to say, ASA-1 and ASA-2 cannot prevent decomposition throughout the entire cycle of use during aging processes due to their poor thermal stabilities, which cannot enhance the interfacial adhesive strengths constantly and are therefore highly unsatisfactory for the service life of the highway.It can be seen in Asphalts modified by anti-stripping materials exhibit no weight loss after RTFOT, based on PG grades after PAV can evaluate the application performances and limitations under both cold and hot environments. As shown in Since splitting failure occurs when the asphalt mixture suffers from moisture damage, the freeze–thaw splitting test of asphalt mixture is used to measure the damage intensity ratio before and after moisture damage, so as to study the water stability of the asphalt mixture. Dynamic elasticity modulus simulates the dynamic effect condition of vehicles; the modulus is measured at a quick loading rate, and can be defined as the ratio between maximum stress and maximum reversible axial strain. The vast majority of pavement materials are viscous-elastic plastic complexes, and they are sensitive to time, temperature, loading conditions, etc. Therefore, it better approaches the actual pavement situation and is more practical than the static modulus of elasticity. The interfacial adhesion mechanism between the asphalt and acidic aggregate is shown in For the asphalt mixture using acidic aggregates, their surfaces are wrapped with hydrogen ions from OMMT/PAR; the N and O in OMMT/PAR can combine with the H from the aggregates’ surface by means of a hydrogen bond. Furthermore, OMMT layers in OMMT/PAR have a parallel composition compared to aggregates, are negatively charged, and can be electrostatically attracted by a positive charge on the aggregates’ surface, according to electronic theory, and the surfaces are coated with OMMT/PAR molecules; H atoms present in asphalt can combine with OMMT/PAR through hydrogen bonds. According to the solubility parameter close principle, macromolecular asphalt completely adheres to aggregate coated with abundant macromolecular OMMT/PAR, and the adhesion between the acidic aggregate and the asphalt is dramatically improved, resulting in a better thermal stability at the interfaces.Asphalt anti-stripping OMMT/PAR composite modified by OMMT showed high decomposition temperature, and enhanced the thermal stability and effectively provided excellent interfacial adhesive properties, although it suffered under high-temperature environments in the mix plant or during transportation.Asphalt modified by OMMT/PAR could reach the 5 grade, compared to the 4 grade (ASA-1) and 3 grade (ASA-2) reached after PAV, respectively, suggesting that asphalt modified by OMMT/PAR is able to keep its effects indefinitely, and shows excellent durability during aging.Asphalt modified with OMMT/PAR and their mixture also showed excellent pavement properties compared with others. Moreover, the moisture damage process and the reasons for OMMT/PAR improving the interfacial adhesion properties were explained by adhesion mechanism analysis.According to our novel anti-stripping OMMT/PAR composite, the interfacial adhesion properties were improved compared to other products. The conclusions are as follows.Based on the study above, OMMT/PAR anti-stripping composites show a successful improvement of the interfacial adhesive property between acidic aggregates and asphalt.


Next, by using a previously reported TDP-43 aggregation model, we have shown that wild-type and an aggregate-prone form of TDP-43 are detected in CMA+ lysosomes isolated from cell cultures. In addition, their protein levels increased in cells displaying CMA down-regulation, indicating that these two TDP-43 forms are CMA substrates in vitro. Finally, we observed that the aggregate-prone form of TDP-43 is able to interact with Hsc70, to co-localize with Lamp2A, and to up-regulate the levels of these molecular components of CMA. The latter was followed by an up-regulation of the CMA activity and lysosomal damage. Altogether our data shows that: (i) TDP-43 is a CMA substrate; (ii) CMA can contribute to control the turnover of physiological and pathological forms of TDP-43; and (iii) TDP-43 aggregation can affect CMA performance. Overall, this work contributes to understanding how a dysregulation between CMA and TDP-43 would participate in neuropathological mechanisms associated with TDP-43 aggregation.TAR DNA binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) are fatal neurodegenerative diseases with the presence of TDP-43 aggregates in neuronal cells. Chaperone Mediated Autophagy (CMA) is a lysosomal degradation pathway participating in the proteostasis of several cytosolic proteins including neurodegenerative associated proteins. In addition, protein oligomers or aggregates can affect the status of CMA. In this work, we studied the relationship between CMA and the physiological and pathological forms of TDP-43. First, we found that recombinant TDP-43 was specifically degraded by rat liver’s CMA+ lysosomes and that endogenous TDP-43 is localized in rat brain’s CMA+ lysosomes, indicating that TDP-43 can be a CMA substrate  Ribonuclear protein TAR DNA binding protein 43 kDa (TDP-43) was first discovered as a transcription factor and a splicing regulator , either to correctly maintain the basal cellular metabolism or to overcome a particular stress  were maintained with water and food ad libitum. All procedures were approved by the Bioethics Committee of Fundación Ciencia and Vida. Subcellular fractions