Idiopathic pulmonary fibrosis (IPF) is characterized by abnormal fibroblast accumulation in the lung leading to extracellular matrix deposition and remodeling that compromise lung function. However, the mechanisms of interstitial invasion and remodeling by lung fibroblasts remain poorly understood. The invadosomes, initially described in cancer cells, consist of actin-based adhesive structures that coordinate with numerous other proteins to form a membrane protrusion capable of degrading the extracellular matrix to promote their invasive phenotype. In this regard, we hypothesized that invadosome formation may be increased in lung fibroblasts from patients with IPF. Public RNAseq datasets from control and IPF lung tissues were used to identify differentially expressed genes associated with invadosomes. Lung fibroblasts isolated from bleomycin-exposed mice and IPF patients were seeded with and without the two approved drugs for treating IPF, nintedanib or pirfenidone on fluorescent gelatin-coated coverslips for invadosome assays. Several matrix and invadosome-associated genes were increased in IPF tissues and in IPF fibroblastic foci. Invadosome formation was significantly increased in lung fibroblasts isolated from bleomycin-exposed mice and IPF patients. The degree of lung fibrosis found in IPF tissues correlated strongly with invadosome production by neighboring cells. Nintedanib suppressed IPF and PDGF-activated lung fibroblast invadosome formation, an event associated with inhibition of the PDGFR/PI3K/Akt pathway and TKS5 expression. Fibroblasts derived from IPF lung tissues express a pro-invadosomal phenotype, which correlates with the severity of fibrosis and is responsive to antifibrotic treatment. Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by alveolar epithelial cell injury, aberrant epithelium repair, fibroblast accumulation and excessive deposition of extracellular matrix (ECM). Fibroblasts and myofibroblasts form fibroblastic foci, the accumulation of which is associated with disease progression and poor prognosis . PharmacFibroblasts are known to promote the aberrant ECM remodeling in several respiratory diseases such as asthma, chronic obstructive pulmonary disease and particularly interstitial lung diseases including IPF . in IPF.First identified in Src-transformed fibroblasts , invadosAlthough the involvement of fibroblasts in the dysregulation of ECM homeostasis is an important event in the development of fibrosis, little is known about the key players involved in this process. We hypothesized that invadosomes, which are known to drive ECM remodeling in cancer, might be assembled by IPF fibroblasts and that these structures could be targeted by first-line treatments. To address this question, fibroblasts were isolated from human and murine fibrotic lungs and assessed for their ability to form invadosomes. In this study, we report the presence of an invadosome-associated gene signature in IPF lungs and fibroblastic foci. Accordingly, IPF lung fibroblasts have an increased capacity to form invadosomes, an event that correlates with the severity of fibrosis observed in the patients’ lungs. Furthermore, nintedanib and pirfenidone decreased invadosome formation by IPF fibroblasts, with nintedanib concomitantly inhibiting TKS5 expression and the PDGFR/Akt axis. These data suggest that invadosomes are associated with IPF pathogenesis and reveal an additional mode of action for some of the current first-line treatments.Given the possible presence and involvement of invadosomes in IPF disease, we first asked whether IPF lungs are characterized by a pro-invadosomal gene expression signature. For this, we used the public gene expression dataset GSE32537 and performed differential expression analysis between control and IPF subsets. The upregulation of fibrosis-related genes in IPF samples was used to validate the dataset. The results indicated that fibroblast-associated genes COL1A1, COL3A1, ACTA2, FN1, HAS2 and TGFB1 were strongly increased in IPF samples compared with the controls A. Next, To assess the ability of IPF fibroblasts to form ECM-degrading invadosomes, we isolated lung fibroblasts from healthy and IPF lung tissues, and the cells were subjected to invadosome assays. Fluorescent images show that IPF fibroblasts form more invadosomes and create larger areas of degraded matrix compared with fibroblasts from healthy donors. The areas of degraded matrix found 20 h after cell seeding often appear beside the cells, suggesting fibroblast motility on the gelatin matrix A. SpecifTo determine whether invadosome formation is also present in a model of induced fibrosis, we next studied lung fibroblasts isolated from mice, 28 days after exposure to bleomycin. Actively degrading cells are 1.7 times more frequent in bleomycin fibroblasts , and theTo define whether fibroblasts from collagen-rich regions of the lung have an increased capacity to form invadosomes, tissues from apical and basal regions of the lung were collected for each IPF patient and processed for histology. Then, high-resolution images of the tissues were taken following Masson’s trichrome staining to quantify collagen. Histologically, these tissues consisted of a heterogeneous distribution of normal tissues as well as immature and mature fibrotic areas, alveolar inflammation, fibroblastic foci and dense areas with collagen deposition, which is entirely consistent with the diagnosis of IPF. In Given that nintedanib and pirfenidone, the two first-line IPF treatments, were shown to slow the rate of disease progression, it was of interest to investigate their effect on invadosome formation. Images of fibroblasts from an IPF patient illustrate the efficacy of nintedanib to impair invadosome-induced gelatin degradation A. Nintedp-cortactin and PI3k/Akt signaling pathways. Nintedanib had no significant effect on Src phosphorylation at the Y416 activation site as well as cortactin phosphorylation at Y421, an Src phosphorylation site [Among the receptor tyrosine kinases (RTK) targeted by nintedanib, PDGFRα/β receptors are activated during IPF due to the strong production of PDGF by alveolar macrophages . Using hion site   comprised of activated fibroblasts and myofibroblasts are a histologic hallmark of IPF and strongly correlate with disease progression and mortality . Cells cSH3PXD2A) expression levels were found to be elevated in tissues, fibroblastic foci and cells derived from patients with IPF, suggesting its involvement in the disease. Using a gelatin degradation assay and a colocalization assay with TKS5, cortactin and f-actin, we identified functional invadosomes in lung fibroblasts derived from IPF patients and bleomycin-exposed mice. Healthy lung fibroblasts could produce invadosome structures and degraded gelatin matrix, but these features were robustly increased in fibroblasts isolated from fibrotic lungs.TKS5, an essential component of invadosomes, acts as a scaffold for the actin polymerization complex and takes part in MMP trafficking and ROS-mediated MMP activation . Here, TMMP14, MMP2 and ADAM12 in the fibroblastic foci, which was consistent with the ability of isolated IPF fibroblasts to degrade ECM in invadosome assays. TGFβ and multiple growth factors are bound to the ECM in a latent form and can be released and activated by MMPs and ADAMs [MMPs with gelatinase activity are augmented in lung samples, BALF and plasma derived from patients with IPF . In addind ADAMs ,41. It ind ADAMs . These fnd ADAMs . These pInvadosomes have been largely studied in vitro, but growing evidence in different organisms indicates their role in vivo in both physiological conditions and malignancies ,44. To fCTTN) expression between the control and IPF tissues. Immunodetection of the Src Y416 phosphorylation site and the Src-mediated cortactin Y421 phosphorylation site also suggested that the activity of these proteins was not modulated by nintedanib used at a concentration sufficient to block invadosome formation. Therefore, nintedanib seemed to inhibit invadosome formation through an Src-independent mechanism.We observed that nintedanib can achieve ~50% inhibition of invadosome formation at a lower concentration (0.5 µM) than pirfenidone (5.0 mM). These concentrations, which are