Realizing the neurological information processing by analyzing the complex data transferring behavior of populations and individual neurons is one of the fast-growing fields of neuroscience and bioelectronic technologies. This field is anticipated to cover a wide range of advanced applications, including neural dynamic monitoring, understanding the neurological disorders, human brain–machine communications and even ambitious mind-controlled prosthetic implant systems. To fulfill the requirements of high spatial and temporal resolution recording of neural activities, electrical, optical and biosensing technologies are combined to develop multifunctional bioelectronic and neuro-signal probes. Advanced two-dimensional (2D) layered materials such as graphene, graphene oxide, transition metal dichalcogenides and MXenes with their atomic-layer thickness and multifunctional capabilities show bio-stimulation and multiple sensing properties. These characteristics are beneficial factors for development of ultrathin-film electrodes for flexible neural interfacing with minimum invasive chronic interfaces to the brain cells and cortex. The combination of incredible properties of 2D nanostructure places them in a unique position, as the main materials of choice, for multifunctional reception of neural activities. The current review highlights the recent achievements in 2D-based bioelectronic systems for monitoring of biophysiological indicators and biosignals at neural interfaces.  The rapid advancements in neuroscience and bioinspired neurotechnologies require the accurate monitoring, reception and decoding of neurological potential signals of the nervous system . In thisThe advent of the first generation of neural interfaces was considered as a key development toward the realization of electrical properties of cell and tissues that enabled in vitro neural signal acquisition. The voltage clamp technology, developed in 1976 by Erwin Neher, enabled us to record single ion channel signals . In 1981Neural signal recording can be divided into two main categories, i.e., intercellular and extracellular recordings. The intracellular technique records the electric signals generated across the cell membranes; therefore, the electrodes are placed inside and outside of the membranes. In a different configuration, extracellular recording is enabled through the employment of recording electrodes in the extracellular fluid for the reception of signals generated by nearby neurons. There are several main differences between intracellular and extracellular recording. The intracellular neural signals are more localized and feature more powerful amplitudes, while the extracellular signals are weaker and originate from a large area of the brain. The extracellular recording of neural signals can be divided into two main categories of invasive and non-invasive methods based on the location of electrodes. In the non-invasive neural interfacing, the signals are collected via electrodes assembled on the top surface of the scalp b to recoThe invasive neural interfaces can be categorized into penetrating and non-penetrating sensors c–e 27,2,29. The From a technical point of view, an efficient and reliable neural interfacing process can be achieved through the development of biocompatible sensors with high signal-to noise-ratio (SNR) characteristics and low and stable impedance values . Figure Studies on multifunctional biosensing probes have fueled a continuous need for new materials. These materials are expected to combine multiple sensing and stimulation functionalities and simultaneously create chronic biocompatible interfaces with neural cells and brain tissue. In this regard, 2D nanomaterials are among the main advanced atomically thin nanostructures that hold great promise for the development of highly active and miniaturized sensors ,50,51,52Graphene, the most famous carbon-based 2D structure, is recognized as a functional material for neural interfacing applications . Its excHigh-temporal-resolution recording of neuron cell activities is one of the main targets of electrophysiology, where optical and cellular imaging techniques are accompanied by electrophysiological signal recordings to complement the performance of each individual technique and then provide a clear image of the neurological behavior ,61,62. HFluorescence microscopy can be combined with invasive graphene-based microelectrodes to visually depict the precise active location of neural signals . Calcium−2 was recorded for a 3D graphene-based electrode that was employed for in vivo stimulation of motor cortex arrays of mouse leg ..45]. ThiIn parallel with microelectrodes, GFETs are being developed for a wide range of biosensing applications, where the transduction of biological signals is amplified locally due to the strong field effects induced ,72,73,74−2 at the 1 kHz frequency), while it showed considerably better charge storage capacity (361 ± 45 mC cm−2) compared with that of the conventional metallic-based filament microelectrodes used in neural interfacing systems. Similar research studies also included graphene as the main sensing component in microfibers [The availability of various types of graphene-based materials, including single-layer CVD films, multilayered exfoliated films, and chemically and mechanically synthesized films, has enabled the development of various types of graphene-based neural interfaces. Despite the fact that a large number of bioresearch activities have been devoted to graphene, the high hydrophobicity of pristine graphene has limited its applications. Functionalized graphene, graphene oxide (GO) and reduced graphene oxide (rGO) 2D structures can be valuable alternatives to graphene in neural interfaces. Two-dimensional GO is an atomically thin structure where oxidation enables the incorporation of oxygen functional groups into the surface of the graphene, thus decreasing the graphene aggregation in aqueous solutions as well as reducing its electrical conductivity. Recently, ultra-large graphene oxide flakes were deposited on binder-free microfibers to replace conventional filament-shaped metallic invasive neural electrodes ,65. In trofibers ,77.Despite its greater functionalization capabilities, GO has highly restricted electron mobility on its surface. GO is technically an insulator; therefore, reduction reactions are carried out to partially remove oxygen and then alter or recover the electrical conductivity of 2D GO films. Consequently, reduced graphene oxide (rGO) with higher conductivity is extensively employed for biosensing and especially non-invasive neural interfacing applications. Typically, 2D rGO nanostructures are developed or grown during the fabrication process for neural interfaces at the heterointerfaces between the sensing components, which in turn facilitate swift charge transfer and low contact impedance at the neural interfaces. Non-invasive graphene-based polymer wearable sensors are well-developed for the characterization and precision sensing of biosignals with high levels of accuracy ,21,22. O−2) [2O3) nanocomposite electrodes as implantable neural interfaces for applications in electrophysiology and neurochemistry. The rGO/Au2O3-modified electrodes displayed significantly improved activities [Due to the facile dispersion of GO and rGO, these structures can be feasibly used for the fabrication of fiber neural interfaces. In such development, the fibers  are dip-coated in GO or rGO dispersions, followed next by thermal treatment. These microfiber neural interfaces showed considerably improved conductivity and low impedance for wearable neural interfacing ,87,88,89−2) . Anothertivities . CurrentTwo-dimensional materials beyond graphene represent atomically thin nanostructures with remarkably novel semiconducting, insulating and metallic characteristics that put them in a distinguished position in comparison with their carbon-based counterparts. The family of 2D materials beyond graphene covers various type of nanostructures, transition-metal dichalcogenides (TMDCs) and MAX phase 2D materials (MXenes). These 2D structures recently found their way into neural interfacing technologies . The fol2 chemical formula, where M is one of the transition metals , and X is one of the chalcogen elements . The electronic characteristics of 2D TMDCs are of particular interest, since they differ dramatically from those of their bulk and multi-layered structures and even their single-layered 2D structures [TMDCs are the well-known and perhaps the most eminent 2D materials beyond graphene . TMDCs aructures ,98,99. Eructures ,101,102.2 and PtTe2, have attracted researchers’ attention due to their electronic characteristics. The ultra-thin few-layered PtTe2 has shown metallic properties attributable to the strong interactions between its Te interlayer chalcogen atoms [2 is generally a semiconductor material that undergoes semiconductor-to-metal transition when the number of its layers is increased [2 and PtTe2 ultra-thin films on polymer-based substrate at temperatures of less than 400 °C has allowed the development of flexible neural interfaces based on these 2D materials [2 and PtTe2 were initially grown by CVD on Si/SiO2 or Kapton substrate at 400 °C. The developed films on Si/SiO2 were afterwards transferred to 200 nm thick poly methyl methacrylate (PMMA) flexible substrate to fabricate wearable sensors  forces between the 2D layers of MoS2 films made it possible to fabricate free-standing 2D MoS2 devices. The weak vdW bonding forces enabled the rotational and sliding motion of 2D layers and, therefore, facilitated the high level of flexibility of thin films. The free-standing vdW 2D films also showed a well-behaved linear relationship and displayed a gain in tensile strength of 43% with a measured Young’s modulus of 47.3 MPa [2 films and underlayer films provided exceptional electronic functionalities for applications on living organisms [2 transistors attained a considerable SNR of 49.8 dB during human motion. Skin-gate transistor neural interfaces