enriched in intact lysosomes were prepared according to Juste and Cuervo (g for 5 min at 4°C. Supernatants (post-nuclear fraction) were collected and centrifuged at 17,000 g for 10 min at 4°C. The remaining fat layer was removed from the tube walls using a clean Kim wipe and pellets were resuspended with a “cold finger” in 3.5 volumes of 0.25 M sucrose/g of tissue and centrifuged again at 17,000 g 10 min at 4°C. Pellets were resuspended in 0.25 M sucrose plus two volumes of 85.6% Nycodenz and loaded on the bottom of an ultra-clear ultracentrifugation tube. Then samples were centrifuged in a discontinuous Nycodenz gradient  at 101,709 g for 1 h at 4°C. Four fractions were obtained from this step: 1, 2, 3 and 4 from the top to the bottom of the tube. The different fractions were collected carefully with a glass Pasteur pipette. Fractions 1 and 2 were considered as lysosomes containing high (CMA+) and low (CMA−) CMA activity, respectively. Fraction 3 was considered as a Mitochondria/lysosomal fraction, whereas faction 4 was obtained as Light Mitochondria fraction. Each sample was washed with 0.25 M sucrose by centrifugation at 37,000 g for 15 min at 4°C. For transport and competitive assays, liver lysosomal fractions were resuspended in 300 μl of 0.25 M sucrose pH 7.2 and used immediately . In the case of brain samples, all fractions were quantified, resuspended in Laemmli buffer and keep at −20°C until their analysis by Western blot.Male Sprague–Dawley rats  in a total volume of 30 μl. Increasing amounts of recombinant TDP-43 protein (Abcam ab140718) were added. The samples were incubated at 37°C for 20 min and then centrifuged at max speed for 20 min at 4°C. Supernatants were discarded and the pellets washed with 100 μl MOPS-sucrose. For the competition assay, different amounts of recombinant glyceraldehyde-3-phosphate dehydrogenase protein  were added to the reaction mix. Finally, pellets were resuspended in 10 μl of loading buffer and analyzed by Western blot.Cell lines expressing the different TDP-43 forms were previously described in Budini et al. . Briefly5 cells were cultured in four 150 mm plates and after washing with cold PBS, they were scrapped with 2 ml of PBS and collected by centrifugation at 500 g for 5 min 4°C. Then cells were washed and finally resuspended in 2 ml of 2.5 M sucrose pH 7.2 and lysed by nitrogen cavitation. Samples were centrifuged at 2,500 g for 15 min 4°C and postnuclear supernatant (PNS) placed on the top of a Nycodenz gradient  using a 14 × 89 mm ultra-clear tube. Samples were spin-down at 20,000 rpm for 35 min 4°C in an SW40.1 rotor. Lysosomal/Mitochondria interphase was collected and placed in a new ultra-clear tube 14 × 89 mm for mixing with 80% Nycodenz to achieve a density of 35%. A new gradient of Nycodenz was generated  and new ultra-centrifugation was performed as described above. Mitochondria and lysosomal fractions were separated  and collected from the interphases generated. Samples were washed with one volume of 0.25 M sucrose and centrifuged at max velocity (4.500 g) for 15 min 4°C. The supernatant was discarded carefully, and pellets were used for Western blot analysis using the following antibodies: anti-TDP-43 (Proteintech 10782-2-AP), anti-Lamp2A (Abcam ab18528), anti-GAPDH (Santa Cruz sc-365062) and anti-Flag (Sigma M2).The protocol for lysosomal isolation from cells was performed according to Storrie and Madden  and Agarg for 5 min 4°C. Pellets were vortexed in 1 ml of hypotonic buffer  supplemented with protease inhibitor cocktail (Thermo Scientific), 0.5 mM DTT, 0.1 mM PMSF; and kept on ice for 10 min. Then, cells were disrupted by nitrogen cavitation and cell lysates centrifuged at 100,000 g for 70 min at 4°C  in the 70.1 Ti rotor. Supernatants were recovered for Western blot analysis.S100 cytosolic fractions were prepared according to the protocol of Schneider et al. . Cells ag at 4°C, supernatants were quantified. One milligram (1 mg) of total protein from cell lysate was incubated with 40 μl of Anti-Flag M2 Affinity Gel (Sigma-Aldrich) overnight at 4°C. Then, beads were centrifuged and washed with PBS three times for 10 min at 4°C and then resuspended in 50 μl of resuspension buffer  plus 20 μl of SDS 5×. Western blot analysis was performed by using anti-Flag , anti-Hsc70  and anti-Lamp2A (Abcam ab18528).Cells were collected in RIPA lysis buffer  supplemented with protease inhibitors (Thermo Scientific A32965) and incubated 30 min on ice. After centrifuging at 500 Proteins were run in 10% SDS-Poly acrylamide gel and transferred to nitrocellulose membrane using a Trans-Blot Turbo System-Bio-Rad . To evaluate samples from lysosomal isolation and cell lysates, 10 μg to total protein was loaded on the gel. The nitrocellulose membrane was incubated with a blocking solution  for 1 h. First antibodies were diluted in a TBS 0.1% Tween-20, 2% BSA solution and incubated overnight at 4°C. After three washes, the membrane was incubated with secondary antibodies in a TBS 0.1% Tween-20 solution for 1–2 h at room temperature. After three washes, the membrane was developed in an Odyssey-FC system (Licor). Antibodies and their working solutions were the follow: anti-TDP-43 , anti-Lamp2A  and anti-GAPDH , anti-Hsc70 , anti-Flag , anti-LC3 A/B , p62 , BAG3 .