commonly used for in vitro studies, correspond to, respectively, ~7 fold and ~60 fold above the concentrations found in human plasma ,49. ConsAmong the RTKs targeted by nintedanib, inhibition of PDGFRβ receptor was shown to markedly attenuate bleomycin-induced lung fibrosis . Our groGiven the role of invadosomes in ECM remodeling, invasion and migration, the observation of an invadosomal phenotype in IPF lung fibroblasts suggests a contribution of invadosomes to the pathogenesis of IPF, the details of which remain to be defined. To further study the role of invadosomes in lung fibrogenesis, the bleomycin mouse model could be used, since invadosomes were more numerous in fibroblasts isolated from lungs of mice with bleomycin-induced fibrosis.In conclusion, this study provides evidence suggesting that invadosome formation is a cellular mechanism by which IPF fibroblasts can promote interstitial remodeling of the lung and perpetuate pulmonary fibrosis. Given that the ability of IPF lung-derived fibroblasts to form invadosomes is strongly associated with fibrotic ECM, the capacity of fibroblasts to form invadosomes is likely to be relevant to numerous diseases with ECM remodeling and a fibrotic component. Despite the availably of two treatments that slow the rate of fibrosis progression, IPF remains a fatal lung disease for which effective treatments are needed. Further studies will be required to define whether the mechanisms leading to the increased capacity of IPF fibroblasts to form invadosomes could become relevant therapeutic targets in IPF.2 in DMEM supplemented with 15% fetal bovine serum, penicillin, streptomycin and glutamine. Additional normal human lung fibroblasts (NHLF) # CC-2512 and IPF disease human lung fibroblasts (DHLF-IPF) # CC-7231 were purchased from Lonza and cultured under the same conditions. The purity of fibroblast cultures was analyzed by flow cytometry   after obytometry . Lung fiytometry  and publCoverslips were coated with Oregon green gelatin and prepared as previously described . Lung fi3) and on two non-serial sections.Lung tissues were fixed with 10% formalin before paraffin embedding. Tissue sections of 4 µm were stained with Masson’s trichrome by the Histology Platform at the Université de Sherbrooke. TKS5 NBP1-90454) immunohistochemical staining was detected using diaminobenzidine and counter-stained with Harris hematoxylin. Microscope slides were scanned at 20× and 40× magnifications with the Hamamatsu NanoZoomer 2.0 RS slide scanner . Collagen quantification was performed as described by Chen et al. [ immunohip values were identified as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.GraphPad Prism 8.01 software  was used to perform the statistical analysis. Each individual value in the histograms corresponds to a single donor. Significance between two groups of non-normally distributed data was measured with an unpaired Mann–Whitney test. A Kruskal¬¬–Wallis test followed by Dunn’s multiple comparisons were used to compare more than two groups. Correlation strength was measured with a two-tailed Pearson’s test. Results are presented as mean ± SEM and


Progressive pulmonary fibrosis is a serious complication in subjects with sarcoidosis. The absence of reliable, non-invasive biomarkers that detect early progression exacerbates the difficulty in predicting sarcoidosis severity. To potentially address this unmet need, we evaluated a panel of markers for an association with sarcoidosis progression . This panel encompasses proteins related to inflammation, vascular injury, cell proliferation, and fibroblast mitogenesis processes.Plasma biomarker levels and biomarker protein expression in lung and lymph nodes tissues  from sarcoidosis subjects with limited disease and progressive (complicated) sarcoidosis were performed. Gene expression of the protein-coding genes included in this panel was analyzed using RNAseq in sarcoidosis granulomatous tissues from lung and lymph nodes.HBEGF and NAMPT among the top dysregulated genes and identified cytokine-related and fibrotic pathways in lung granulomatous tissues from sarcoidosis.Except for IL-8, plasma levels of each biomarker—eNAMPT, IL-1RA, IL-6, ANG-2, and HBEGF—were significantly elevated in sarcoidosis subjects compared to controls. In addition, plasma levels of HBEGF were elevated in complicated sarcoidosis, while eNAMPT and ANG-2 were observed to serve as markers of lung fibrosis in a subgroup of complicated sarcoidosis. Genomic studies corroborated These findings suggest HBEGF, eNAMPT, and ANG-2 may serve as potential novel indicators of the clinical severity of sarcoidosis disease. HBEGF gene as differentially expressed in sarcoidosis compared to healthy controls and accurately discriminated subjects with limited sarcoidosis from subjects with progressive lung sarcoidosis , a member of the EGF growth factor family expressed in the bronchial epithelium, smooth muscle, CD4 + T cells, and cardiac muscle. HBEGF signaling is a critical contributor to repair and regeneration processes that restore tissue homeostasis following injury, promoting wound healing, angiogenesis, and adipogenesis  and subsequent NF-κB activation (IL-1a SNP was significantly overrepresented in sarcoidosis subjects (compared to controls)  . IL-6 haontrols) .Based upon these genomic and epigenomic studies identifying potentially novel markers in sarcoidosis and other inflammatory lung disorders –25, the n = 62). Complicated sarcoidosis was assigned to those exhibiting lung involvement with parenchymal lung disease by CT scan or radiographic Scadding stages III and IV; Forced vital capacity (FVC) < 50%; and cardiac or neurological involvement in addition to lung involvement, as previously described (n = 19) and analyzed this subphenotype independently. For those with missing data for chest involvement, we used the ICD-9 and ICD10 codes to define the complicated status. Written informed consent was obtained from all participating subjects with protocols approved by the Institutional Review Board (IRB) at each participating institution. University of Arizona (IRB #1509097312R001), University of Miami (IRB# 20150612). De-identified samples were also obtained from a sarcoidosis cohort from the Instituto Nacional de Ciencias Medicas y Nutricion in Mexico City (Ref.#1711).A total of 127 plasma samples from sarcoidosis and 82 healthy controls were analyzed. The clinical characteristics and demographic information are presented in escribed . We also°C until biomarker measurement was conducted. 150 μl of plasma was used to quantify HBEGF levels using Quantikine® ELISA Human HBEGF immunoassay (R&D Systems®), quantitative sandwich enzyme immunoassay technique. Plasma levels of eNAMPT, IL-6, IL-8, Ang-2, and IL-1RA, were measured in 50 μl of plasma utilizing an electrochemiluminescent multiplex immunoassay predesigned panel from MesoScale  , samples were stored at ≤ –70y, MSD®) –29  . Supplemn = 11) and healthy controls (n = 5) in compliance with their respective IRB. Lung (n = 8) and lymph node (n = 8) tissues fixed in 10% formalin underwent H&E and IHC staining for three different biomarkers  using anti-Human HBEGF antibody , ANG-2 recombinant rabbit mAb , and rabbit anti-human NAMPT pAb  as previously described  was required to consider statistical significance. All analyses were performed with Stata v.17 , and Graphpad Prism v. 8.0 software .Numerical variables were analyzed using non-parametric Mann–Whitney test and Kruskal–Wallis test when parametric assumptions were not met. Group comparisons were performed using Spearman correlation. Sensitivity and specificity of the variables were calculated to evaluate the area under the curve (AUC) of the Receiver Operator characteristics (ROC) in each of the plasma markers. Cut-off values were selected using J statistics (Youden index). A significance of 0.05% (n = 127) and healthy control groups (n = 82) are presented in p = 0.37). The sarcoidosis group exhibited a higher proportion of females and African Americans compared to controls (p < 0.01). Radiographic or chest CT scan data indicating lung involvement stage was available on 71 of the 127 sarcoidosis subjects, with pulmonary fibrosis reported in 19 (15%) of total sarcoidosis subjects. 