developed on the basis of 2D MoS2 films demonstrated versatile performance for biosensing, biopotential amplified signals and motion analyzing/processing targets [Recently, two types of Pt-based TMDCs, i.e., PtSeen atoms ,104,105.ncreased . Apart faterials ,109,110.aterials  develope sensors b. The ne sensors c. Au sof sensors c. The he sensors d. The no8.6 Ω·cm e were reectrodes f. It wasectrodes g of natudicators . In a reDC films . These 247.3 MPa . The vdWrganisms . The dev targets .n+1AXn, where M belongs to the group of early-transition metals , A is an element from group 12–16 of the periodic table, and X is either carbon or nitrogen, or both of them. The “n” in the atom indices can be 1, 2 or 3. While the bulk of MXenes represent metallic conductive characteristics, the 2D MXene films show various electronic properties, ranging from dielectric to semi-metal behavior. By implementing these tunable chemical characteristics, it is possible to orchestrate a combination of surface characteristics originated from the presence of surface-terminating functional groups on the 2D MXene films similar to hydroxyl (−OH), oxygen (−O), or fluorine (−F) groups. This prepares these 2D nanostructures for facile surface functionalization and modification. The hydrophilicity characteristics of 2D MXenes provide an excellent opportunity for solution-based processing techniques, and consequently, the need for functionalization treatments by super-acids and surfactants is eliminated [MXenes are novel 2D materials representing a broad range of atomically thin nanostructures composed of early-transition metal carbides, nitride and carbonatites. These 2D materials are synthesized from the selective etching of MAX phase materials . The geniminated ,119,120.iminated ,122.3C2 films demonstrated outstanding volumetric capacitance (1500 F/cm3 for Ti3C2Tx) [4 S/cm for Ti3C2Tx) [3C2 is by far higher than that of graphene (100 F/cm3) and graphene gel films (~260 F/cm3). A research study reported a fabrication approach for the development of high-resolution neural interfaces, where the electrode arrays were fabricated based on 2D Ti3C2. This solution-processing method was scalable and reliable for the fabrication of various types of non-invasive neural interfaces for the monitoring of EEG signals [3C2Tx acted as the active sensing material. The 2D Ti3C2Tx films were spray-coated and patterned on parylene C substrates. The measurement of on-skin sensing capabilities of the micro sensors showed impedance values of ~29.78 ± 8.1 kΩ.cm2 at the frequency of 1 kHz during the identification of muscle activation. The measured SNR of this sensor was in the range of 39–16 dB [Among the various types of MXenes, the Ti-based MXenes are the most investigated 2D nanostructures. In particular, 2D TiTi3C2Tx)  and elecTi3C2Tx) . The vol signals ,123. The39–16 dB . Specifi39–16 dB .3C2 MXene nanosheets were recently developed for the high-resolution sensing of brain signals [3C2 nanosheets was used to fabricate the sensors on flexible parylene substrates  and perform faultlessly in the human body; therefore, it is necessary to satisfy various requirements during the design of neural interfaces and selection of materials . Due to 2) was tangibly higher than that on a glass substrate (178 2) due to the stronger adhesive characteristics of graphene compared with those of glass substrate [The biocompatibility of 2D materials for neural interfacing applications needs to be exploited extensively. So far, most researchers have studied the biocompatibility of graphene neural interfaces. Their studies mostly focused on cell-to-graphene direct interactions. In a typical study, the cellular survival rates on 2D graphene-based substrates were investigated and then compared with other common substrates for neural interface applications ,132,133.ubstrate . An inteubstrate .The biostability issues in electronic neurotechnologies are of less concern, since graphene 2D nanosheets are not freely in contact with bio-tissues and are mainly encapsulated by the polymer-based insulating structures. The main group of neural interfaces is composed of multilayer graphene 2D flakes that are randomly oriented into 3D scaffold structures. In this group of neural interfaces, biocompatibility has a vital role in functional applications ,137,138.3C2 films showed no significant difference after 7 days in an in vitro experiment. In a comparative measurement between Ti3C2 and polystyrene substrates, the widespread formation of neural networks on both substrates was confirmed by immunocytochemistry [3C2 and polystyrene substrates yielded equivalent neuronal viability. These early observations indicate the capacity of Ti3C2 substrate for the formation and growth of neuronal networks with negligible cytotoxicity effects. However, the mentioned study showed that the neuron cultures on Ti3C2 experienced a reduction in neuron density compared with that of the polystyrene substrate. This indicates that neural adhesion on the Ti3C2 substrate decreased during the 7 days experiment. It also suggests that further optimization processes are required to promote neuronal adhesion on the surface of Ti3C2 as an atypical MXene film [Regarding the hydrophilicity properties of 2D materials, recently introduced MXene-based neural interfaces have been found to be capable platforms for the adhesion of neural cells. Recent experiments showed promising biomedical applications for MXene sensors in cancer theranostics , diagnoshemistry . Overallene film .e-skins. Specifically, recently fabricated e-textrode neural sensors based on graphitized electro-spun fiber/monolayer graphene clearly demonstrated their stability after washing up to 10 times. Graphene-coated textiles are well-known flexible nanostructures for fabrication of on-skin sensor electrodes for long-term monitoring of EEG signals [Another concern is the durability of 2D nanostructures on various substrates. The materials in neural interfaces are prone to delamination, corrosion, breakage, and failure of interconnections. For instance, 2D graphene-based neural interfaces suffer from delamination of 2D graphene nanosheets from the flexible polymer substrates. The Si/SiC substrates were found to be the most stable for long-term performance in bioactive media. However, rigid substrates are not desired for functional neural interfacing applications in the human body. Soft substrates or mesh-like morphologies are possibly the most appropriate options for biosensing and neural interfacing applications. Water-soluble polymer supporting layers (similar to PVA) decorated with functional 2D materials for biosensing are one of the preferable options for the next generation of on-skin dry neural interfaces. Therefore, there is a great opportunity in the fabrication of polymer-based e-skins based on 2D materials for the long-term performance of neural interfaces on human skin. Reusability is another important detail for the desired applications of novel  signals ,87,88. T signals ,143. ThiGenerally speaking, the present article provided an overview of recent advancements in the fabrication and development of various types of neural interfaces based on 2D nanomaterials. Both non-invasive and invasive neural interfaces based on 2D materials were introduced, and their functionalities were demonstrated. The multi-functionalities of 2D materials and their physicochemical properties bring both unexpected properties and improved benefits to active nanointerfaces. Recent advances have enabled rapid progress in the fabrication of micron-sized sensors with bifunctional capabilities. Graphene and its family are by far the most employed 2D structures in neural interface technology. Recent advancements in applications of 2D carbon-based nanostructures have enabled the successful monitoring of neural activities and human motion. Apart from graphene-based neural interfaces, researchers have recently focused on other types of 2D materials beyond graphene. Currently, only a few research studies have reported on the application of TMDC and MXene 2D structures in neural interface technology. Therefore, this area represents a great opportunity for the further development and progress of bioelectronic technologies. For successful development in this area of bioelectronics, it will be necessary in the future to address the challenges of fabrication technology, durability and biocompatibility of neural interfaces.


TAR DNA-binding protein 43 kDa (TDP-43), a nuclear protein, plays an important role in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS). The long-disordered C-terminal region (CTR) of TDP-43 is known to be aggregation-prone and a hotspot for ALS mutations, so elucidation of the physiological function of CTR will provide insights into the pathogenesis of ALS. The CTR has two Gly, aromatic, and Ser-rich (GaroS) segments and an amyloidogenic core divided into a hydrophobic patch (HP) and a Gln/Asn (Q/N)-rich segment. Although TDP-43 lacking the CTR is known to be unstable, as observed in knock-in mice, it is unclear which of these segments contributes to the stability of TDP-43. Here, we generated 12 mouse lines lacking the various sub-regions of CTR by genome editing and compared the embryonic lethality of homozygotes, and protein and mRNA expression levels of TDP-43. We demonstrated the functional diversity of the four segments of CTR, finding that the presence of the Q/N-rich segment greatly restored the protein stability of TDP-43. In addition, we found that the second GaroS deletion did not affect protein stability and mouse development. TDP-43 mainly localizes in the nucleus and is involved in RNA metabolism, including splicing, that promotes cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 skipping3. Moreover, TDP-43 is essential for mouse development6. In ALS, cytoplasmic aggregation of TDP-43 is a pathological feature that is often accompanied by nuclear loss of TDP-438. Because the amount of nuclear TDP-43 is strictly regulated by binding of TDP-43 to the 3′-untranslated region