−ΔΔCt).Total RNA was extracted by using Trizol (Invitrogen) according to the manufacturer’s instructions, treated with DNase I (Invitrogen) and then quantified. Reverse transcription was performed using 1 μg of total RNA and random primers (Thermo Scientific) in 20 μl of the total volume of the reaction mixture using the reverse transcription reagents kit (Invitrogen). For PCR, KAPA SYBR FAST qPCR (KAPABIOSYSTEMS) kit was used. Reactions were made in triplicate using 2 μl of cDNA and 0.4 μM final concentration of each primer in 25 μl final volume. Primers were the following: β-actin, sense (5′-GATCTGGCACCACACCTTCT-3′) and antisense (5′-GGGGTGTTGAAGGTCTCAAA-3′); hHsc70, sense (5′-GGAGGTGGCACTTTTGATGT-3′) and antisense (5′-AGCAGTACGGAGGCGTCTTA-3′); hLamp2A, sense (5′-TATGTGCAACAAAGAGCAGA-3′) and antisense (5′-CAGCATGATGGTGCTTGAGA-3′). Reactions were subjected to dissociation curve analysis to exclude the possibility of nonspecific amplification. Changes for each gene were calculated using the mean of the change in Ct values (ΔCt) normalized to the Ct values of β-actin for each sample (2Cells were fixed in cold methanol (−80°C) for 5 min on ice, washed with PBS and then incubated with a blocking solution (2% BSA in PBS 1X) for 30 min. Primary antibodies were incubated in blocking solution at the following concentrations: anti-Flag, 1:250 (Sigma M2); anti-Lamp2A 1:200 (Abcam); anti-Galectin 3 1:50 (Santa Cruz sc-23938). After washing with PBS 1X, cover-glasses were incubated with secondary antibodies 1 h with anti-mouse-Alexa Fluor 594 (A21203) or anti-rabbit-Alexa Fluor 488 (A21444). Finally, Hoechst 33342 was used for cell nuclei. Samples were imaged with a Nikon Eclipse Ti-E microscope and Fiji software.post hoc test to determine statistical significance (p < 0.05). Numerical results are reported as mean ±SE. GraphPad software 8 was used to perform the statistical analysis. For Western blot densitometric analysis, the ImageJ software was used. ns: no significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Data analysis was performed from at least three independent experiments, obtained separately from different sets of cell cultures or animals. Different statistics methods were applied. When one group was analyzed, one-sample t and Wilcoxon test were used and numerical results reported as ±SEM. When more than two groups were compared, one or two-way ANOVA were used, followed by the Bonferroni in vitro.Aberrant forms of different neurodegenerative associated proteins that have been recognized as CMA substrates are able to alter the activity of this pathway. To study whether this is the case for TDP-43, we first wanted to confirm if this protein is a real CMA substrate. Although TDP-43 has been proposed to be a CMA substrate  that was thinner . The anaThe previous experiments confirmed that TDP-43 is a CMA substrate. However, up to date, nothing is known about the relationship between TDP-43 aggregation and CMA. To study this aspect, we used a cellular TDP-43 aggregation model previously reported. Briefly, this model is a tetracycline-inducible HEK293 cell line overexpressing a specific form of TDP-43 prone to aggregate  and evaluated the turnover of the different forms of TDP-43 indicated in The previous experiments suggested that Flag-TDP-43 WT and the aggregated-prone form Flag-TDP-12xQ/N can be CMA substrates. To confirm this, we addressed whether both TDP-43 forms were present in CMA lysosomal fractions isolated from their respective cell lines. As shown, lysosomal fractions from both cell lines were enriched with Lamp2A and GAPDH proteins, indicating a high content of CMA positive lysosomes , line 5.Then, we characterized whether wild type or TDP-43 aggregated-prone form were able to interact with the CMA the component Hsc70. For this, the control cell line, or the ones expressing Flag-TDP-43 WT or Flag-TDP-12xQ/N, were induced with tetracycline and then subjected or not to serum deprivation . We used serum deprivation condition as a CMA activity activator  after aggregates induction. Increased Lamp2A and Hsc70 mRNA levels were observed in the cell line harboring TDP-43 aggregates after 24 h . InteresTo confirm whether the presence of TDP-43 aggregates can enhance the CMA activity, we investigated GAPDH protein levels, a well-known CMA substrate, in cells expressing TDP-43 aggregates during 24 and 72 h. Compared with the control cell line, a reduction in GAPDH protein levels was observed in the cells expressing aggregates . FurtherUnder a CMA activating stimulus, lysosomes are recruited to the perinuclear zone of the cell  and CMA activity, but also causes a partial damage of Lamp2A positive lysosomes, suggesting a general dysfunction of the pathway.This work clearly demonstrates that TDP-43 is a CMA substrate Although nothing is known regarding the state of CMA in ALS or FTLD patients, where TDP-43 aggregation is implicated, some reports have shown that Lamp2A and Hsc70 protein levels are altered in other neurodegenerative diseases (Papagiannakis et al., The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by Bioethics Committee of Fundación Ciencia and Vida.FO performed most of the experiments and helped with writing and discussion. JH helped with lysosomal purification and lysosomal transport assays experiment, and helped with discussion. FR performed qPCR experiments. JM performed and analyzed the perinuclear localization of lysosomes. JR performed the lysosomal damage experiment. AC contributed to the discussion. AA contributed with writing, editing and discussion. IA contributed with reagents, experimental design, image analysis, writing and discussion. MB wrote the article, made the figures, guided the experimental approaches and project. All authors read and approved the final manuscript.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.