62 subjects (49%) of the sarcoidosis cohort were classified as complicated sarcoidosis; documented parenchymal lung disease by CT scan or radiographic Scadding stages III and IV; FVC < 50%; and/or cardiac or neurological involvement. The characteristics of the complicated and uncomplicated groups are presented in The patient characteristics in the sarcoidosis , followed by ANG-2 . eNAMPT, IL-6, and HBEGF exhibited similar AUC values (0.74–0.78)  and a moderate correlation between IL-1RA and IL-6 levels . A predictive model with regression analysis was applied to examine the correlation between eNAMPT and IL-1RA and showed IL-1RA values to significantly predict eNAMPT levels . This significance was not observed for IL-6 levels after adjusting for regression standard error  . Spearmard error .p = 0.007) (p = 0.017), with significant discriminatory power . We next assessed the ANG-2 cut-off values predictive of pulmonary fibrosis and identified a range of 2.4 ng/ml and 2.8 ng/ml of ANG-2 in plasma to produce a sensitivity of 72 and 66% and a specificity of 65 and 75%, respectively . These analyses identified HBEGF as the sole biomarker to significantly differentiate these two groups, with higher mean HBEGF levels in the complicated group, 47.8 pg/ml ± 109.6 vs. 21.4 pg/ml ± 50.5  . We next= 0.007)  compared= 0.007) . ANG-2 wectively . ANG-2 lp = 0.000038) and a three-fold increase in expression in sarcoidosis lung compared to normal lung tissues (p = 0.00004). Similarly, ANG-2 expression in sarcoidosis lymph node tissues (p = 0.0001) and lung tissues (p = 0.0001) was significantly higher compared to healthy tissue. Finally, NAMPT expression in sarcoidosis lymph node (p = 0.0009) and lung tissues (p = 0.005) was significantly higher compared to healthy tissue  using our previously reported publicly available gene expression dataset (GSE157671) generated by NextGen sequencing of micro-dissected sarcoidosis granulomas within lung tissues  comprised of cytokine-cytokine receptor interaction, MAPK signaling, IL-17, Jak-STAT signaling, chemokine signaling, NF-kappa B, and TNF signaling pathways  accurately discriminated between healthy controls and sarcoidosis subjects, with an optimal threshold > 74%. Our plasma eNAMPT results strongly contrast with a previous report indicating no difference between sarcoidosis and healthy controls , possiblANG-2 and HBEGF proved to be useful biomarkers in assessing disease severity with ANG-2 differentiating sarcoidosis pulmonary fibrosis (stage IV) from sarcoidosis without pulmonary involvement (stages I/II). We noted that when Stage III was incorporated in the analysis, ANG-2 levels were no longer discriminatory, an observation potentially related to the insensitivity of CXRs for assessment of early fibrotic changes of the parenchyma compared to HRCT or PET scans . HBEGF pHBEGF gene expression in PBMCs sarcoidosis . AXL targeting reduces fibrosis development in experimental renal fibrosis and in human intestinal organoid models . Chemokine (C-X-C motif) ligand 2 (CXCL2) also called macrophage inflammatory protein  can be found The studies involving human participants were reviewed and approved by the University of Arizona (IRB #1509097312R001) and University of Miami (IRB# 20150612). De-identified samples were also obtained from a sarcoidosis cohort from the Instituto Nacional de Ciencias Medicas y Nutrición Salvador Zubirán in Mexico City (Ref. #1711). The patients/participants provided their written informed consent to participate in this study.NC, CB, JG, MM, and BS: conception and design of the work, the analysis and interpretation of data for the work, the drafting and revision of the manuscript, and approval of the final version to be published. VR-H, TB, TG, GH-M, JS, BS, SS, JF, and MM: collection and analysis of data, revision of the manuscript, and approval of the final version to be published. VR-H, TB, MH, TG, SS, BS, JF, RO, and SC: data collection and assistance with processing and manuscript production and revision. All authors contributed to the article and approved the submitted version.


Drawing on Weberian ideal type, I outline the use of an innovative blended-methods approach to sample the communities in which CEnR practitioners would collect in-depth data. To complement typical practices of entering a community without preconceived ideas, understanding how communities in the sampling frame relate to one another is important for equigenic  implementations. The selection of neighborhood communities from quadrants in 2x2 matrices allows pertinent concepts to emerge and relevant solutions to be drawn from thriving communities to aid program co-creation and implementation in other communities. For example, this has led to the identification of communities in British Columbia with differing socioeconomic status, social capital, and coping during COVID-19. This methodological innovation is congruent with asset-based community development (ABCD) to minimize arbitrariness in sampling decisions and advance health equity in our cities.


Sensors, such as optical, chemical, and electrical sensors, play an important role in our lives. While these sensors already have widespread applications, such as humidity sensors, most are generally incompatible with flexible/inactive substrates and rely on conventional hard materials and complex manufacturing processes. To overcome this, we develop a CNT-based, low-resistance, and flexible humidity sensor. The core–shell structured CNT@CPM is prepared with Chit and PAMAM to achieve reliability, accuracy, consistency, and durability, resulting in a highly sensitive humidity sensor. The average response/recovery time of optimized sensor is only less than 20 s, with high sensitivity, consistent responsiveness, good linearity according to humidity rates, and low hysteresis (− 0.29 to 0.30 %RH). Moreover, it is highly reliable for long-term (at least 1 month), repeated bending , and provides accurate humidity measurement results. We apply the sensor to smart-wear, such as masks, that could conduct multi-respiratory monitoring in real-time through automatic ventilation systems. Several multi-respiratory monitoring results demonstrate its high responsiveness (less than 1.2 s) and consistent performance, indicating highly desirable for healthcare monitoring. Finally, these automatic ventilation systems paired with flexible sensors and applied to smart-wear can not only provide comfort but also enable stable and accurate healthcare in all environments.The online version contains supplementary material available at 10.1186/s40580-022-00326-6. Wearing of face masks by the general population has become a common and ubiquitous daily lifestyle worldwide. The daily wearing of personal protective equipment such as masks creates moisture inside owing to sweat and differences in internal and external temperature. Electrostatics-based masks are especially vulnerable to humidity, reducing the performance of the mask, and causing not only discomfort and nervous temperament to the wearer but also harm to the skin , 2.Recently, many researchers have made great efforts to eliminate this inconvenience, including sensors and ventilation systems with additional filters. However, most of the early wearable sensors added to various accessories have been focused on function and performance rather than user convenience , 4. In aTo overcome this bottleneck, many researchers have been striving to develop more flexible and smaller bands, patches, clothing, and contact lenses that are easier for users to wear , 9. ReceAmong various materials to overcome the above limitations, the core–shell structural material has the most efficient structure competent of forming organic–inorganic nanocomposites that have attracted attention in various fields, including sensors . Core–shHere, we aim to develop a flexible humidity sensor that can measure precise humidity intuitively and stably by enabling humidity measurement with very fast and consistent resistance changes depending on the atmospheric humidity. To facilitate this, we employed a complex of chitosan (Chit) and PAMAM dendrimer G3 (PM) as an organic phase that acted as an adhesive to coat individual carbon nanotubes (CNTs) uniformly. The materials formed were characterized by a core–shell structure encased in numerous amines of Chit and PM, and these amines allowed for flexible properties and quick reactions with moisture through self-assembly. When applied to smart wear, such as face masks, we confirmed that real-time multi-respiratory monitoring and ventilation systems showed very accurate and consistent outcomes.Pristine multi-walled carbon nanotubes  were purchased from Carbon Nanotech Co. . CNTs were refluxed in a 5 N HCl solution for 1 d prior to use and washed in deionized water. All chemicals, chitosan , PAMAM dendrimer, ethylene core, generation 3.0 solution (PM), and including organic solvents, which were purchased from Sigma-Aldrich , were of analytical grade and used without further purification.In order to fabricate CNT@CPM nanocomposites, each CNT was wrapped with PM-embedded Chit molecules in a core–shell structure that was prepared as follows: First, 45 mg of Chit was dissolved in 10 ml of acetic acid (1 v/v%) to make a Chit solution. 