of its own mRNA, thereby forming an autoregulatory negative feedback loop13, nuclear loss of TDP-43 can induce the upregulation of TDP-43 mRNA and produce a vicious cycle that leads to the perturbation of TDP-43 homeostasis15. This imbalance of aggregation-prone TDP-43 plays a role in the pathological mechanism of ALS.TAR DNA-binding protein 43 kDa (TDP-43), a member of the heterogeneous nuclear ribonucleoprotein family, has emerged as a key player in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS)2, is also thought to be important for the stability of TDP-43 and its resistance to degradation. The CTR can be divided into four sub-regions, including a first Gly, aromatic, and Ser-rich (GaroS1) segment; a hydrophobic patch (HP); a Gln/Asn (Q/N)-rich segment; and a second GaroS2 segment  residues 311–360 has been identified to be critical for aggregation16. In addition, the CTR is a hotspot for ALS-causing TARDBP mutations. These mutations accelerate aggregate formation, with the result that fragmented and phosphorylated C-termini of TDP-43 accumulate in the affected tissues of ALS patients20. Recently, TDP-ΔC (aa 274–414) knock-in mice were generated, and they showed embryonic lethality of homozygous mice, and that TDP-43 lacking CTR is unstable21. However, it is unclear which of the segments of the CTR contribute to the stability of TDP-43, indicating the importance of analyzing mice with deletions of more localized regions within the CTR. In this study, we generated 12 mouse lines lacking the various sub-regions of the CTR by genome editing and examined their effects on embryonic lethality of homozygotes, TDP-43 levels and subcellular localizations, and Tardbp mRNA levels reflecting autoregulation to clarify the functional diversity of the C-terminal sub-regions.A long-disordered C-terminal region (CTR), known as a prion-like domainich GaroS segment;21, we established 12 mouse lines using the zinc finger nuclease (ZFN) and CRISPR/Cas9 systems, which were used to target the Q/N-rich (N345–Q346) and GaroS2 (Y374) segments, respectively , λpolyA , Tardbp exon 3 to 4 (Exon 3–4), Tardbp exon 5 to 6 (Exon 5–6), and Tardbp exon 6 (WT Exon 6). Exon 3–4 and Exon 5–6 recognize both WT and mutant alleles, whereas one of the WT Exon 6 primers targets deleted sequences induced by genome editing, thus WT Exon 6 recognizes the WT allele alone. In Group 1 homozygous mice, the total expression levels of Tardbp mRNA did not change in comparison with those of WT mice  compared to those of WT mice  flanked by two intrinsically disordered regions (IDR1 and IDR2)26. Hydrophobic residues  in the IDRs are distributed at conserved intervals across vertebrate TDP-43 homologues. These regions are cooperatively involved in liquid–liquid phase separation (LLPS) and forming membraneless organelles29. The self-association of CTR is synergistic and forms a continuum of physical states, which is proposed to be mainly facilitated by four interacting regions in the GaroS1-HP-Q/N-rich segment, α-helices (aa 321–330), a low complexity aromatic-rich kinked segment , a steric zipper (aa 328–333), and a fibril core (aa 331–360)31. In contrast, the GaroS2 segment is not necessary for full-length TDP-43 aggregation32. These observations suggest that the basic elements necessary for functional LLPS are the GaroS1-HP-Q/N-rich segment.The protein structure in solution exhibits two canonical α-helices (321–330 and 335–343) in the HP segment33. These reports support our results showing no apparent phenotype in CRY9B/Δ29 mice lacking the GaroS2 segment. However, there are several mutations in the GaroS2 segment that cause familial ALS, and the Y374X mutation, similar to the CRY9B/Δ29 mutation, has been found in sporadic ALS34. Furthermore, two steric zippers (aa 370–375 and 396–402) are present in the GaroS2 segment30. It is noteworthy that the GaroS2 segment is subjected to phosphorylation at a minimum of seven serine residues 20, which may serve to prevent excessive aggregation31. Considering that disordered proteins are generally regulated by post-translational modifications (PTMs)35, it may be that the GaroS2 segment is not essential for basic TDP-43 activity, but rather regulates its own nuclear function including TDP-43 activity and stability, presumably due to PTMs including phosphorylation.It has been shown that removing the GaroS2 segment has no apparent effect on either the TDP-43 activity of CFTR exon 9 skipping or intra-cellular localization in cultured cells23. Recently, TDP-43 was reported to demix in the cytoplasm as well as the nucleus using CTR36. The demixing of RNA-free TDP-43 in the nucleus induces anisotropic intranuclear liquid spherical shells (anisosomes), and the role of the demixing is speculated to tether RNA binding proteins, including TDP-43, near the site of transcription37. Furthermore, a nuclear export signal (aa 239–250) in TDP-43 appears to be non-functional, and a significant amount of TDP-43 is exported to cytoplasm through passive diffusion39. These observations raise the possibility that TDP-43s with large CTR deletions, including TDP-43 lacking the GaroS1-HP segment in Group 5 mice, can be imported to the nucleus, but they are dysfunctional and leak out during biochemical fractionation due to insufficient nuclear retention. Although further analysis and mouse models are required to understand the full functional diversity of the TDP-43 C-terminal sub-regions, we expect that the results presented here will help elucidate the pathogenesis of ALS and perhaps lead to the development of new ALS treatments.We used western blotting to show that full-length human TDP-43 with larger CTR deletions localized mainly in the cytoplasmic fraction, whereas the protein constructs were present in the nuclei of living cells when imaged using fluorescence microscopy. In a pioneering study using U2OS cells and CTR deletion constructs, the observed immunofluorescence patterns of subcellular localizations were variable, whereas biochemical fractionation showed that cytoplasmic localization was dependent on the length of the CTR deletionTardbp exon 6 and manufactured as ZFN binding (uppercase) and cutting (lowercase) sites (GTTAGCCAGCCAGCAGaaccagTCGGGCCCATCTGGGA). The ZFN mRNAs were microinjected into the pronuclei of B6C3F1/Jcl fertilized oocytes to generate ZE lines according to the manufacturer’s instructions. Next, a ssDNA oligonucleotide  were designed to target Tardbp exon 6 encoding the N-terminal region of the GaroS2 segment. The gRNA was synthesized commercially . The gRNA and CAS9 protein  were electroporated in fertilized zygotes of C57BL/6JJcl mice using previously described methods with minor modifications40. Electroporation was performed in HEPES-buffered Whitten’s medium using platinum plate electrodes on tempered glass . The pulse conditions of CUY21EDIT II (BEX) were 30 V  × 7 times. After electroporation, zygotes were incubated in Whitten’s medium for 24 h at 37 °C and 5% CO2, and the surviving two-cell-stage embryos were transferred into the oviducts of pseudo-pregnant female mice.To generate GaroS2-deficient mice, the guide RNA (gRNA) target sequence  was designed to target https://arriveguidelines.org). All mice were maintained under specific pathogen-free conditions with free access to CE-2 standard food  and ultrafiltered water. For the mouse lines where embryonic lethality was expected, including ZM39P/Δ295, the analysis at E12.5 was prioritized to reduce the number of mice.The genome-edited founder mice were crossed with C57BL/6JJcl mice to obtain heterozygous mouse lines. The genotyping conditions are described in Supplementary Table 41. The blots were subsequently incubated with the following antibodies: TDP-43 , α-tubulin , or p84 . For subcellular fractionation of cultured cells, the Pierce NE-PER reagents (Thermo Fisher Scientific) were applied according to the method used by Nonaka et al.24, and antibodies for p84  and GFP  were used for detection.Cerebrum samples from eight-week-old mice were homogenized and divided into four subcellular fractions enriched with cytoplasmic (C), membrane (M), nuclear (N), or insoluble (I) proteins using EzSubcell Extract . The fractions , 3 µg per lane, were subjected to western blotting as described previously+ RNA-A) from an External Standard Kit for qPCR  into the cerebral homogenate at a concentration of 1.8 × 106 copies/mg brain tissue, and simultaneously extracted total RNA from the cerebrum using RNAiso Plus (Takara). First-strand cDNA was synthesized using a SuperScript VILO cDNA synthesis Kit . Real-time qRT-PCR was performed using SYBR Premix Ex Taq II and a Thermal Cycler Dice Real-time System (Takara). Each cDNA sample was measured in duplicate applying the ΔΔCT method. Statistical analyses were performed using the REST2009 program (http://www.gene-quantification.de/rest-2009.html). The primer sets are listed in Supplementary Table To monitor the measurement process, we spiked external RNA . Immunofluorescence images were obtained using Alexa Fluor 488 goat anti-rabbit IgG  and a LSM710 confocal laser microscope .Immunohistochemical analysis was performed as described previously43. Deletion constructs were generated with a PrimeSTAR Mutagenesis Basal Kit (Takara) using the following primers: for dGaroS1, 5′-TGCCGAAATTAATCCAGCCATGATG-3′ and 5′-GGATTAATTTCGGCATTGGATATATG-3′; for d261CTR, 5′-TGCCGAACCATCGGGTAATAACCAA-3′ and 5′-CCCGATGGTTCGGCATTGGATATATG-3′. The dCTR construct was obtained from multicopy insertions of primer oligonucleotides during d261CTR construction. These deletion constructs were purified with an EndoFree Plasmid Maxi kit  and transfected in HeLa cells using Polyethylenimine MAX . After transfection, HeLa cells were cultured in DMEM supplemented with 10% fetal bovine serum in 5% CO2 at 37 °C for 48 h. The cells were then harvested for subcellular fractionation or subjected to fluorescence imaging using an inverted microscope, Axio Vert.A1FL-LED (Carl Zeiss).WT human TDP-43 cloned into the pEGFP-C3 vector  was constructed previouslySupplementary Information.