HLA-DPB1 in CBD and several HLA-DRB1 alleles in sarcoidosis. One of the defining features of both CBD and sarcoidosis is an infiltration of activated CD4+ T cells in the lung. CD4+ T cells in the bronchoalveolar lavage (BAL) of CBD and sarcoidosis patients are highly Th1 polarized, and there is a significant increase in inflammatory Th1 cytokines present in the BAL fluid. In sarcoidosis, there is also a significant population of Th17 cells in the lungs that is not present in CBD. Due to persistent antigen exposure and chronic inflammation in the lung, these activated CD4+ T cells often display either an exhausted or anergic phenotype. Evidence suggests that these T cells are responding to common antigens in the lung. In CBD there is an expansion of beryllium-responsive TRBV5.1+ TCRs expressed on pathogenic CD4+ T cells derived from the BAL of CBD patients that react with endogenous human peptides derived from the plexin A protein. In an acute form of sarcoidosis, there are expansions of specific TRAV12-1/TRBV2 T cell receptors expressed on BAL CD4+ T cells, indicating that these T cells are trafficking to and expanding in the lung in response to common antigens. The specificity of these pathogenic CD4+T cells in sarcoidosis are currently unknown.Pulmonary sarcoidosis and chronic beryllium disease (CBD) are inflammatory granulomatous lung diseases defined by the presence of non-caseating granulomas in the lung. CBD results from beryllium exposure in the workplace, while the cause of sarcoidosis remains unknown. CBD and sarcoidosis are both immune-mediated diseases that involve Th1-polarized inflammation in the lung. Beryllium exposure induces trafficking of dendritic cells to the lung in a mechanism dependent on MyD88 and IL-1α. B cells are also recruited to the lung in a MyD88 dependent manner after beryllium exposure in order to protect the lung from beryllium-induced injury. Similar to most immune-mediated diseases, disease susceptibility in CBD and sarcoidosis is driven by the expression of certain MHCII molecules, primarily  Mycobacteria sp. and various fungal organisms, while non-infectious etiologies include hypersensitivities, particulate exposures, and autoimmune reactions, among others [reviewed in  resulted in the release of IL-1α and DNA into the airways, which subsequently acted as damage-associated molecular patterns (DAMPS) by engaging IL-1R1 and TLR9 to induce migration of DCs into lung-draining lymph nodes and the subsequent generation of memory CD4+ T cells -induced injury . Using aAlthough there is no evidence of B cells playing a direct role in disease pathogenesis in sarcoidosis, several studies have reported altered antibody responses in these patients. Particularly in patients with pulmonary sarcoidosis, there is a direct correlation between the number of T cells in the BAL and the proportion of BAL cells that secrete IgG, which is not apparent in control subjects . Additio4) in culture, secreting Th1 cytokines, including IFN-γ and TNF-α and lesser quantities of IL-2  are currently unknown, genetic studies are important to determine common disease characteristics. However, many genetic studies involving sarcoidosis patients demonstrate that even commonalities among subsets of patients differ between ethnic and regional groups, suggesting that there are multiple factors involved in the progression of sarcoidosis. A large-scale ACCESS study that collected patient data in the United States from 1996 to 1999 demonstrated that siblings of sarcoidosis patients are about 5-fold more likely to develop the disease than unrelated control subjects , suggestcoidosis . The moscoidosis . The maj 2 years . SpecifiAs discussed in the previous section, specific MHCII expression is crucial for disease development in both CBD and sarcoidosis. CD4+ T cells recognize peptide epitopes in an MHCII-restricted manner . In CBD,HLA-DRB1*03:01 (HLA-DR3) (Although the inciting antigen(s) in sarcoidosis is unknown, there is strong evidence suggesting that the disease is driven by antigen-specific CD4+ T cells in the lungs of patients. The most conclusive evidence comes from Löfgren's syndrome. The majority of Löfgren's syndrome patients in Sweden express HLA-DR3) , and expHLA-DR3) . AdditioHLA-DR3) –79. ImpoHLA-DR3) . Using dHLA-DR3) . AdditioOne of the most active areas of sarcoidosis research is focused on determining the etiologic antigens that drive the recruitment of CD4+ T cells to the lungs of sarcoidosis patients. One problem hindering this endeavor is the lack of understanding of the origin of the inciting antigens, whether it is a foreign or self-antigen, or even a product of environmental exposure. Although there is some evidence for each of these categories, nothing has been conclusive enough to determine a cause of disease. The most detailed studies involving an autoantigen focus on vimentin, a filamentous protein of which the cytoskeleton is composed . VimentiMycobacteria sp. Several meta-analyses demonstrate that the odds of finding mycobacterial DNA by PCR in the lungs of sarcoidosis patients is ~5–20% greater than in control subjects   . Additio (mKatG) . Further (mKatG) , and IgG (mKatG) . DespiteCutibacterium acnes (previously Propionibacterium acnes) infections are also associated with sarcoidosis. C. acnes is a commensal bacteria that is commonly found on the skin of healthy individuals. By quantitative PCR, DNA from C. acnes is found in the majority of lymph nodes obtained from sarcoidosis patients (C. acnes is also found in a large percentage of lymph nodes isolated from healthy control subjects (C. acnes in sarcoidosis patients is a consequence of the disease or normal colonization of commensal bacteria.patients . Howeversubjects , making In addition to infectious agents, it has also been postulated that there are environmental factors involved in the progression of sarcoidosis. Stage I sarcoidosis occurs more frequently in the Spring, and the lowest incidence of disease diagnosis occurs in the Winter, suggesting that airborne allergens or other seasonal particulates may be involved in disease progression . There aThe immunology of sarcoidosis and CBD are quite similar, based on a predominance of CD4+ T cells in the lung, a Th1 polarized immune response, and a pathologic hallmark of granulomatous inflammation. As such, the presence of a known antigen in CBD can further our understanding of the driving factors involved in sarcoidosis pathogenesis. In CBD, our group has performed unbiased antigen discovery approaches that focused on the related and expanded CD4+ T cell subsets in the BAL of patients with active disease and have delineated several beryllium-dependent T cell ligands. It is possible that a similar unbiased antigen discovery approach utilizing expanded CD4+ T cell clones in the lungs of sarcoidosis patients may lead to the discovery of the inciting antigens that drive CD4+ T cell alveolitis and granulomatous inflammation in sarcoidosis.SG, SA, and AF wrote the article. SA contributed to designing the figure and table for the article. All authors contributed to article revisions.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.