15 mg of CNT and each mol% of PM  was dropped on a gold electrode and dried at 60 °C. The resulting CNT@CPM nanocomposite-coated electrodes were thoroughly rinsed with water and absolute ethanol to remove any small molecules, including inorganic side products.−1 at 4 cm−1 resolution. Raman spectroscopy  was repeated 16 times under probing conditions over a spectral range of 50–3500 cm−1 at a resolution of 2 cm−1 using 532 nm laser excitation wavelengths, and X-ray photoelectron spectroscopy (XPS) was performed using an Electron Spectroscopy for Chemical Analysis II system  with a monochromatized aluminum K anode  at the National Center for Interuniversity Research Facilities (NCIRF) at Seoul National University. Atomic field microscopy (AFM) was used to show these characteristics, and the measurement conditions were as follows. The sample size was 5-μm diameter square, and analysis was conducted in contact mode using a cantilever with a resolution of 0.2 nm for the X/Y axis and 0.01 nm for the Z axis. The Brunauer–Emmett–Teller (BET) surface of the samples (100 mg) was determined from triplicate analysis using the BJH equation N2-desorption method . The contact angles were measured by dropping 20 μL of water droplets onto the sample surface. The relative amine number was calculated by calculating the molar amount of PM added in method parts. The morphological analysis of the CNT@CPM nanocomposite samples was performed using high-resolution transmission electron microscopy  with a resolution of 0.17 nm and field emission scanning electron microscopy . The FE-SEM studies were conducted using samples that had been sputter-coated with a 5-nm Pt layer prior to analysis. Energy dispersive X-ray spectroscopy  and line-scan analysis were conducted under the resolution conditions of 125 eV and SDD type.The Chit, CNT, PM, and CNT@CPM nanocomposites were characterized through qualitative analysis using Fourier transform infrared spectroscopy , which was repeated 16 times under potassium bromide pellet conditions over a spectral range of 400–4000 cmA humidity and temperature controller  was used to measure relative humidity (RH) at 25 °C. Resistance was measured with an LCR meter  at 1 kHz, 1 V, and 25 °C. The resistance variation graph for humidity was analyzed every 10 s by measuring the resistance of each humidity every 10 s when the relative humidity was adjusted every 10 percent from 30%RH to 100%RH. The hysteresis curves were repeated 5 times from 30%RH to 100%RH and again to 30%RH, and dynamic transient response curves were measured by changing from 30%RH up to each RH. The long-term stability test of each CNT@CPM flexible sensor was conducted by measuring the resistance once every 3 d for over 1 month while sensor was maintained at 50, 70, and 90%RH. The hysteresis extracted the average of the relative humidity differences  between the adsorption and desorption graph at the same resistance value in range of 30–100%RH [3Fe(CN)6 (0.5 M KCl) and 2.0 mM K4Fe(CN)6 (0.5 M KCl). Impedance measurements (EIS) were performed after each sample in the impedance analyzer  was for 30 min per humidity range in the 25 °C humidity chamber, and the measurement conditions were increased and decreased by 0.01 V per second from − 1.0 V to 1.0 V at frequencies (61 = 10/dec.).All electrochemical analyses were performed directly using the CNT@CPM flexible sensors. The current–voltage (I-V) curves were measured using a Voltage sag protector (VSP) potentiostat , the measurement conditions were increased and decreased by 50 mV per second from − 1.0 V to 1.0 V at 25 °C and 30%RH, and the average value was used after three repeated measurements. A three-electrode system was used with Ag/AgCl (3.0 M KCl) from Cypress systems  as a reference electrode, a 0.5 mm Pt wire counter electrode, and 4 mm diameter CNT@CPM flexible sensors as the working electrode. Cyclic voltammetry (CV) curves were measured in a mixed solution of 2.0 mM KAll experiments were repeated at least three times and data are presented as means ± standard deviation (S.D.).A schematic illustration summarizing the overall progress of our present paper is presented in Fig. ) also showed the same results as those of the HR-TEM. Obviously, the nanopores between the nanofiber strands shown in CNT@CPM-1 became thicker and were being blocked owing to the increasing PM ratio. Strong interaction with density changes according to fiber thickness can allow the flexible PI-based sensor to be strongly and reliably attached. The AFM images were measured to determine the change in the hydrophilic properties according to the ratio of PM. The AFM data  are compared in Fig. 0 wt% areata Fig. c, d showata Fig. d increasThese results were also confirmed using BET measurements of the surface area. The results of measuring the surface area according to the PM ratio of CNT@CPM are summarized in Table 2) that can act as proton donors and acceptors, providing a possible proton transport mechanism with humidity [+ ions generated through the dissociation of humidity molecules [+ ions through the humidity phase that is physiosorbed, as shown in Additional file p-type semiconductor [S) values of the CNT@CPM nanocomposite sensors to humidity were calculated using the following equations [0 = (RRH − R0)/R0 is the resistance change, R0 is the initial resistance value (30 RH%) of the device, and RRH is the steady-state resistance when exposed to different RH values. The sensor response, curve slope, and linearity of the sensors resulting from the sensor materials with different PM concentrations are shown and summarized in Fig. 2 value of 0.998 (CNT@CPM-3), because the increase in PM concentration caused an improvement in the sensor response by efficient hydrogen bonding due to an efficient reaction even at low humidity. At 10 wt%, the R2 value was reduced to 0.968 because the reactivity was too high in the low humidity range. EIS measurements for our sensors according to humidity showed that %RH was related to the direct resistivity of the sensor itself  in the RH range of 30–100%, which was superior to conventional electric resistance humidity sensors. Moreover, we apply the sensor to smart-wear that could perform accurate human health monitoring of multiple types of respiration . These flexible sensors not only exhibited the ability to detect several organic gases, but it also showed consistent and stable results even after prolonged exposure to physical stress, such as bending. Taken together, novel CNT@CPM flexible sensor, which demonstrated stability and high and accurate response times, is a promising and convenient human healthcare monitoring system.Additional file 1. Information on the characterization of CNT@CPM nanocomposites and the flexible sensor coated with CNT@CPM, including shell thickness distribution graph, schematic illustration, water contact angle results, XPS test, FT-IR spectra, Raman spectra, EDS results, humidity sensor traits table, EIS curves, I-V characteristics, CV curves, long-term stability results and actual performance videos of humidity sensing.Additional file 2. Video S1, The practical and actual application video of humidity sensor.Additional file 3. Video S2, Actual performance video of humidity sensing and automatic ventilation system.