As proof-of-concept, long-term wireless respiration monitoring is demonstrated during sleep to identify symptoms of sleep apnea in wearers.A flexible sensor that can be attached to the body to collect vital data wirelessly enables real-time human healthcare management. One potential application for home-use healthcare devices is monitoring of sleep conditions to diagnose sleep apnea syndrome. Such data are not readily gathered using conventional tools, owing to the bulk and cost of instrumentation. In order to monitor respiration at home, it is necessary to improve sensing performance and long-term stability of the sensors without sacrificing wearability and comfortability. To build a platform for wireless home-use respiration monitoring, this study develops a mask-borne flexible humidity sensor using ZnIn  •A highly stable humidity sensor•Wireless wearable respiration monitoring•Per-form for sleep apnea monitoring Clinical measurement in health technology; Bioelectronics; Materials in biotechnology Continuous, real-time vital information allows detection of abnormal vital sign changes that may be caused by a disease. If such signs can be detected at an early stage, there is a greater chance of successful treatment and of saving a life. However, monitoring must measure continuous vital data during daily life in order to identify small changes. Because conventional medical instrumentation is unsuitable for home monitoring owing to bulky and expensive sensors, wearable sensors have the potential to revolutionize vital monitoring. Moreover, to enhance their wearability and comfort, wearable flexible sensors have been widely reported for healthcare applications , 2021b. 2S4 (ZIS) nanosheet-based humidity sensor  film, laser-induced graphene (LIG) electrodes, a ZIS stacking layer, and a humidity pass filter A. A wire3O+ ions and water molecules ) is relatively stable even after 20 cycles A. To ens0, where ΔR is the difference between initial resistance, R0, at 30°C in the flat state and the set temperature or bending radius, respectively. The resistance change ratio of the ZIS humidity sensor remains essentially invariant at temperatures from 30°C to 50°C  is a breathing disorder that causes human breathing to slow or stop for brief periods during sleep. If this sensor system can monitor SAS during sleep at home, it will be useful to detect the syndrome in its early stage. A wearable wireless sensor system on a mask was employed to monitor breathing rate and movement during sleep as a proof-of-concept for home-use SAS diagnosis. Three volunteers were invited to monitor their respiration rates during sleeping. They wore the mask with the sensor system, and real-time and continuous breathing rate and body motion during sleep were recorded using a smartphone A. FigureNext, reproducibility of this measurement was confirmed by repeating the monitoring from this and two other volunteers. First, breathing rate and body motion during sleep could be repeatedly monitored during sleep regardless of the volunteer , althougIn this study, a wireless, flexible humidity sensor system for real-time monitoring of sleep respiration was developed by optimizing a humidity sensor design and successfully monitoring respiration during sleep. We found that a smaller LIG interdigital electrode distance and thinner ZIS nanosheets for humidity sensing show greater stability. After confirming the stability of the sensor, wireless, real-time breathing rate and movement were monitored in three volunteers during sleep, based upon wireless humidity sensor system on a mask. This wearable humidity sensor and a smartphone app successfully recorded respiration including normal, deep sleep, and identified symptoms of SAS in a volunteer with sleep apnea. Based on real-time BPM data from the humidity sensor, trends of REM, non-REM, and SAS symptoms were clearly monitored. Although many challenges remain to replace conventional medical SAS tools, this mask-based wireless sensor system may be a good platform to advance home-use medical and healthcare diagnostic electronics.In this study, we have discussed the stability factor of the stacked ZIS nanosheet film, concluding that interface between nanosheets affects the stability. However, we did not find the specific material or structure at the interface caused for an instability factor. For the physiological trial, we only showed the possibility to find sleep apnea syndrome from respiration monitoring. For the practical use, clinical trials compared to the conventional methods will be required.takei@pe.osakafu-u.ac.jp or takei@omu.ac.jp).Further information and requests for resources and reagents should be directed and will be fulfilled by the lead contact, Professor Kuniharu Takei , InCl3·4H2O , and thioacetamide . The detailed process has been previously reported9. In this work, a ZIS nanosheet was formed using a solution of 10 mg mL−1.The ZIS solution was prepared from ZnCl2 laser ablation  on a PI film (thickness: 50 μm). The width of the electrodes was 0.4 mm, and the distance between interdigitating electrodes was varied from 0.1 mm to 1.1 mm in order to find the optimal distance. Subsequently, 75 μL of the ZIS nanosheet solution was drop-cast and annealed at 100°C for 20 min. To connect this humidity sensor to a measurement system, silver (Ag) electrodes were printed with a dispensing machine and annealed at 70°C for 30 min. Finally, the surface of the humidity sensor was covered with a humidity pass filter  to protect the ZIS nanosheet film from contact with water.LIG electrodes of interdigitating pattern were formed using COFundamental experiments with the ZIS humidity sensor were performed in a humidity and temperature controllable oven . All were recorded with a multi-channel data logger . As controls, commercial humidity and temperature sensors were also used to check humidity and temperature in the oven.The humidity sensor with a wireless circuit board was placed in a mask pocket and worn by volunteers  during sleep. Humidity and acceleration sensor outputs were automatically recorded by a smartphone linked via Bluetooth. Human subject experiments were performed in compliance with a protocol approved by the ethics committee at Osaka Prefecture University. Informed consent was obtained from all volunteers to record and use all data.Our study does not include statistical analysis or quantification.


The impact of phenolic acid on the antioxidant activity of SSPS was evaluated. Then, films were prepared by using phenolic acid-grafted SSPS. The physical, mechanical and biological performances of phenolic acid-grafted SSPS films were further investigated. The results indicated that an ester linkage was formed between the SSPS and phenolic acid. The grafting ratio of para-hydroxybenzoic acid, protocatechuic acid and gallic acid-grafted SSPS was 29.45, 31.76 and 30.74 mg/g, respectively. Phenolic acid endowed SSPS with improved antioxidant properties. Gallic acid (GA)-grafted SSPS possessed the best DPPH radical scavenging ability and reducing power, which may be related to the three phenolic hydroxyl groups in GA. Phenolic acid-grafted SSPS films showed increased moisture content and decreased water solubility compared to SSPS film. The phenolic acid-g-SSPS decreased the mechanical properties but enhanced the water vapor barrier property, and antioxidant and antibacterial properties of SSPS film. Meanwhile, the para-hydroxybenzoic acid-grafted SSPS film showed the lowest water vapor permeability (3.70 × 10−7 g mm/h cm2 Pa), and the GA-grafted SSPS film exhibited the best antioxidant and antibacterial activities.Three kinds of phenolic acid-grafted soluble soybean polysaccharide (SSPS) with similar grafting ratios were prepared, and their structure was characterized by FT-IR, UV-vis and  Petroleum-derived plastic materials are excessively consumed and are difficult to degrade, causing serious environmental pollution . Thus, eSoluble soybean polysaccharides having a structural resemblance to pectin is an acidic polysaccharide. SSPS possesses a galacturonan skeleton of rhamnogalacturonan and homogalacturonan  with a side chain of α-1,3- or α-1,5-arabinan and β-1,4-galactan . PreviouIn recent years, chitosan (CS) has been frequently grafted with various phenolic acids to enhance its water solubility and antioxidant and antibacterial activities ,11, whicIn this work, three kinds of phenolic acid-grafted SSPS (phenolic acid-g-SSPS) with similar grafting ratios were prepared. Structural characterization of phenolic acid-g-SSPS was carried out. Then, the phenolic acid-g-SSPS was further prepared into films. Finally, the physical and mechanical properties and biological performances of phenolic acid-g-SSPS films were studied.Soluble soybean polysaccharide , para-hydroxybenzoic acid (PPA), gallic acid (GA), protocatechuic acid (PA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), gelatin, Folin-Ciocalteu-reagent, N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were all bought from Yuanye Biological Co., Ltd., Shanghai, China.A series of phenolic acid-g-SSPSs were prepared, and the three kinds of phenolic acid-g-SSPSs with similar grafting ratios were selected for further evaluation. Details are as follows. v/v), followed by the addition of EDC . Then, NHS  was added to the reaction system and stirred for 2 h in the ice bath (4 °C). Afterward, the reactive system was mixed with the prepared SSPS solution, and the solution was stirred at room temperature for 48 h after stirring in the ice bath for 4 h. Then, the reaction solution was centrifuged  to remove residual GA. The final product was obtained by dialyzing against distilled water for a three-day period , lyophilized for 24 h and named GA-g-SSPS. Similarly, PPA-g-SSPS and PA-g-SSPS were synthesized according to the aforementioned method when the PPA  and PA  were substituted for GA. The yields of PPA-g-SSPS, PA-g-SSPS and GA-g-SSPS were 70.59%, 51.70% and 58.53%, respectively.Phenolic acid-g-SSPS was synthesized by the EDC/NHS reaction according to Shreiber’s method . SSPS  from 600–4000 cmf 4 cm−1 . UV-vis spectra of sample (1 mg/mL) were recorded (200–400 nm) using a U-3900 spectrophotometer (Hitachi (China) Ltd., Tokyo, Japan) .2O (0.55 mL). Then, 1 H NMR spectra were recorded at 25 °C at 400 MHz by using a Bruker Avance III HD spectrometer   reagent  and sodium phosphate buffer solution  were mixed; then the mixture was incubated in water  after evenly mixing in a vortex oscillator. Trichloroacetic acid (TCA)  was mixed with the reaction solution and centrifuged for 10 min. The supernatant of the mixture (2.5 mL), deionized water (2.5 mL) and FeCl3 solution  were mixed, and the mixture was allowed to stand for 0.5 h. At last, the absorbance was obtained at 700 nm.The reducing power was tested according to Oyaizu’s method . PhenoliPhenolic acid-g-SSPS films were obtained in accordance with Liu’s way with some changes . PhenoliA spiral micrometer  was used to test the thickness of films at any of 10 locations (as dispersed as possible) .W0), then the film was placed in an oven (105 °C) and dried to a constant weight (W1). Then, the film was soaked in 50 mL of deionized water and stirred slowly for 6 h. The remaining film was removed and placed into the oven again, and dried to