This review aims to showcase the current use of graphene derivatives, graphene-based nanomaterials in particular, in biopolymer-based composites for food packaging applications. A brief introduction regarding the valuable attributes of available and emergent bioplastic materials is made so that their contributions to the packaging field can be understood. Furthermore, their drawbacks are also disclosed to highlight the benefits that graphene derivatives can bring to bio-based formulations, from physicochemical to mechanical, barrier, and functional properties as antioxidant activity or electrical conductivity. The reported improvements in biopolymer-based composites carried out by graphene derivatives in the last three years are discussed, pointing to their potential for innovative food packaging applications such as electrically conductive food packaging. Non-sustainable food production and consumption leads to many serious health and environmental problems. It is estimated that one third of the total foodstuffs produced worldwide, 1.3 billion tons, equivalent to one trillion dollars, is wasted. The United Nations (UN) 2030 sustainable development agenda addresses this problem with Goal 12—Ensure sustainable consumption and production . See, foBiodegradable biopolymers have been gaining societal, scientific, industrial, and economic importance to develop sustainable food packaging bioplastics, alternatives to conventional non-biodegradable fossil-fuel-based plastics ,13. In rd--linked glucopyranosyl units, and the highly branched amylopectin, α-d--linked glucopyranosyl units partially substituted by α-d- linkages units -Starch has been used to form edible or biodegradable films and granulates through casting from aqueous solutions, film blowing, forming, injection molding, blow molding, and extrusion ,20,21,22Plasticizers, along with temperature (for gelatinization) , allow tIn a laboratorial environment, other interesting future scalable approaches have been explored to improve starch-based materials. Gonçalves et al.  used oilCellulose is a linear polymer of several hundred to many thousands of (β1→4) linked D-glucose units ; it is tA notable material derived from acid hydrolysis of cellulose is the nanocrystalline cellulose (CNC). It has been widely explored in numerous areas in food packaging  39,40,4,440,41. d-glucoses attached by (β1→4) linkages (2) that can be protonated to NH3+ and readily form electrostatic interactions with anionic groups in an acid environment. This characteristic allows the incorporation of a variety of chemical groups [Chitosan (CS) is a linear polysaccharide of 2-amido-2-deoxy-β-linkages , obtainel groups   and letl groups .2 and O2, it has been investigated as edible coatings for fruit packaging applications. For this particular use, it delays the rate of respiration, decreases both weight loss and ethylene production, inhibits postharvest diseases, and increases the antioxidant process, allowing to extend the product shelf life while preserving the fruits overall quality. These results were achieved just by using CS as a packaging solution [This biopolymer has been widely studied due to its potential in areas such as material science  and pharsolution . When apsolution ,70. Howesolution ) to oversolution ,73.d-mannuronic acid (M) and α-l-guluronic acid (G) monomers  structure designed as an “eggbox” model, has been explored for the production of biodegradable and edible films [Alginate is the general name given to the family of linear polysaccharides consisting of binary copolymers, made up of (1–4) linked β-monomers , occurriruginosa . The algle films . The maile films . Alginatle films ,77 and ale films . Modifiele films , sodium le films ,81, or cle films  are freqle films . The comle films . In the le films . Inorganle films . Similarp-(α1à4)Glcp-(α1à6)Glcp trimer  [Currently, the term ‘‘pullulan” is used in the literature to mean not only the ‘‘polymaltotriose”, the maltotriose -(α1à4)Glcp trimer , produceousonni, ,85,86 bustrains) .This microbial extracellular polysaccharide is a biodegradable, biocompatible, non-mutagenic, nontoxic, hygroscopic (meaning that depending on the relative humidity at which it stored it can absorb water), non-carcinogenic, and edible polymer ,89. To cGelatin is derived from the fibrous insoluble protein called collagen  and is Gelatin is abundant, low-cost, and biodegradable . It has To overcome these issues, several approaches have been explored, namely addition of different molecules such as crosslinkers (to reduce solubility), plasticizers, and antimicrobial or antioxidant compounds ,111; comZein, seen in Zein is an option for food packaging because it has excellent oxygen and carbon dioxide barrier properties and high thermal resistance ,126. FurTo improve zein films, several different options have been explored: adding plasticizers  ,129, addLactobacillus which by polymerization originates the polylactic acid  (PHBV), seen in As mentioned in the previous section, biopolymers by themselves do not present all the necessary requirements intended for food packaging, namely in terms of mechanical resistance and water/gas barrier properties. Furthermore, there is an ongoing trend towards active and intelligent packaging, i.e., approaching packaging that is able to interact with food to actively extend its shelf life, and to inform the consumer about the safety state of the food products, respectively . In this2 hybrids bonds [2 and sp3 configurations [2 configuration, thus approaching the graphene configuration [Graphene is one of the most promising nanomaterials which is constituted by a flat monolayer of carbon atoms in a two-dimensional (2D) hexagonal lattice, held together by a backbone of overlapping spds bonds . This stds bonds , withoutds bonds , impermeds bonds , and optds bonds , among ods