Fzd7 decreased proliferation, differentiation, and expression of myogenic regulatory genes: myoblast determination factor-1 and myogenin in both lines. Both lipid accumulation and expression of adipogenic regulatory genes: peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-β, and neuropeptide-Y were suppressed with the Fzd7 knockdown. The RBC2 line was more dependent on the Fzd7-mediated Wnt/PCP pathway for proliferation, differentiation, and lipid accumulation compared to the NC line. Thus, thermal stress may affect poultry breast muscle growth potential and protein to fat ratio by altering function and fate of SCs through the Fzd7-mediated Wnt/PCP pathway in a growth-dependent manner.Satellite cells (SCs) are a heterogeneous population of multipotential stem cells. During the first week after hatch, satellite cell function and fate are sensitive to temperature. Wingless-type mouse mammary tumor virus integration site family/planar cell polarity (Wnt/PCP) signaling pathway is significantly affected by thermal stress in turkey pectoralis major (p. major) muscle SCs. This pathway regulates the activity of SCs through a frizzled-7 (Fzd7) cell surface receptor and two intracellular effectors, rho-associated protein kinase (ROCK) and c-Jun. The objective of the present study was to determine the effects of thermal stress, growth selection, and the Fzd7-mediated Wnt/PCP pathway on proliferation, myogenic differentiation, lipid accumulation, and expression of myogenic and adipogenic regulatory genes. These effects were evaluated in SCs isolated from the p. major muscle of 1-week faster-growing modern commercial (NC) line of turkeys as compared to SCs of a slower-growing historic Randombred Control Line 2 (RBC2) turkey line. Heat stress (43°C) increased phosphorylation of both ROCK and c-Jun with greater increases observed in the RBC2 line. Cold stress (33°C) had an inhibitory effect on both ROCK and c-Jun phosphorylation with the NC line showing greater reductions. Knockdown of the expression of  Birds are homeotherms, and thus, maintain body temperature within a limited range . Newly hThe number of myofibers is fixed by the time of hatch . Muscle in vitro studies, faster-proliferating SCs synthesize more lipid than slower-proliferating SCs  signaling pathway was greatly altered by both hot and cold thermal stress in turkey p. major muscle SCs . Among tkey line . In micekey line . A recenvia its effector c-Jun  ras homolog gene family member A (RhoA)  through or c-Jun . In mammor c-Jun . The Fzdignaling . Migratiignaling . In addiignaling  and adipignaling  in mouseignaling . In myogignaling  and SCs ignaling , the priWnt genes in SCs compared to non-growth-selected turkeys  in SCs isolated from modern commercial (NC) line turkeys and Randombred Control Line 2 (RBC2) turkeys representing slower-growing turkeys of the 1960s  was synthesized as a stealth siRNA duplex , and was designed using Invitrogen Block-iT siRNA designer . The Fzd7 siRNA targets the Fzd7 open reading frame from 1522 to 1546 with the following sequence: sense strand: 5′-CCG GAC UUC ACA GUC UUC AUG AUC A-3′; anti-sense strand: 5′-UGA UCA UGA AGA CUG UGA AGU CCG G-3′. A stealth siRNA with 48% guanine and cytosine content was used as the negative control siRNA (Thermo Fisher Scientific).Small interfering RNA targeting Fzd7 siRNA, p. major muscle SCs from each line were plated  in 24-well gelatin-coated plates  in 500 μl of Dulbecco’s Modified Eagle’s Medium  transfection medium supplemented with 10% chicken serum  and 5% horse serum (Gemini Bio-Products), and incubated at 38°C in a 95% air/5% CO2 incubator (Thermo Fisher Scientific) for 24 h allowing for cell attachment. After 24 h, cells in each well were transfected with 20 pmol/μl of either the Fzd7 siRNA or the negative control siRNA with 1 μl of lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. After 12 h of transfection, the medium was replaced with McCoy’s 5A growth medium (Sigma Aldrich) containing 10% chicken serum (Gemini Bio-Products), 5% horse serum (Gemini Bio-Products), 1% antibiotics-antimycotics , and 0.1% gentamicin  for 72 h of proliferation with the medium changed every 24 h. At 72 h post transfection, cells were removed from the incubator and total RNA was extracted for gene expression analysis by Real-Time Quantitative PCR (RT-qPCR) as described in Fzd7 siRNA.To determine the knockdown efficiency of the 2 incubator. After 24 h, the plating medium was replaced with the growth medium. Satellite cells from both lines were then randomly assigned to proliferate at 33°C, 38°C, or 43°C for 72 h, with the growth medium changed every 24 h. After 72 h of proliferation, the growth medium was replaced with a DMEM differentiation medium containing 3% horse serum, 1% antibiotics-antimycotics, 0.1% gentamicin, 0.1% gelatin, and 1 mg/ml bovine serum albumin . The differentiation medium was changed every 24 h. At 24-h intervals during differentiation, one plate in each group was removed from incubation and rinsed with phosphate buffered saline  for protein extraction.The RBC2 and NC line SCs were plated in 24-well plates  in a DMEM plating medium containing 10% chicken serum, 5% horse serum, 1% antibiotics-antimycotics, and 0.1% gentamicin. The plated cells were allowed to attach for 24 h while incubated at 38°C in a 95% air/5% COFzd7 siRNA transfection, cell culture and transfection procedures were the same as described in For 3VO4, and protease and phosphatase inhibitors (Thermo Fisher Scientific)] was added to the 24-well plates and incubated for 15 min on ice. Cells were scraped from each well, and the lysate was incubated on ice for another 15 min. A syringe with a 26 G needle was used to aspirate the cell lysate. The cell lysate was centrifuged at 10,000 rpm for 15 min at 4°C, and supernatant was collected. Protein concentration was measured using the Bradford method , 150 mM NaCl, 1 mM EDTA, 1 mM Nad method  and sampd method . Fold chCell culture and transfection procedures were the same as described in Proliferation assay was conducted according to the method of 2 incubator. After 24 h of attachment, cells in each well were transfected with 20 pmol/μl of either Fzd7 siRNA or negative control siRNA with 0.5 μl of Lipofectamine 2000. After 12 h of transfection at 38°C, the transfection medium was replaced with growth medium, and cells were randomly assigned to a 33°C, 38°C, or 43°C incubator for 72 h, with the growth medium changed daily. After 72 h of proliferation, the growth medium was replaced with differentiation medium for 72 h of differentiation with the media changed every day. One plate from each treatment group was removed at 24 h of intervals, rinsed with PBS, and stored at −70°C for the differentiation assay.For transfection at the beginning of proliferation, 11, 000 cells were plated in each well of 48-well gelatin-coated plates in 500 μl of transfection medium and incubated at 38°C in a 95% air/5% COFzd7 siRNA or the negative control siRNA with 0.5 μl Lipofectamine 2000 per well. After 12 h of transfection, the growth medium was replaced with differentiation medium for 72 h of differentiation. Differentiation medium was changed every 24 h. One plate from each treatment group was removed every 24 h, rinsed with PBS and stored at −70°C for the differentiation assay.For transfection at the beginning of differentiation (at 0 h of differentiation), 9,000 cells were plated in each well of 48-well gelatin-coated plates in plating medium at 38°C. After 24 h of attachment, plating medium was replaced with growth medium, and cells from both lines were randomly assigned to a 33°C, 38°C, or 43°C incubator for 72 h of proliferation. The growth medium was changed every 24 h. At 72 h of proliferation, the cells were transfected with 20 pmol/μl of either the Satellite cell differentiation was determined by measuring creatine kinase activity using a modified method of Cell culture and transfection procedures were as described in Cell culture and transfection procedures were the same as described in Lipids were stained with AdipoRed lipid fluorochrome . In brief, after removing the medium, each well was rinsed twice with PBS. Another 1 ml of PBS containing 30 μl of AdipoRed was added to each well with the cultured cells and one blank well without any cells in. After incubating for 15 min at room temperature, the AdipoRed optical density (OD) of each well was measured in the Fluoroskan Ascent FL plate reader at a wavelength of 485 nm. The final OD of each well was the OD of each well minus the measured OD of the blank well. The AdipoRed assay was independently repeated in two cultures with four replicate wells per treatment per culture.MyoD) and myogenin (MyoG) with the former a proliferation marker (PPARγ) (NPY) (C/EBPβ) (Myogenic regulatory genes measured in this study include myoblast determination factor 1 (n marker  and the n marker . Adipoge (PPARγ) , neuropeγ) (NPY) , and CCA(C/EBPβ) .C/EBPβ, MyoD, MyoG, NPY, PPARγ, and an internal control gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were previously designed, and the specificity was confirmed in this lab as reported by Fzd7 were designed using primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and the specificity of the Fzd7 primers was confirmed by DNA sequencing the PCR product . The RT-qPCR reaction was performed in a DNA Engine Opticon 2 real-time machine (Bio-Rad) and included: 1) denaturation at 94°C for 15 min; 2) amplification for 35 cycles with each cycle including denaturation