a stable weight (W2) [The sample film was cut into 3 × 3 cm squares and weighed on an analytical balance (ght (W2) . The moiM indicates the increasing weight of the weighing bottle (g), d indicates the thickness (mm), t indicates the time period of weight measurement (h), A indicates the circular area covering the mouth of the weighing bottle (cm2) and The WVP of the film was measured by using the method of Gomez-Estaca . The filF was the stress at film break (N), d was the film thickness (mm), h was the width of the film (mm), 0L was the initial length of the film (mm).The mechanical performance of the film was evaluated on a DCP-KZ300 experimental machine . The rectangular film (150 × 15 mm) was placed on the measuring equipment with a spacing of 60 mm and a stretching speed of 50 mm/min . The tenThe released phenol content and DPPH radical scavenging ability were determined by Liu’s method . BrieflyStaphylococcus aureus and Escherichia coli was evaluated [6 CFU/mL) was evenly dispersed on an agar plate. The filter paper was removed from the bottle and put on the agar plate to incubate at 37 °C for 1 day. The diameter of the inhibition circle was then recorded with vernier calipers. Sterile water and potassium sorbate were considered the blank and positive control, respectively.The antibacterial activity of phenolic acid-g-SSPS films against valuated . First, p < 0.05.Three parallel tests were performed for each group, and the values were stated as mean ± standard deviation. The values were subjected to Duncan’s multiple range test, and analysis of variance (ANOVA) with SPSS 25.0 software , and a significant difference between the values was considered obtained if −1 were assigned to the O–H stretching vibrations, C–H and COO– stretching vibrations [−1 corresponded to the bending vibration of C–H. In addition, some typical peaks of polysaccharides were observed in the range of 1000–1200 cm−1, which are due to ring vibrations overlapped with C–OH stretching vibrations and C–O–C glycosidic bond vibrations [−1 is assigned to the β-glycosidic linkage in SSPS [−1 in phenolic acid-g-SSPS, which may be due to the ester groups (C=O stretching vibrations) formed between the hydroxyl group of SSPS and carboxyl groups of phenolic acid. A new absorption band at 1731 cm−1 was also noted in gallic acid-grafted chitosan, indicating the formation of an ester bond [As presented in brations . The peabrations . The abs in SSPS . The aboter bond .UV-vis spectra of SSPS and phenolic acid-g-SSPS are represented in 1 H NMR spectra can also provide structural support for phenolic acid-g-SSPS in The The grafting ratio of phenolic acid-g-SSPS was evaluated by the Folin-Ciocalteu method. The grafting ratio of PPA-g-SSPS, PA-g-SSPS and GA-g-SSPS was determined to be 29.45, 31.76 and 30.74 mg/g, respectively. Studies have shown that the biological activities of phenolic acid-grafted chitosan and the physical, mechanical and biological activities of phenolic acid-grafted chitosan films were related to the grafting ratio and the structure of phenolic acid . In our DPPH is extensively used to assess the antioxidant ability of samples because it is a stable free radical . As showThe reducing power of phenolic acid-g-SSPS is presented in As seen in The water resistance of a film can be assessed by two indicators, water solubility and moisture content. The moisture content of phenolic acid-g-SSPS films was higher than that of SSPS film . It migh−7 to 3.70 × 10−7 g mm/h cm2 Pa. The WVP of PA/GA-grafted CS films was lower than that of CS films [A key role of food packaging film is to prevent or reduce as much as possible, the transfer of moisture from the environment to the food . Thus, lCS films ,15. CovaCS films . In addiEnough mechanical strength can guarantee the integrity of the film and protect the film against small flaws, so TS and EB are two key indicators of the film’s mechanical properties . TS and Staphylococcus aureus and Escherichia coli than SSPS film. The order of antibacterial ability is as follows: GA-g-SSPS film > PA-g-SSPS film > PPA-g-SSPS film. The result exhibits that the antibacterial ability of phenolic acid-g-SSPS films is positively correlated with the released phenol content. The GA-g-SSPS film that has the highest released phenol content possesses the best antibacterial ability. In addition, due to differences in the structure of the bacterial outer membrane, all films exhibit stronger inhibitory ability against Staphylococcus aureus than that of Escherichia coli [As listed in hia coli . Comparehia coli .1 H NMR spectroscopy revealed that the reaction had successfully occurred. Phenolic acid endowed the SSPS with antioxidant properties. GA-g-SSPS possessed the best DPPH radical scavenging ability and reducing power, which could be linked to the three phenolic hydroxyl groups in GA. Meanwhile, phenolic acid-g-SSPS can decrease the water solubility and WVP and enhance the biological activities of the SSPS film. Since GA-g-SSPS film showed the best antioxidant and antibacterial performances, it can be expected to be a progressive material for food-active packaging. In subsequent studies, the influence of GA-g-SSPS film on the freshness of foods such as vegetables, fruits and meats should be assessed. The results in this study have important insights into the performance of active food packaging materials and their possible applications for the future.In this work, PPA, PA and GA were grafted onto SSPS by the EDC/NHS coupling method through ester bonding, respectively. FT-IR, UV-vis and


The valve number in AS-treated +/−Foxc2 mice was significantly higher than that of the vehicle-treated +/−Foxc2 mice. Furthermore, since β-catenin upregulates the expression of Foxc2 and Prox1 during lymphatic valve formation, and AS1842856 treatment increased the level of active β-catenin in both cultured human LECs and in mouse mesenteric LECs in vivo, we used the mouse model with constitutive active β-catenin to rescue loss of lymphatic valves in Foxc2+/− mice. Foxc2+/− mice have 50% fewer lymphatic valves than control, and rescue experiments showed that the valve number was completely restored to the control level upon nuclear β-catenin activation. These findings indicate that pharmacological inhibition of FOXO1 can be explored as a viable strategy to resolve valve defects in congenital lymphedema.Mutations in many genes that regulate lymphatic valve development are associated with congenital lymphedema. Oscillatory shear stress (OSS) from lymph provides constant signals for the growth and maintenance of valve cells throughout life. The expression of valve-forming genes in lymphatic endothelial cells (LECs) is upregulated by OSS. The transcription factor FOXO1 represses lymphatic valve formation by inhibiting the expression of these genes, which makes FOXO1 a potential target for treating lymphedema. Here, we tested the ability of a FOXO1 inhibitor, AS1842856, to induce the formation of new lymphatic valves. Our quantitative RT-PCR and Western blot data showed that treatment of cultured human LECs with AS1842856 for 48 h significantly increased the expression levels of valve-forming genes. To investigate the function of AS1842856  FOXC2 gene causes lymphedema-distichiasis syndrome in human patients  from lymph flow. VE-cadherin, an adherens jucntion protein, on the membrane functions as a key component in the mechanotransduction signaling pathway to transmit the external OSS signal into the lymphatic endothelial cells (LECs) through cytoplasmic AKT activation . The OSSve cells . Similarntenance . Mutatiopatients . The actelopment . In contpression . Howeverpe level . In thisFoxo1 is expressed in many cell types in the body, including lymphatic endothelial cells (LECs). We have previously reported that FOXO1 acts as a repressor for lymphatic valve formation. Deletion of Foxo1 in LECs induces the growth of new valves in vivo. More importantly, ablation of Foxo1 in a mouse model for human disease lymphedema–distichiasis, the Foxc2 heterozygous mice, completely resolves both the loss of valves and the leakiness of valves in this disease model. Mechanistically, knockdown of FOXO1 using shRNA in human dermal LECs (hdLECs) markedly upregulates the expression of valve-forming genes, including FOXC2, GATA2, and KLF4. Furthermore, FOXO1 mediates the repression activity by directly binding to the promoter region of the FOXC2 gene and negatively regulates FOXC2 expression  have been shown to be essential for lymphatic valve formation. Deletion of any of these three genes leads to the absence of lymphatic valve formation during embryonic development  and GJA4 (connexin37) are highly expressed in the valve leaflets. Loss-of-function of either gene results in defective valves  injected AS1842856 into Foxc2+/CreERT2 pups. Instead of using wildtype pups as control, we used Prox1-GFP mice as control and for visualizing the lymphatic vasculature  injection; 2) Prox1-GFP pups with AS1842856 injection; 3) Foxc2+/CreERT2; Prox1-GFP pups with DMSO injection; and 4) Foxc2+/CreERT2, Prox1-GFP pups with AS1842856 injection. DMSO and AS1842856 injections were performed every other day from P7 to P21  valves/mm was significantly higher in Foxc2+/CreERT2 pups with AS1842856 injection than those in Foxc2+/CreERT2 pups with DMSO injection; 3) valves/mm was still significantly lower in Foxc2+/CreERT2 pups with AS1842856 injection than those in Prox1-GFP pups with DMSO injection; and 4) there was no significant difference in valves/mm between Prox1-GFP pups with DMSO injection and Prox1-GFP pups with AS1842856 injection  mice, in which the serine and threonine encoded by exon 3 of the β-catenin gene (Ctnnb1) and phosphorylated by GSK3β are deleted to prevent the degradation of β-catenin in the destruction complex (Foxc2+/CreERT2 mice with the Ctnnb1lox(ex3) mice and the LEC reporter line, Prox1-GFP, for visualizing the lymphatic vasculature Ctnnb1; Prox1-GFP mice. As mentioned previously, the open reading frame in one allele of the Foxc2 gene is replaced with the cDNA for CreERT2 in the Foxc2+/CreERT2 mice making it heterozygous for Foxc2 and carrying tamoxifen-inducible Cre activity to recombine loxP sites. We injected tamoxifen (TM) at postnatal day (P) 1 and analyzed the mesenteric lymphatic vasculature at P8 and P14 in the +/CreERT2Foxc2; Prox1-GFP and +/CreERT2Foxc2; lox(ex3)Ctnnb1; Prox1-GFP and the control lox(ex3)Ctnnb1; Prox1-GFP mice ; Prox1-GFP mice ; Prox1-GFP mice ; Prox1-GFP mice at P14  mesentery expressed the same valve genes as the controls. The same TM protocol was performed as in Foxc2+/CreERT2, and the Foxc2+/CreERT2; Ctnnb1lox(ex3) mice. Considering that we do not have a method to distinguish the pre-existing valves from the new valves induced by β-catenin, we imaged multiple areas in the mesenteric lymphatic vessels in the Foxc2+/CreERT2; Ctnnb1lox(ex3) mice and compared the expression pattern of those valve genes to the control and Foxc2+/CreERT2. While all the valves in both the control and the Foxc2+/CreERT2; Ctnnb1lox(ex3) mesentery expressed high levels of Prox1, Foxc2, and Itga9, 1% of Prox1high valve areas in Foxc2+/CreERT2 mice lacked the expression of Itga9, indicating that these valve areas were missing the valve leaflets, which is consistent with our previous findings   . TherefoFoxc2+/CreERT2, and Foxc2+/CreERT2; Ctnnb1lox(ex3) mice at P14. Our SMA staining showed that SMCs were recruited to the collecting lymphatic vessels in all three genotypes, and the increased nuclear activity of β-catenin in the nucleus did not affect SMC recruitment  coverage . Loss ofruitment . Thus, tFoxo1 in the lymphatic endothelium induces new lymphatic valves to grow, which rescues the lymphatic valve defects in the Foxc2 heterozygous mice, a mouse model of lymphedema-distichiasis, by increasing the number of valves and by enhancing the function of valves. It was the first study to show that valve defects in congenital lymphedema can be restored after the impaired lymphatic vasculature has formed, which drove us to further investigate the function of FOXO1 inhibition in treating lymphedema. In this study, we tested the effect of a FOXO1-specific inhibitor AS1842865 in hdLECs and found that AS1842865 treatment upregulated the expression of valve and flow-responsive genes, including FOXC2, GATA2, KLF4, ITGA9, GJA4, and NOS3, on both the mRNA and protein levels. Moreover, i.p injection of AS1842865 increased the valve number in Foxc2 heterozygous mice in vivo. Intriguingly, Western blot showed that AS1842865 treatment increased the level of non-phosphorylated β-catenin, the active form of β-catenin, which was visualized by immunostaining in the nucleus of both cultured hdLECs and mouse mesenteric LECs in vivo. These data indicated that AS1842865 treatment enhanced the nuclear activity of β-catenin in LECs. To further investigate the function of nuclear β-catenin in vivo, we genetically increased the activity of β-catenin in the nucleus of LECs in Foxc2 heterozygous mice, a mouse model of lymphedema-distichiasis. Strikingly, enhanced nuclear β-catenin restored the valve loss in Foxc2 heterozygous mice to the wildtype level. Therefore, in addition to deletion of Foxo1, activation of β-catenin in the nucleus of LECs induces new lymphatic valves to grow.Previously, we showed that deletion of in vivo to begin valve development, and they cooperatively regulate each other in a feed-forward mechanism  expression is regulated by Foxc2  from lymph . It has echanism . Despiteng genes . These dby Foxc2 , the uprby Foxc2 .shFOXO1 to knockdown FOXO1 in cultured hdLECs and obtained the same increase in non-phospho β-catenin in shFOXO1 LECs compared to the cells treated with scramble control (data not shown). These unexpected results led to a question that we currently do not have an answer for: how, specifically, has AS1842865 increased the translocation of β-catenin into the nucleus? To investigate this, we examined the protein levels of GSK3β, the key component of the β-catenin destruction complex and its inactive form phospho-GSK3β in the AS1842865-treated LECs. Since our results showed that non-phospho β-catenin was increased, meaning less β-catenin was degraded in the destruction complex, we should observe an increased level of phospho-GSK3β, the inactive form of GSK3β, and decreased level of GSK3β. However, the expression level of phospho-GSK3β was decreased and the expression level of GSK3β was increased, which was the opposite to what we expected. Since the increased level of nuclear β-catenin was not due to GSK3β activity, we will analyze the activity of other components of the destruction complex and the activity of Wnt ligands in future studies. Nevertheless, the increased nuclear β-catenin added one more explanation for the upregulation of FOXC2 expression because β-catenin directly binds to the promoter region of the FOXC2 gene to activate FOXC2 expression. In summary, AS1842865 treatment potentially triggered the activation of a signaling cascade that regulates flow response and lymphatic valve formation without the presence of flow.Moreover, AS1842865 treatment did not upregulate the mRNA level of β-catenin but increased the nuclear localization and the activity of β-catenin on the protein level. In addition, we used Foxc2 heterozygous mice have been characterized as the mouse model for human lymphedema-distichiasis disease  at P1, P3, and P5 in the Foxc2+/CreERT2 mice to completely delete Foxo1, but the same protocol cannot be used in this study. We discovered that three injections of TM induced severe vessel dilation due to the excessive amount of β-catenin (data not shown). Thus, we reduced the amount of TM with one injection at P1. Our data showed that the mesenteric lymphatic vessels were no longer dilated, and the valves were still induced by the sufficient amount of β-catenin from exon 3 deletion. Therefore, in addition to Foxo1 deletion, moderately increased β-catenin activity can rescue the valve loss in Foxc2 heterozygous mice, the mouse model for lymphedema-distichiasis. However, activation of β-catenin at the organismal level is still not desired due to the function of β-catenin in inducing cancer cell proliferation.In addition to AS1842865 treatment, we showed that increased nuclear β-catenin activity completely resolved the valve loss in the The patients of primary lymphedema are susceptible to developing other comorbidities in addition to poor quality of life, such as metabolic disorders and infections, which could be caused due to dysregulated Foxo1 signaling. Elevated levels of Foxo1 have long been implicated in the pathogenesis of metabolic disorders and drug resistance in certain types of cancers, and small molecule inhibitors of Foxo1 have been proposed as a form of pharmacologic treatment . Hence, Prox1-GFP mouse line was from Dr. Young Kwon Hong from the University of South California (USC), LA. The Foxc2+/CreERT2 mouse line was obtained from Dr. Sathish Srinivasan at the Oklahoma Medical Research Foundation. The Ctnnb1lox(ex3) mice were procured with Dr. Makoto Taketo from Kyoto University, Tokyo. The FOXO1 inhibitor, AS1842856, was obtained from EMD Millipore Corporation, United States, and in vivo inhibition was achieved by intraperitoneal injections (i.p.) of AS1842856 (10 mg/kg) in wildtype and Foxc2+/CreERT2 pups every other day from P7 to P21 and AS1842856 (10 mg/kg) every other day from P7 to P11 or P7 to P14 for whole-mount immunostaining. For nuclear Ctnnb1 activation experiments, tamoxifen (TM) was injected subcutaneously into P1 Ctnnb1lox(ex3); Prox1-GFP, Foxc2+/CreERT2; Prox1-GFP, and Foxc2+/CreERT2; Ctnnb1lox(ex3); Prox1-GFP pups.All mouse husbandry and experiments were performed following the guidelines of the University of South Florida (USF) Institution of Animal Care and Use Committee (IACUC). Male and female mice were used for all experiments, and all mouse models were maintained on the C57BL/6J background and obtained through the material transfer agreement (MTA). The in situ under a Zeiss V16 fluorescent microscope, and the GFP images were analyzed using Fiji (ImageJ). Each lymphatic branch in the small intestine has a defined collecting lymphatic vessel that is connected to the lymph nodes and the pre-collecting lymphatic branches formed next to the gut wall. The segmented line tool in Fiji was used to measure the length of the collecting lymphatic appearing from the lymph node all the way to the branched pre-collecting lymphatics near the gut wall. The multi-point tool in Fiji was used to count the GFP high valves on the length of the measured collecting vessel. At least four controls and four KO animals were used for every experiment.TM-administered postnatal pups were euthanized using an IACUC-approved method, and the colleting lymphatics were analyzed at the experimental timepoints. Since the small intestine contains the majority of the lymphatic vasculature in the mesentery, collecting lymphatics from the small intestine right before the colon were imaged Tissues were collected and fixed in 2% paraformaldehyde (PFA) overnight on an orbital shaker  at 4℃. The next day, the fixed tissues were washed with phosphate-buffered saline (PBS) three times for 10 min each time, followed by permeabilization in 1X PBS + 0.03% Triton (X-100) (PBST) for 1 h. The tissues were blocked using 3% donkey serum in 1X PBST for 2 h and then incubated overnight with primary antibodies in 1X PBST on an orbital shaker at 4℃. The following day, the tissues were washed with 1X PBST (four times for 10 min each) to remove primary antibodies and then incubated with secondary antibodies in 1X PBST for 90 min at room temperature on an orbital shaker  after which the tissues were washed with 1X PBST  and incubated with conjugate antibodies in PBST for 60 min at RT. The tissues were then washed with 1X PBST  to remove secondary antibodies and treated with DAPI (Invitrogen) in PBS for 10 min on an orbital shaker at 4℃ for nuclear visualization. The DAPI was washed with PBS (5 min), and the tissues were mounted with ProLong Diamond Antifade Mountant (Invitrogen) onto glass slides  and stored at 4℃ until further use. The slides were imaged using the Leica SP8 confocal microscope, and the figures were created using Adobe Photoshop.The human dermal lymphatic endothelial cells (hdLECs) purchased from PromoCell (C-12216) were cultured in the EBM-V2  medium on fibronectin (20 μl/well)-coated 6-well plates. hdLECs were treated under four different conditions: DMSO (1 μM), AS1842856 (EMD Millipore Corporation) 0.1 μM, 0.5 μM, and 1 μM for three different time points: 24 h, 48 h, and 72 h for qRT-PCR. For the Western blot, hdLECs were treated with 0.5 μM AS1842856 for 48 h.® RT-for-PCR kit (Takara) according to the manufacturer’s guidelines. qPCR was performed in a QuantStudio 6 Real-Time PCR System (Applied Biosystems) with TaqMan® probes , and the Cq value of each gene was normalized to the those of GAPDH.The RNeasy Plus Mini Kit (Qiagen) was used to isolate total RNA from hdLECs in accordance with the manufacturer’s instructions. cDNA synthesis was performed using the Advantage™, Thermoscientific) was added. A cell scraper was used to scrape the hdLECs off the 6-well plate. hdLECs were pipetted and submerged in the lysis RIPA solution. The lysate was collected in a 1.5-ml protein LoBind tube and incubated for 15 min in ice on an orbital shaker  for complete lysis. The lysates were then centrifuged for 20 min at 18,300 RPM at 4℃, and the supernatant was collected in a separate Protein LoBind tube without disturbing the pellet. A bicinchoninic acid assay  was performed to calculate protein concentration according to manufacturer’s instructions, and 25 μg of the total protein was used to perform the Western blot. The Invitrogen™ mini gel tank apparatus was used to run the Western blot on a 4%–12% Blot™ Bis Tris Plus polyacrylamide gel. Protein transfer to a PVDF membrane was carried out using an iBlot 2 dry blotting system  under the P0 module. The iBind Flex Western System (Thermo Fisher Scientific) was used to process the mini blots using an incubation time of > 2.5 h, after which the blots were imaged using a Bio-Rad ChemiDoc imaging system.hdLECs were washed three times with 1X PBS and 1X RIPA buffer, and 100 X phosphatase inhibitor  unsaturated Western blot images (.scn) were opened with Image Lab 6.1 (Bio-Rad); 2) using the “lane and bands” tool on the left sidebar of Image Lab, the lanes were defined and adjusted manually; 3) using the “lane and bands” tool, the bands were identified manually with “add” bands option; 4) the “lane profile” tool was used to omit background; 5) the standard and sample lanes were labeled on the “image info” tool; 6) the adjusted volume (Int) value was obtained using the “lane analysis” tool on Image Lab; 7) each protein’s band volume was normalized to the band volume of the housekeeping protein (β-Actin); and 8) relative expression of each protein was calculated by normalizing the band volume of the experimental group (AS 0.5) to its control (DMSO).t-test was used to determine significant differences between two groups. One-way ANOVA with Tukey’s multiple-comparison test was used to determine significant differences for data with one independent variable. Two-way ANOVA with Tukey’s multiple-comparison test was performed to determine significant differences for data with two independent variables. p < 0.05 was considered significant. All data are represented as means ± SEM. GraphPad Prism software (version 6) was used for all statistical analyses and to plot quantitative data.The unpaired two-sided Student’s