bonds . When shds bonds . Graphends bonds . Thus, Gurations . The higurations ,160. Glourations . Thus, Gguration ,163,164.Interestingly, graphene derivatives have been widely used as polymer reinforcement and are well known to impact on several properties of the final nanocomposite, namely on its mechanical, thermal, electrical, conductive, and fire retardancy properties, to name just a few ,155,156.The use of graphene derivatives in biopolymer-based composites for food packaging applications is still lightly explored. The Web of Science search of the topic “biopolymers food packaging composites” displays 234 results, but when refined including the word “graphene”, only nine articles are displayed (accessed on 22 September 2020). However, given the potential of these nanomaterials to improve determining properties of biopolymeric composites for food packaging application, this will certainly be an expanding area with high impact in the future. Hereafter, some of the most relevant properties obtained for biopolymer-based composites including graphene derivatives will be reviewed.π–π electrostatic interactions between nanosheets or nanotubes, and/or their common weak dissolvability in water and organic solvents during biocomposite fabrication [The first step in the development of graphene-based biopolymer composites is to guarantee the uniform dispersion of graphene materials into a polymer matrix, which might not be easy given the high propensity for self-agglomeration of graphene-based materials, as a result of the strong van der Waals forces and rication . The solrication . Howeverrication . Sonicatrication ,169,170.rication  or the crication ,167 are rication . After trication ,174,175.rication .2 which, in the presence of water, form carbonic acid that can interact with the biocomposites and influence their resistance [The preparation of biopolymers nanocomposites with GNP ,178,179,sistance .The thermal stability of the biocomposites can also be improved with the inclusion of graphene derivatives into biopolymer-based formulations. The temperature of degradation is delayed by 5 °C up to 20 °C, when compared with the biopolymer film without these nanofillers ,167. TheGraphene-based biocomposites exhibit great barrier properties against gases, water vapor, and UV light ,173,179.Surface hydrophobicity of biopolymer-based materials is also enhanced by the incorporation of graphene derivatives. As described in the The development and optimization of graphene-based biocomposites is ongoing and it is important to prove that the biodegradable properties of natural polymers are not compromised. The biodegradation of polymers occurs as a result of the activity of specific microorganisms to hydrolyze and oxidize these molecules in a short time after their disposal. The strong interaction between graphene fillers and the biopolymer can improve the mechanical stability, as previously mentioned; however, the degradation rate can be slightly reduced by delaying the diffusion of water into the polymeric matrix . Lyn et Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Staphylococcus aureus, Staphylococcus haemolyticus, and Bacillus subtilis). The surface modification of graphene derivatives with essential oils or other metal compounds such as Ag, ZnO, or TiO2 has been adopted as strategy to enhance the inherent antimicrobial potential of these nanostructures [Aspergillus niger, Cryptococcus neoformans, Candida tropicalis, Candida albicans, Botrytis cinereas, and Rhizopus spp. have also been described [Nowadays, in addition to the desirable biodegradable packaging, there are global research interests for the development of active food packaging to extend shelf life, enhance safety, and maintain the organoleptic properties. Biocomposites with graphene derivatives can exhibit antioxidant, antimicrobial, and antifungal activity as extensively reviewed by Carvalho et al. . The radructures . Recentlructures . Antifunescribed .2WO6 hybrid system was shown to be 4.4 times more effective on ethylene degradation than pure Bi2WO6 when incorporated into starch-based films [2WO6 composites as active packaging materials of highly perishable fruit can be an opportunity to decrease fruit losses and wastes [Alternatively, the delocalized conjugated electron structure of graphene derivatives has been recently explored for production of photocatalytic hybrid systems with application in organic compounds’ degradation ,193. OrgTherefore, the use of graphene derivatives into biopolymer-based composites reveals to be a promising approach to develop active food packaging, enhancing the preservation of food products.Nano- and micro-particulated clay minerals could be incorporated as fillers leading to biopolymer–clay nanocomposites (bionanocomposites) based on the assembly of polysaccharides, proteins, polyesters, etc., with layered silicates, such as smectites and vermiculites, as well as fibrous clays like sepiolite and palygorskite ,200,201.®). It is well known that the dispersion of carbon NP—for instance, carbon nanotubes—in aqueous medium [Clays could be assembled to carbon particles producing graphene-like materials of variable composition and properties, including adsorption ability and electrical conductivity, mainly afforded by the clay component and the graphene derivative component, respectively. These carbon–clay materials could be synthetized following two main processing approaches: (i) top-down, by crushing together both components, and (ii) bottom-up, by growing carbon on clay minerals used as supports ,203. Anos medium ,207,208 s medium . For inss medium . Based os medium .