for 30 s at 94°C, annealing for 30 s at 55°C , at 58°C (MyoD and MyoG), or at 60°C (C/EBPβ), and elongation for 30 s at 72°C; and 3) final elongation at 72°C for 5 min. A standard curve of each gene was generated using serial dilutions of purified PCR products  and the concentration of each sample was quantified by a spectrophotometer . Reverse transcription was performed with Moloney Murine Leukemia Virus Reverse Transcriptase  to produce cDNA from total RNA. Real-Time Quantitative PCR (RT-qPCR) was conducted with DyNAmo Hot Start SYBR Green qPCR kit (Thermo FisherScientific). Information of all the primers is listed in products . Arbitrap ≤ 0.05 was considered as statistically significant.Data from proliferation, differentiation, and AdipoRed assays, myotube measurement, and gene expression analysis were analyzed as a mixed model at each sampling time in SAS . Two fixed effects of temperature and cell line, an interaction effect between temperature and cell line, and a random effect of repeat experiment were included in the model. The statement of lsmean in the MIXED procedure was used to determine each mean value and the standard error of the mean (SEM). Differences between each mean were separated with the Pdiff option. Line effect within each treatment group and temperature effect within each cell line was determined with the SLICE option at each sampling time. For AdipoRed assay, the REG procedure was used to evaluate the linear relationship between sampling times and the OD of AdipoRed for each cell line in each treatment group. Difference in the linear response was determined with the contrast statement. p < 0.001) at all sampling times (p < 0.001). At both 38°C and 43°C, ROCK phosphorylation was greater in the NC line than RBC2 line at 72 h of proliferation, and 24 and 72 h of differentiation (p < 0.001). During the cold stress of 33°C, line effects were not significant at any sampling times.Phosphorylation of ROCK showed a significant interaction effect between temperature and cell line (ng times . In the p < 0.001) between temperature and cell line was observed for c-Jun phosphorylation at all sampling times (p < 0.001). The NC line showed greater c-Jun phosphorylation at both 38°C and 43°C from 72 h of proliferation to 48 h of differentiation (p < 0.001). A significant line effect was not observed at 33°C at any sampling time.Similarly, a significant interaction effect  and 5.31-fold (p < 0.001) in the RBC2 and NC Fzd7 knockdown groups compared to the control groups, respectively (p < 0.001) among the effects of Fzd7 knockdown, temperature, and line was observed in phosphorylation of both ROCK and c-Jun (Fzd7 decreased phosphorylation of ROCK (p ≤ 0.013) and c-Jun (p < 0.001) in both lines at all the temperatures. However, the NC line showed greater reductions in phosphorylation of both ROCK and c-Jun compared to the RBC2 line independent of temperature.Knockdown efficiency of ectively . At 24 hnd c-Jun . KnockdoFzd7 knockdown, temperature, and line effects at any sampling time during SC proliferation (p = 0.003), 48 h (p < 0.001), and 72 h (p < 0.001). The factors of temperature and Fzd7 knockdown significantly interacted at 72 h (p < 0.001). In both the RBC2 and NC control groups, heat stress (43°C) showed a positive effect on the proliferation of SCs  while cold stress (33°C) had a negative effect  on proliferaton at 72 h. Knockdown of Fzd7 decreased (p < 0.001) proliferation of both lines at 38°C  and 43°C  when measured at 72 h. At 33°C, no significant reduction in proliferation was observed in either line at any sampling time.No significant interaction was observed among feration . A signip ≤ 0.001). Knockdown of Fzd7 decreased (p < 0.001) the regression slope in both lines at 38°C  and 43°C .From 0 to 72 h of the proliferation assay, cell proliferation linearly increased as a function of sampling time in both the RBC2 and NC control groups at all the temperatures. The slope of the linear regression was always greater in the NC line compared to the RBC2 line (p < 0.001) among the effects of Fzd7 knockdown, temperature, and line at 0, 24, and 72 h of differentiation (p < 0.001) was only observed between line and temperature and between temperature and Fzd7 knockdown. Within each temperature, differentiation of the NC control group was greater (p < 0.001) than that of the RBC2 control group at 0, 24 and 48 h of differentiation. In the control groups, differentiation was greater at 43°C (p ≤ 0.002), and less at 33°C (p ≤ 0.030) at all sampling times compared to 38°C. Differentiation was linearly increased in both the RBC2 and NC control groups from 0 to 48 h of differentiation at 38°C and 43°C. The NC control group had a greater slope as shown by linear regression compared to the RBC2 line only at 38°C (p = 0.003).There was a significant interaction (ntiation . HoweverFzd7 decreased (p < 0.001) differentiation of SCs of both lines at 38°C and 43°C with a greater reduction observed in the RBC2 line compared to the NC line at all sampling times. At 33°C, knockdown of Fzd7 caused no significant reduction at 24, 48 and 72 h. Differentiation changed linearly with the knockdown of Fzd7 and the slope of the regression line decreased (p < 0.001) in both line at 38°C  and 43°C . The linear relationship between differentiation and sampling time was absent in either line at 33°C.Knockdown of Fzd7 also had a significant effect on myotube diameter. A significant interaction (p < 0.001) was observed among the effects Fzd7 knockdown, temperature, and line at 48 h of differentiation (Fzd7 decreased (p < 0.001) the myotube diameter in both lines at 38°C  and 43°C . At 33°C myotube diameter only decreased in the NC group (p = 0.040).Knockdown of ntiation . MyotubeFzd7 knockdown, temperature, and line effect was significant (p < 0.001) at all sampling times (p < 0.001) compared to the RBC2. Heat stress increased SC differentiation in both line at 0 and 72 h (p < 0.001), and cold stress showed a significant inhibitory effect (p < 0.001) on both lines at all sampling times. At 38°C, differentiation was lower (p < 0.001) in the RBC2 and NC Fzd7 knockdown groups compared to the controls at 48 h  and 72 h . Knockdown of Fzd7 also decreased (p < 0.001) SC differentiation at 43°C, with 1.21 and 1.46-fold reductions observed at 72 h in the RBC2 and NC lines, respectively. At 33°C, the knockdown effect was only significant in the RBC2 line at 72 h (p = 0.045). From 0 to 48 h, there was a linear increase in SC differentiation in the control and Fzd7 knockdown groups of both lines at 38°C and 43°C. Knockdown of Fzd7 at the beginning of differentiation did not significantly change the slope of the linear regression in either line at either 38°C or 43°C. A linear increase was absent in either line at 33°C.The interaction among ng times . At 38°CFzd7 was knocked down at the beginning of differentiation  interaction at 48 h of differentiation. Heat stress increased myotube diameter (p < 0.001) while cold stress decreased the diameter (p < 0.001) in both lines. However, at 38°C myotube diameter decreased only in the NC line, with the knockdown of Fzd7 . Both lines had reductions (p < 0.001) in myotube diameter with the knockdown of Fzd7 at 43°C . No significant knockdown effect was observed in either line at 33°C.Effects on myotube diameter were also seen when ntiation . The effp < 0.001) between temperature and line, between line and knockdown, and between temperature and knockdown at 72 h of proliferation (MyoD increased at 43°C (p < 0.001) and decreased at 33°C (p < 0.001) in both lines compared to the control (38°C) (Fzd7 decreased (p < 0.001) MyoD expression in both lines, with the RBC2 line showing greater reductions (fold change) compared to the NC line at 33°C and 43°C (Interaction effects were significant (feration . Expressl (38°C) . Knockdoand 43°C .Fzd7 knockdown, temperature, and line at 48 h of differentiation (p < 0.001) MyoD expression in both lines while cold stress (33°C) had an inhibitory effect  while cold stress (33°C) showed no significant effect in either line (Fzd7 decreased expression of MyoG in the RBC2 and NC groups only at 43°C (A significant interaction was observed among her line . Knockdo at 43°C .MyoG expression was increased at 43°C (p < 0.001) and decreased at 33°C (p < 0.001) in both lines (Fzd7 decreased MyoG expression (p < 0.001) in both lines at both 38°C and 43°C with the NC line having a greater reduction than the RBC2 line  for lipid accumulation was observed among Fzd7 knockdown, temperature and line effects at all sampling times (p < 0.001) with heat stress (43°C) and decreased (p < 0.001) with cold stress (33°C) in both line control groups at 48 and 72 h. At 48 and 72 h, the NC line had higher (p ≤ 0.007) lipid levels compared to the RBC2 line regardless of temperature. With knockdown of Fzd7, lipid content at 24, 48, and 72 h was significantly reduced (p ≤ 0.002) in both lines at all temperatures.During proliferation, a significant interaction (ng times . Lipid cp ≤ 0.023). Heat stress increased the slopes of both lines while cold stress decreased the slopes (p < 0.001). Knockdown of Fzd7 decreased (p < 0.001) the slope of both lines at all temperatures .From 24 to 72 h of proliferation, lipid content in both lines linearly increased at all temperatures . Within Fzd7 knockdown, temperature and line effects was significant (p < 0.001) at all sampling times (p < 0.001) only in the NC line at 24 and 48 h. A significant decrease (p < 0.001) in lipid content was