In adults, meningioma is the most common primary tumor of the brain. It is classified into three clinical grades of aggressiveness. Whereas disease recurrence after surgery and survival are associated with grade, it is worth investigating proliferation at a molecular level to identify markers capable of improving the clinical management of meningioma. In this study, we explore the DNA methylation profiles of 48 tumors of various grades and conduct statistical analyses on several proliferation indices and markers, such as mitotic index, grade, and Ki-67 or MCM6 expression levels. We identify differential methylation profiles between grades, loci highly correlated with cell growth and division, and a specific methylation signature of regulatory regions persistently associated with proliferation indices, grade, and survival. Finally, we report candidate genes under the control of these regions with potential prognostic and therapeutic value and deserving clinical evaluation.SMC4, ESRRG, PAX6, DOK7, VAV2, OTX1, and PCDHA-PCDHB-PCDHG, i.e., the protocadherin gene clusters. This study highlights the crucial role played by epigenetic mechanisms in shaping dysregulated cellular proliferation and provides potential biomarkers bearing prognostic and therapeutic value for the clinical management of meningioma. Meningiomas are the most common primary tumors of the central nervous system. Based on the 2021 WHO classification, they are classified into three grades reflecting recurrence risk and aggressiveness. However, the WHO’s histopathological criteria defining these grades are somewhat subjective. Together with reliable immunohistochemical proliferation indices, other molecular markers such as those studied with genome-wide epigenetics promise to revamp the current prognostic classification. In this study, 48 meningiomas of various grades were randomly included and explored for DNA methylation with the Infinium MethylationEPIC microarray over 850k CpG sites. We conducted differential and correlative analyses on grade and several proliferation indices and markers, such as mitotic index and Ki-67 or MCM6 immunohistochemistry. We also set up Cox proportional hazard models for extensive associations between CpG methylation and survival. We identified loci highly correlated with cell growth and a targeted methylation signature of regulatory regions persistently associated with proliferation, grade, and survival. Candidate genes under the control of these regions include  TERT promoter mutation or a homozygous deletion of CDKN2A and/or CDKN2B as new criteria for the recognition of grade 3 meningiomas. The standard of care is as follows: when deemed adequate, most patients undergo surgery, whereas adjuvant therapy is not systematic; for grade 2 and grade 3 meningioma, after surgery, conformational radiotherapy is recommended, particularly in cases of incomplete resection in grade 2 and in all grade 3 cases , than for OS, with p-values in the range of [1.69 × 10−3–2.8 × 10−2]  than with OS  . No such associations were found with OS. From these top univariate hits, we derived multivariate associations with PFS  . Furtherwith PFS  and OS . ARHGDIA codes for a Rho GDP dissociation inhibitor protein with antiapoptotic activity and is involved in cellular processes, such as cell proliferation, cell cycle progression, and cell migration [DOK7 was also reported to be correlated with the Ki-67 index and may function as a tumor suppressor gene, which is consistent with our findings. However, in breast carcinoma, significant DOK7 promoter hypomethylation implicated its role in early tumorigenesis [DOK7 expression was associated with lower survival. In in vitro studies, DOK7 was reported to inhibit proliferation and migration by downregulating the PI3K/AKT/mTOR pathway [DOK7 was found to be downregulated in glioma, and its overexpression inhibited both in vitro and in vivo proliferation of glioma cells [DNMT1 (DNA methyltransferase 1). Its downregulation correlates with poor clinical outcome in gastric cancer, where it acts by suppressing cell growth and tumorigenesis. It also antagonizes Wnt signaling [ESRRG promoter hypermethylation was used as a diagnostic and prognostic biomarker in laryngeal squamous cell carcinoma [OTX1 overexpression with tumorigenesis and growth in cancers [When considering mitotic activity in meningiomas, we found negative correlations with DNAm for igration . Its expigration ,58,59. Iigration  and an aigration . DOK7 waigenesis ,62. In l pathway ,64. Simima cells . ESRRG iignaling . ESRRG parcinoma , where iarcinoma . Few stu cancers ,71, and TBR1 DNAm is positively correlated with the Ki-67 and MCM6 indices. TBR1 is a transcriptional repressor involved in multiple aspects of cortical development, including neuronal migration and axonal projection. Recurrent variations of TRB1 have been described in medulloblastoma, and a high frequency of its copy-number loss has been detected in glioblastoma, suggesting a possible involvement in tumorigenesis or progression [Between DNAm and Ki-67 levels, we report negative correlations for VAV2, a guanine exchange factor playing an important role in angiogenesis. Its recruitment by phosphorylated EPHA2 is critical for EFNA1-induced RAC1 GTPase activation, as well as vascular endothelial cell migration and assembly. Interestingly, we also found EFNA1 DNAm to be correlated with MCM6 expression. VAV2 is overexpressed in numerous cancers ,73,74, bgression ,78. To tGMNN- (geminin) and EFNA1- linked DNAm. GMNN was shown to inhibit DNA replication by preventing the incorporation of MCM complexes into prereplication complexes (pre-RC) [EFNA1 codes for the receptor protein-tyrosine kinase Ephrin A1, which is known to mediate developmental events and is involved in migration, repulsion, and adhesion during neuronal, vascular, and epithelial development in the nervous system and in erythropoiesis. In the context of tumor biology, it was shown to be regulated by hypoxia, and its main function involves angiogenesis and tumor neovascularization [With respect to MCM6 levels, we found negative correlations in (pre-RC) ,81,82,83rization . The recWhen correlating proliferative indices  with DNAm levels, we found that the positive and negative correlations exclusively occurred in the hyper- and hypomethylated DMRs differentiating WHO grades. This interrelation hints at a tight epigenetic control of cell proliferation. Thus, either on islands or in open-sea regions, non-concerted modification of methylation does not seem to be the key determinant driving cell division in meningioma. Moreover, fewer overlaps than expected were found between hits with our three chosen markers of proliferation, suggesting their complementing diagnostic value. As expected, because these markers are expressed during different stages of the cell cycle with different magnitudes of expression level, Ki-67 was found to perform well with high-grade and highly proliferative meningiomas, whereas MCM6 expression better delineated low-grade, low-proliferation tumors. Overall, the mitotic index was progressively correlated with demethylation in our proliferation signature.MSI1 regulatory region. This gene encodes an RNA binding protein that could be involved in the maintenance of stem cells in the central nervous system and in cell proliferation. In glioblastoma, it was found to promote the expression of stem cell marker CD44 by impairing miRNA function [Due to the limited number of samples in our dataset, the survival statistics were capped to a threshold of associations incompatible with genome-wide multitesting corrections. To overcome this, we first focused on the subset of 310 CpGs with proliferation-dependent DNAm and found that all CpGs were associated with PFS or OS. We next looked at genome-wide results and found that PFS associations with DNAm were the strongest, with unexpectedly numerous hits of high significance in broad genomic regions such a chromosomes 1q and 20. Results with PFS also confirmed the directionality of our DNAm proliferation signature, with progressive demethylation associated with worse progression. The lack of such an observation in OS indicates that no high adequation exists between these proliferation markers and occurrence of death; however, this could also be explained by a smaller number of events and the inclusion of deaths due to another pathology . OS may be more strongly associated with independent or indirectly linked loci, such as the function . In thesfunction , anotherfunction .SMC4, DOK7, PAX6, ARHGDIA, ESRRG, VAV2, and OTX1, as well as the three protocadherin gene clusters PCDHα, β, and γ, may be particularly relevant and deserve further preclinical and clinical investigations. In the present study, given the importance of proliferation in the prognosis of meningiomas, we designed an original approach focusing on the correlation between DNAm and cell proliferation. Proliferation is one of the main mechanisms of tumorigenesis and involves a considerable number of pathways shared by multiple types of tumors. However, epigenetic regulation is only a part of the mechanisms by which cells alter their gene expression. Although we do not have evidence of how DNAm changes affect gene expression, DNAm could potentially regulate downstream genes expression, which may contribute to tumorigenesis or progression. To better dissect the underlying molecular pathways of aberrant cellular growth in meningiomas, integrative and multi-omics studies are needed. As a first step, in this pilot mono-omic study integrating molecular and histological indices, we identified a proliferation signature encompassing hundreds of regulated genes, with several candidates serving as potential predictive and prognostic biomarkers or new therapeutic targets. Most notably, In conclusion, this study highlights the crucial role played by epigenetic mechanisms in shaping dysregulated cellular proliferation in meningioma. It provides molecular biomarkers with potential to revamp the current prognostic classification, as well as new druggable targets, adding therapeutic value to clinical management. The reported findings are novel and show the additional value of DNAm evaluation in diagnosis and prognostication for patients with meningioma.


Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease with high mortality and unclear etiology. Previous evidence supports that the origin of this disease is associated with epigenetic alterations, age, and environmental factors. IPF initiates with chronic epithelial lung injuries, followed by basal membrane destruction, which promotes the activation of myofibroblasts and excessive synthesis of extracellular matrix (ECM) proteins, as well as epithelial-mesenchymal transition (EMT). Due to miRNAs’ role as regulators of apoptosis, proliferation, differentiation, and cell-cell interaction processes, some studies have involved miRNAs in the biogenesis and progression of IPF. In this context, the analysis and discussion of the probable association of miRNAs with the signaling pathways involved in the development of IPF would improve our knowledge of the associated molecular mechanisms, thereby facilitating its evaluation as a therapeutic target for this severe lung disease. In this work, the most recent publications evaluating the role of miRNAs as regulators or activators of signal pathways associated with the pathogenesis of IPF were analyzed. The search in Pubmed was made using the following terms: “miRNAs and idiopathic pulmonary fibrosis (IPF)”; “miRNAs and IPF and signaling pathways (SP)”; and “miRNAs and IPF and SP and IPF pathogenesis”. Additionally, we focus mainly on those works where the signaling pathways involved with EMT, fibroblast differentiation, and synthesis of ECM components were assessed. Finally, the importance and significance of miRNAs as potential therapeutic or diagnostic tools for the treatment of IPF are discussed. Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, and devastating interstitial lung disease of unclear etiology with few therapeutic options and an average survival of 3–5 years after diagnosis . NumerouThe mitochondria plays an important role in the generation of ROS and in the apoptosis process; it has also been implicated in IPF disease . RecentlER stress is another proposed mechanism for the onset and progression of fibrotic diseases, including IPF . EvidencHypoxia is another prominent clinical feature of IPF disease. However, its real role in this pathology is still poorly understood. Mechanistically, hypoxia stimulates the proliferation of fibroblasts through hypoxia-inducible transcription factor alpha (HIF-1α), and HIF-2α . It has On the other hand, the contribution of endothelial mesenchymal transition (EndMt) process to some pathologies like IPF has been recognized lately. The first evidence showing that lung capillary endothelial cells could originate fibroblast through EndMT in a BLM-fibrosis mice model was performed by Hashimoto et al. in 2010 . It was All the mechanisms mentioned above have been associated with IPF’s pathogenesis, and for a more complete evaluation of these, we recommend reviewing the excellent work of Giang Phan et al. . NeverthIn addition to the mentioned, the incidence and prevalence of IPF increase with age, and the disease is more common in male patients above 65 years of age . InitialmiRNAs are a group of single-stranded non-coding RNA with a length of 19–25 base pairs. The miRNAs are transcribed in the cell nucleus by RNA polymerase II as primary miRNAs (pri-miRNAs) . Then, uInterestingly, in 2010 differences in the miRNAs expressed in lung tissues of IPF patients were demonstrated for the first-time, in comparison with miRNAs found in the lung of healthy subjects ,48. A yeIn this review, we carried on searches on PubMed by using the following cues: “miRNAs and idiopathic pulmonary fibrosis (IPF)”; “miRNAs and IPF and signaling pathways (SP)”; and “miRNAs and IPF and SP and IPF pathogenesis”. Through this search method, we mainly analyzed articles published between 2016 and 2021. However, because of their relevance, articles published in other years were mentioned too, although we focused principally on the articles obtained through the method described above. Moreover, we analyzed mainly all articles evidencing the impact miRNAs have on cell-signaling pathways involved with the IPF pathology.The manuscript was organized to consider both the expression direction of each miRNA (up or down-expressed) as well as its pro-fibrotic or anti-fibrotic properties. In addition, the main signaling pathways were summarized, highlighting those linked to increased extracellular matrix (ECM) synthesis and to cell regulatory mechanisms. Moreover, the potential utility of each miRNA as a target for IPF treatment was also discussed.EMT regulates different processes during the early stages of development of most organisms. However, it also plays a role in adult organisms and in the pathology of certain diseases such as organ fibrosis and cancer ,59. EMT In addition to the above, an increase in both expression and synthesis of matrix metalloproteinases has been reported, with a consequent augment in extracellular matrix degradation that favors cell invasion processes .At present, numerous evidences indicate that aberrant activation of different developmental pathways are associated with the pathology of IPF ,67,68,69Smad proteins intervene in the cell signaling acting as transcription factors. Eight Smad proteins have been reported in vertebrates (Smad1 to Smad8), and these are divided into three groups: Receptor-Smads (R-Smads), Common Smad (Co-Smad) and Inhibitory Smads (I-Smads) ,79. R-SmAlthough three TGFβ isoforms have been identified in mammalians , it is known that TFGβ1 is the most closely related to the development of IPF ,84. It iThe distinction between Smad-dependent and non-Smad-dependent pathways can be difficult to explain because there may be cross-talk between these pathways with non-Smad proteins . Non-SmaThe mitogen-activated protein kinase (MAPK) family includes three subfamilies: the extracellular signal-regulated kinases (Erk1 and Erk2), p38 MAP kinases, and c-Jun N-terminal kinases (JNKs) . All theThe phosphatidylinositol-3-kinase (PI3K) pathway is a non-Smad pathway required for EMT during TGFβ-induced fibrosis. P13K induces mainly the profibrotic pathway of Akt/mammalian target of rapamycin (mTOR) . mTOR isThe PI3K-Akt pathway is involved in cell growth, proliferation, survival and antiapoptotic processes. For its activation, PI3K stimulates the synthesis of phosphatidylinositol-3,4,5-triphosphate (PIP3), which recruits Akt and PIP3-dependent kinase that phosphorylates and activates Akt ,104. A mThis group consists of regulatory proteins of cytoskeleton organization and cell migration such as Rho, Rac and Cdc42. TGFβ regulates RhoA activity at the tight junctions of epithelial cells through the interaction with the TGFβRI/Par6 complex, allowing the phosphorylation of partitioning-defective protein (Par6) by TGFβRII in a specific serine. This process enhances RhoA ubiquitination by Smurf1 and degradation at tight junctions ,108. On 1-FZD10). In addition, some co-receptors are also required, such as the low-density-lipoprotein-related protein5/6 (LRP5/6) [2) pathway [2+ pathway is regulated by heterotrimeric G proteins which control Ca2+ signaling [Using gene expression microarrays, the upregulation of several genes of the Wnt signaling pathways in the lungs of patients with IPF, in comparison with control lungs, was demonstrated for the first-time . By then(LRP5/6) ,114. Whe(LRP5/6) ,115,116.(LRP5/6) . The non pathway . And, whignaling . Variousignaling ,110. In ignaling . In compignaling .However, and in spite of all advances in our comprehension of idiopathic pulmonary fibrosis, current therapeutic options still have great limitations ,122,123.Data obtained from the bleomycin (BLM) mice model or from fibroblasts stimulated with TGFβ showed pro-fibrotic activity of some miRNAs. An example is miR-21, which was the first miRNA studied in the BLM-induced fibrosis mice model in 2010 . In thisCurrently, it is well known that Smad7 blocks the function of Smad2/3, generating a negative effect on TGFβ1/Smad signaling . For exaMost of the recently reported miRNAs are within this group, since almost all are associated with the signaling pathways linked to TGFβ or with the activation of some of their components. A characteristic of this group of miRNAs is that they are important inducers of EMT, and are therefore considered the main contributors to lung fibrosis pathogenesis. In the middle of 2016, Ge et al.  showed sAlthough the accumulation of apoptosis-resistant fibroblasts is a hallmark of IPF lungs, the precise mechanisms by which these cells acquire this characteristic have not been fully elucidated yet . For insIdiopathic pulmonary fibrosis is considered the most lethal of the interstitial lung diseases with an unknown etiology. This illness is characterized by the focal accumulation of activated fibroblast and myofibroblasts derived mainly from EMT, which have the ability to overproduce ECM proteins and alter the epithelial architecture. During these processes, several signaling pathways associated with cell differentiation, migration, and transition are activated. Today, several studies have shown that the dysregulated expression of miRNAs could have an important role in the pathogenesis of IPF. Therefore, updating our knowledge about how these miRNAs intervene in or affect the onset or progression of IPF is an important issue of research. Until now, it has been demonstrated that human miRNAs listed in public databases represent only a small fraction of the total human miRNA repertoire ,40,175; Therefore, the absence of actual therapies highlights the need to evaluate other possibilities to treat IPF. Although the use of miRNAs as therapeutic drugs is a possibility yet to be verified in clinical trials, at present, more investigations have focused on the therapeutic role of miRNAs derived from extracellular vesicles (EVs). More studies have investigated the use of miRNAs from mesenchymal stem cells (MSC-EVs) from chronic respiratory diseases; these studies have shown promising results ,179,180.An important topic of research involves updating our knowledge on the role of miRNAs, as well as evaluating their association with the signaling pathways involved with the pathogenesis of Idiopathic Pulmonary Fibrosis. The goal is to improve our understanding of these pathways and molecular mechanisms, still poorly understood, and this improvement could help us suggest both new diagnostic biomarkers and promising strategies for the treatment of IPF disease.