−1), which increases by the additional incorporation of MWCNT favoring percolation reaching values up to 2500 S m−1 [Clay–graphene materials dispersed in diverse biopolymers are fillers of great interest because they can introduce significant improvement on the resulting biocomposites, increasing their barrier properties and conferring electrical conductivity. In fact, synergistic effects of clay minerals and graphene-based materials are observed for the use of clay–graphene systems as fillers of polymers. For instance, GNP/sepiolite can be used as fillers of alginate leading to films showing simultaneously reinforcing properties together marked in-plane conductivity  is one of the most promising emerging non-thermal technologies. The PEF treatment consists in the application of short high voltage pulses to food products to inactivate enzymes and microorganisms, while maintaining their sensorial and nutritional properties ,212,213.on level .−1 and 2.1 × 10−5 S m−1, in-plane and through-plane, respectively. CS-based flexible films containing rGO-Fe3−xO4 were also suggested for electrically conductive food packaging [−1 with 50 wt% rGO-Fe3−xO4. In both works [−1. The MWCNT were the most used fillers due to their high electrical conductivity capacity, typically required for electronic applications [−1, while the same procedure using ascorbic acid lead to an electrical conductivity of 0.83 S m−1. The films thermally reduced (TR) at 450°C achieved the highest conductivity of 23.42 S m−1, but this method weakened the mechanical properties [After the PEF proof of concept, it is necessary to develop electrically conductive materials suitable for food packaging. Biopolymer-based composites containing graphene derivatives can be a solution. In this regard, CS-based biocomposites containing rGO were recently suggested for electrically conductive food packaging . In thisackaging . These sth works ,170, theth works ,218. Filth works . PLA hasications ,218,220.ications . GNP andications . Howeverications ,223,224.operties .−1 [The food packaging materials for PEF treatment in-pack should have a through-plane electrical conductivity close to the electrical conductivity of the packaged food, which is typically between 0.1–2 S m−1 . Many of−1 ,210. Thu−1 . TherefoThe returns of the combination of graphene derivatives with biopolymers were reviewed. The electrical conductivity provided by the graphene derivatives opens the way for the development of novel and auspicious materials and technologies, namely electrically conductive packaging materials for food processing by PEF.The electrically conductive biocomposites show great potential to be used in intelligent packaging as flexible platforms to incorporate specific sensing molecules since the target detection would also promote changes in the electrical conductivity of the biocomposites. In this context, a PLA-based sensor containing a layer of paper coated with GO was recently described . This se2 or ZnO on the clay surface [The presence of clays assembled to graphene derivatives is very attractive for the preparation of multifunctional nanoplatforms with potential applications in food packaging. As reported above , the bar surface . This st surface , given t surface ,236,237.Prior to the application of these materials, it is important to understand the risks of their migration into food. Until now, there are few literature evidences, and those studies indicate that there is no migration of graphene derivatives into food, or that the migration occurs far below the international migration limits ,169. Add


In the case of talc, we obtain κ ∝ n3 for n ≥ 5, indicating no interlayer sliding upon folding, at least in this thickness range. In contrast, tip-enhanced Raman spectroscopy measurements on edges in folded graphene flakes, 14 layers thick, show no significant strain. This indicates that layers in graphene flakes, up to 5 nm thick, can still slip to relieve stress, showing the richness of the effect in 2D systems. The obtained interlayer adhesion energy for graphene (0.25 N/m) and talc (0.62 N/m) is in good agreement with recent experimental results and theoretical predictions. The obtained value for the adhesion energy of graphene on a silicon substrate is also in agreement with previous results.Folds naturally appear on nanometrically thin materials, also called “2D materials”, after exfoliation, eventually creating folded edges across the resulting flakes. We investigate the adhesion and flexural properties of single-layered and multilayered 2D materials upon folding in the present work. This is accomplished by measuring and modeling mechanical properties of folded edges, which allows for the experimental determination of the bending stiffness (κ) of multilayered 2D materials as a function of the number of layers ( These 2 bubbles  or the c bubbles –14. The  bubbles . The qua bubbles , and ult bubbles . It is i bubbles .After the successful synthesis of graphene in 2004 , many otd of the folded 2D material. In principle, the model describes any 2D material, and its predictions are corroborated by comparison with classical molecular dynamics simulations and to results of previous investigations on graphene and talc. Because folds naturally occur in flakes of varying thickness, corresponding to multilayers with a different number of primitive layers, the proposed method provides a direct way to investigate the bending stiffness of 2D materials as a function of the flake thickness . In the case of talc, we obtain κ ∝ h3 for materials thicker than five layers, indicating no interlayer sliding upon folding, at least in this thickness range. This result implies that layers in folds might be either compressed or stretched, leading to changes in their vibrational properties relative to a flat flake.In this work, we present a method to obtain interlayer and 2D material/substrate adhesion energies and the bending stiffness of 2D materials by experimentally probing the mechanical response of folded edges to deformation. A folded edge is defined as an edge region of the 2D material where it folds over itself during the exfoliation process. Our method is based on AFM measurements of the geometry and mechanical response of folded edges, and on the fitting of the experimental data by an analytical continuum model parameterized solely by α, κ, and the total thickness H, its total thickness d, and its layer thickness h (see the inset in R0 = (H − h)/2 and the flake thickness h for nine talc folds. The measured values of thickness h, from 1.2 to 30.0 nm, indicate that the measured folds involve materials from monolayer talc to approx. 