observed in both lines under cold stress (33°C) but only at 24 h of differentiation. Knocking down Fzd7 significantly decreased (p ≤ 0.007) lipid content at 24 and 48 h in both lines at all temperatures, except at 33°C for the NC line (p = 0.247).During differentiation, an interaction among the effects of ng times . Within p < 0.001) was negative . At 38°C and 33°C, lipid content was lower (p < 0.001) in both line control groups s at 48 and 72 h, compared to the 24 h.From 24 to 72 h of differentiation, lipid content showed a linear decrease at 43°C in the control groups for both lines . The sloFzd7 knockdown, temperature, and line at both 72 h of proliferation  in both lines with a greater reduction observed in the NC line compared to the RBC2 line at 72 h of proliferation (PPARγ was greatly increased with the cold stress (33°C), with the NC line showing a greater increase (PPARγ expression increased (p < 0.001) in both lines at 43°C but decreased at 33°C in the RBC2 line (p < 0.001) (Fzd7 downregulated (p ≤ 0.002) expression of PPARγ in both lines at both sampling times, with greater reductions (fold change) in the RBC2 line compared to the NC line at both 43°C and 33°C (A significant interaction was observed among the effects feration . In contincrease . At 48 h< 0.001) . Knockdoand 33°C .Fzd7 knockdown, temperature, and line significantly interacted with each other at both sampling times  NPY expression in the RBC2 control group while cold stress (33°C) had a positive effect  in both lines at all temperatures during proliferation with the RBC2 line having greater reductions compared to the NC line at 38°C and 33°C (NPY was upregulated at 43°C (p < 0.001) and downregulated at 33°C (p ≤ 0.013) in both the RBC2 and NC compared to 38°C , with the NC SCs showing greater reductions than the RBC2 SCs  in the RBC2 control group at both 43°C and 33°C (C/EBPβ expression only at 43°C (p < 0.001) , while cold stress downregulated the C/EBPβ only in the NC control group  in both the RBC2 and NC Fzd7 knockdown groups, compared to the controls at all temperatures with the NC line having greater reductions than the RBC2 line (There was a significant interaction among the ntiation . At 72 hand 33°C . The NC < 0.001) . KnockdoBC2 line .Wnt genes, Wnt7a, which regulates the Wnt/PCP pathway through the Fzd7 receptor  and elevated (43°C) temperatures. The effect of the knockdown was not significant under cold stress since the proliferation of SCs had already been suppressed. These results imply that the Wnt/PCP pathway through the Fzd7 receptor acts in the proliferation of turkey SCs only at higher temperatures. The SCs of the growth-selected NC line were less dependent on the Fzd7-mediated Wnt/PCP pathway in maintaining proliferation compared to the RBC2 line. It is possible that other signaling pathways which support the proliferation of SCs have also been affected by growth selection. As reported by With regard to SC proliferation, the mitotic activity of poultry SCs peaks during the first week after hatch . During d stress . InhibitFzd7 decreased the activity of both ROCK and c-Jun. If Fzd7 knockdown occurred at the beginning of proliferation, SC differentiation as well as myotube diameter was reduced to a greater extent compared to Fzd7 being knocked down at the beginning of differentiation. This suggests that the Fzd7-mediated Wnt/PCP pathway affects myogenic differentiation of SCs mainly through regulating SC proliferation and migration prior to subsequent fusion to existing myofibers.With the progression of myogenic differentiation, SCs in cell cultures gradually fuse to form multinucleated myotubes . The actFzd7 significantly decreased both lipid accumulation and the expression of PPARγ, C/EBPβ, and NPY during later proliferation and early differentiation. The commercial NC line SCs were less dependent on the Fzd7-mediated Wnt-PCP pathway in maintaining lipid accumulation compared to the RBC2 line. Together, these findings suggest thermal stress-induced changes in SC adipogenesis is regulated, in part, by the Fzd7-mediated Wnt/PCP pathway in a developmental time- and growth-dependent manner.Satellite cell adipogenic potential depends on the developmental stage of the p. major muscle SCs . Due to in vivo is associated with altered intramuscular fat deposition (Additional pathways associated with adipogenesis may also be affected by growth selection. For example, activity of the mTOR pathway is higher in the NC line SCs compared to the RBC2 line independent of temperature . The incposition . Heat stposition , and resFzd7 suppressed the proliferation, differentiation, and lipid accumulation in both lines of SCs. The reduction in these processes were greater in the commercial NC line compared to the non-selected RBC2 line. At normal temperature or during heat stress, the proliferation, differentiation, and adipogenesis of the NC line SCs, were overall less responsive to the knockdown of Fzd7 compared to the non-selected RBC2 line. Furthermore, knockdown of Fzd7 at the beginning of proliferation decreased the differentiation and myotube diameter to a greater extent in both lines compared to the knockdown of Fzd7 at the beginning of differentiation. These results indicate that Fzd7-mediated SC myogenesis was initiated during proliferation. Hence, thermal stress during the period of SC peak mitotic activity may affect the structure and composition of turkey p. major muscle, in part, through the Fzd7-mediated Wnt/PCP pathway. During heat stress, increased SC proliferation and differentiation is associated with myofiber hypertrophy whereas intracellular lipid production will result in more intramuscular fat depots. Muscle fibers resulting from excessive hypertrophic growth will occupy the available connective tissue spacing and come in contact with each other (NPY (MEF2C (In conclusion, the results from the current study indicate that thermal stress affects proliferation, differentiation, lipid accumulation, and expression of myogenic and adipogenic regulatory genes in turkey p. major muscle SCs through the Fzd7-mediated Wnt/PCP pathway in a growth-dependent manner. Specifically, heat stress promotes proliferation, differentiation, and lipid synthesis by stimulating the phosphorylation of both ROCK and c-Jun in both the NC and RBC2 line SCs, while cold stress showed an inhibitory effect. During cold stress, knockdown of the expression of ch other . This wiher (NPY  and MEF2Y (MEF2C  are alte


A graphene-based terahertz negative-conductivity metasurface based on two types of unit cell structures is investigated under the control of an external bias voltage. Electrical characterization is conducted and verification is performed using a finite-difference time-domain (FDTD) and an optical-pump terahertz (THz)-probe system in terms of simulation and transient response analysis. Owing to the metal-like properties of graphene, a strong interaction between the metasurface and monolayer graphene yields a short-circuit effect, which considerably weakens the intensity of the resonance mode under passive conditions. Under active conditions, graphene, as an active load, actively induces a negative-conductivity effect, which enhances the THz transmission and recovers the resonance intensity gradually because of the weakening of the short-circuit effect. The resulting resonance frequency shows a blue shift. This study provides a reference value for combining graphene exhibiting the terahertz bias-driven negative-conductivity effect with metasurfaces and its corresponding applications in the future. A graphene-based terahertz negative-conductivity metasurface based on two types of unit cell structures is investigated under the control of an external bias voltage. THz waves avoid the application risks associated with high-frequency waves (such as X-rays) with high energy and strong destructive force, solve the problem of severe Rayleigh scattering in intermediate frequency waves (such as visible light and near-infrared light) with short wavelengths, and compensate for the low spatial resolution attributed to long microwave wavelengths. On the basis of these factors, THz waves are widely used in remote sensing, homeland security, astronomy, nonlinear applications, material diagnostics, broadband wireless communications, and many other fields.6–11 Compared with fully three-dimensional metamaterial structures, metasurfaces occupy less physical space, thus offering the possibility of low-loss structures.6 Because resonant metasurfaces can interact with the incident electromagnetic field, obvious resonant absorption can be realized in the THz frequency range by adjusting the parameters of a metasurface unit cell, such as its period, linewidth, gap width, and medium.12–14 However, once the parameters of metasurface unit cells are determined, the THz resonance absorption becomes stable and unchangeable, considerably limiting the applications of metasurfaces. Therefore, metasurfaces are typically combined with other materials to form a hybrid metasurface for achieving the flexible control of THz waves by applying external control conditions to the device.15–18Metamaterials are a group of artificial materials with a regular array of periodic subwavelength structures composed of metal or dielectric materials in the entire space area. When resonant metamaterials interact with incident electromagnetic fields, they show electromagnetic response characteristics not possessed by natural materials. These characteristics include a