30-layer talc. The corresponding values of the radius R0 range from 2.15 to 162 nm, that is, an increase of two orders of magnitude. The figure also shows a fitted curve R0 = ahb, where b = 1.75 and a = 0.38 (m−3/4).A folded 2D material deposited on a substrate exhibits a cross-section geometry similar to that indicated in R0 (red arcs) and the radius r0 of a half-soliton-like region (black arcs) [R0, r0, and d0), and the adhesion (α) and flexural (κ) properties can be obtained variationally. The variational procedure within the model consists of the minimization of an energy functional that contains two terms, that is, the curvature energy Ec = ∫κ/(2R2)dS where R is the local curvature radius and κ is the bending stiffness. The adhesion energy Ea = αSa, where Sa is the contact area and α is the adhesion energy per unit area between the 2D material and the precursor layered material. As a result of the variational procedure, we obtain  . Within tain see :andr0 and R0. In the case of the folded edge in three-layered graphene, we used values of R0 = 0.81 nm and d0 = 1.01 nm from MD simulations to obtain  = 1.88 nm. Considering α the same for both folded edges of graphene, we thus found κ = 1.3 aJ for three-layered graphene, which is roughly six times the value reported for a graphene monolayer (κ = 0.231 aJ). Thus, MD results indicate that the change of κ with the number of layers in multilayered graphene is non-linear. The change of κ in a real 2D material will be discussed below.R0 and d0. Q =  =  as a function of 1/h for the nine measured talc samples. h is a directly measured quantity , will solely depend on the behavior of κ as a function of h. In the limit of thick materials, we would expect that adjacent layers do not slide relative to each other upon an elastic bending deformation. In this non-sliding limit, we expect that κ ∝ h3, as predicted by the classical Euler–Bernoulli beam theory. In another limit, which we will call the sliding limit, we will assume the possibility that adjacent layers freely slide upon bending deformation. In this limit, which implicitly includes the monolayer case, we obtain κ ∝h. Both limits have been recently considered in the analysis of experimental profiles of bubbles in 2D materials [Q leads to two asymptotic limits as a function of 1/h, that is, Q ∝ 1/h in the non-sliding limit and Q = constant in the sliding limit. Both limits are indicated in tity see , and , aaterials . In our From the above, we conclude that sufficiently thick talc flakes behave like rigid objects without interlayer sliding. Therefore, upon fold formation, the layers at the inner part of the fold will have a compressive in-plane strain, and those at the outer part will have a tensile in-plane strain. The compressive and tensile strains could modify the vibrational properties of a fold compared to those of the (flat) bulk of the flake. This should be applicable to any 2D material and not restricted to talc. This hypothesis was investigated employing a near-field tip-enhanced Raman spectroscopy (TERS) setup –37 that R0 and d for uncompressed folds. As already discussed, the quantity κ/α allowed us to verify a prevalence of non-sliding behavior. However, one might be also interested in obtaining the absolute values of κ and α from the AFM measurements. In fact, to the best of our knowledge, no experimental measurement of α for talc has been reported so far. We will show that it is possible to obtain the value of α from AFM force-curve measurements on folded edges. According to our model  as shown in p = 0.40 N/m are similar to those obtained from the fit in where R0 = 4.5 nm and d = 5.25 nm, obtained from the height profile, and r0 = 11.7 nm, obtained through p = 0.24 N/m) is consistent with the values reported in the literature .p = 0.24 N/m) is consistent with the values reported in the literature (0.28 and 0.37 N/m [h3) consistent with the Euler–Bernoulli beam theory, indicating that layers in sufficiently thick flakes do not slip to relieve strain. In contrast, TERS measurements on edges in folded graphene flakes that were 5 nm, or 14 layers, thick shows no significant strain indicating that layers in graphene flakes up to 5 nm thickness can still slip to relieve stress. Even though it is applied here to homolayers, the present methodology could also bring invaluable insights about the interlayer interaction in the growing field of heterolayered 2D materials, probing the mechanical properties of typical interfaces such as graphene/hBN, graphene/TMDs, hBN/TMDs, or any other technologically relevant two-dimensional heterostructure.We have shown that it is possible to obtain the interlayer adhesion energy and the bending stiffness of layered 2D materials by fitting results of AFM force curves on naturally occurring folded edges to an expression predicted by a simple model for those edges. The obtained interlayer adhesion energy for graphene (0.25 N/m) and talc (0.62 N/m) are in good agreement with recent experimental results ,30 and t0.37 N/m –41). TheSupporting information features the theoretical models for deposited and compressed folded edges, the experimental methods  and the computational details of the MD simulations.File 1Models, experimental part and computational details.