negative refractive index, artificial magnetism, and superfocusing to achieve the flexible control of the incident light phase, amplitude, and polarization.2-hybridization honeycomb crystal plane. Since its first successful preparation in 2004,19 monolayer graphene has shown high carrier mobility, broadband flat optical response, tunable bandgaps, high electronic and optical conductivities, and many other unique electrical and optical properties, thus garnering extensive research attention.20–23 The conductivity of graphene can be effectively regulated via chemical doping or by applying externally excited light or an external bias voltage, possibly enabling the combination of graphene and metasurfaces.24–26Graphene is a single-atom form of carbon with a spi.e., RMRR/graphene/quartz and RSRR/graphene/quartz) with increasing applied voltage, the THz transmission gradually increases, the intensity of the resonance peak of the metal dipole is gradually recovered, and the resonance frequency shows a blue shift. Therefore, the stable control of the device is realized under an external bias voltage. The conductivity of the device is calculated using the equivalent transmission line theory for elucidating the enhancement in THz transmission. A finite-difference time-domain (FDTD) and an optical-pump THz-probe system are used to verify the abovementioned experimental results.In this work, a graphene-based terahertz negative-conductivity metasurface device is proposed in which the unit cell structure of the metasurface is designed as a rectangular split-ring resonator (RSRR) and a rectangular metal-rod resonator (RMRR). Without an external bias voltage, a very weak resonance peak of the metal dipole appears under THz transmission. When an external bias voltage is applied to both electrodes of the samples  g = 5 μm, and period P = 150 μm. Finally, the center line of the sample is considered as the symmetrical axis and two parallel gold electrodes with a length, width, thickness, and spacing of 2 cm, 5 mm, 200 nm, and 3 mm, respectively, were plated on both sides of the center line of the sample via vacuum thermal evaporation. Thus, the complete sample structure required for the experiment was prepared.The experimental measurement system is a THz time-domain spectroscopic (THz-TDS) system . The enl3.i.e., the RMRR/graphene/quartz and RSRR/graphene/quartz samples) decreases considerably and the resonance frequency shows a substantial red shift. An external bias voltage is applied to the electrodes of the RSRR/graphene/quartz and RMRR/graphene/quartz samples. With increasing external bias voltage, the THz transmission gradually increases, the resonance intensity is recovered, and the resonance frequency undergoes a certain blue shift. The resonance frequency of the RMRR/graphene/quartz metasurface sample undergoes a blue shift from 519 to 540 GHz  and Enonexcited(ω) denote the electric field intensities of the THz waves passing through the samples with and without the application of external control conditions, respectively. Moreover, d denotes the sample thickness, ω represents the angular frequency of the THz waves, Z0 denotes the free-space impedance, and n represents the refractive index of the quartz substrate. The complex conductivity of the sample calculated using the equivalent transmission line theory is shown in The real and imaginary parts of the film conductivity are then obtained as follows:σ = σ − σ0, where σ and σ0 represent the complex conductivities of the sample with and without the application of external control conditions, respectively. Then, the aforementioned results show that when an external bias voltage is applied to the sample with monolayer graphene/quartz structures, Δσ < 0, corresponding to the negative-conductivity effect.34,35 Therefore, the negative-conductivity effect of graphene enhances the THz transmission under the application of an external bias voltage to the samples.The conductivity of both the real and imaginary parts of the monolayer graphene film decreases with an increase in the external bias voltage. The THz transmission is enhanced by the decrease in the conductivity of the monolayer graphene film. Consider the change in conductivity is ΔR–T) and current–voltage (I–V) curves of the sample are obtained  until saturation is reached, and this phenomenon becomes increasingly prominent with increasing external bias voltage. Based on R–T and I–V curves of the graphene/quartz sample are highly consistent with the variation trend of the sample conductivity obtained using the equivalent transmission line theory.To validate the variation trend of the sample conductivity calculated using the equivalent transmission line theory, Keithley's 2450 semiconductor test system is used to evaluate the graphene/quartz sample and the resistance–time , the resistance Rg in parallel with the equivalent capacitance C increases. As Rg increases, the inhibition effect of monolayer graphene on the electric field in the capacitance gap decreases; thus, the short-circuit effect is weakened and the intensity of the dipole resonance mode is recovered. Alternatively, under the applied external bias voltage, numerous carriers are injected into the device, affecting the dipole resonance mode and inducing a blue shift of the resonance frequency.Under the applied external bias voltage, in addition to the obvious enhancement in THz transmission, the intensity of the dipole resonance mode is gradually recovered and the resonance frequency undergoes a certain blue shift. Therefore, for the equivalent circuit models of the two metasurface samples  solver is used for simulation verification. −1. With decreasing conductivity of monolayer graphene, the resonance intensity is gradually recovered and the resonance frequency undergoes a blue shift. Clearly, the resonance frequency of the RMRR metasurface sample shows a blue shift from ∼575 to ∼634 GHz and that of the RSRR metasurface sample shows a blue shift from ∼485 to ∼535 GHz. Notably, as the conductivity of monolayer graphene decreases, the resonance intensity in the simulation results is gradually recovered but ultimately does not recover to that of the sample without monolayer graphene; these results are consistent with the experimental results.For the two types of metasurface resonators with monolayer graphene, the conductivity of monolayer graphene decreases in the simulation using finite-difference time-domain to a value equivalent to that obtained when applying an external bias voltage in the experiment . Compare−1, respectively. At 1000 S m−1, the field strength at the capacitance gap of the adjacent RMRR is very weak. Alternatively, at 100 S m−1, the field strength at the capacitance gap improves considerably. This explains the gradual recovery of the resonance intensity of the dipole resonance mode with decreasing conductivity of monolayer graphene. Similarly, −1, respectively. At 1000 S m−1, the field strength at the capacitance gap and on the metal arm opposite to the capacitance gap of the RSRR is very weak. However, at 100 S m−1, the field strength at the capacitance gap and on the metal arm opposite to the capacitance gap increases substantially. This also explains the gradual recovery of the resonance intensity of the dipole resonance mode with decreasing conductivity of monolayer graphene.T and T0, respectively), thus confirming the pump-induced change in THz transmission ΔTr = T − T0. The enlarged image in via the stable control and electrical characterization. Since the conductivity of graphene can be changed by bias voltage or photoexcitation, the proposed structure can be used to fabricate active resonance tunable terahertz metasurface switches, which will enrich the design and application of terahertz functional devices.The optical-pump THz-probe system is used to evaluate the transient response of the samples. 4.via the electrical characterization of the graphene/quartz samples. A finite-difference time-domain and an optical-pump THz-probe system are used to verify the negative-conductivity metasurface. The combination of graphene's negative-conductivity effect and metasurfaces is expected to promote the further development of THz active hybrid metasurface devices based on graphene.Graphene-based terahertz negative-conductivity metasurfaces with two types of unit cell structures—RSRR and RMRR—under the control of an external bias voltage are investigated. With the addition of monolayer graphene, the resonance intensity of the metasurface decreases considerably and the resonance frequency shows an obvious red shift. When an external bias voltage is applied to the sample, the THz transmission increases gradually, resonance intensity increases gradually, and resonance frequency undergoes a blue shift. The physical mechanism of THz transmission enhancement for the actively induced negative-conductivity effect of graphene is elucidated The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.The authors declare no conflict of interest.


Since the groundbreaking discovery of graphene by Geim and Novoselov in 2004, there has been continuous research focused on the utilization of graphene (GR) and graphene-related materials (GRms) in technologically high-impact applications, spanning from electronics, sensing, and spintronics, to catalysis, energy storage, and environmental remediation. The one-atom-thick two-dimensional crystal of conjugated carbons arranged in a honeycomb lattice results in remarkable physicochemical properties, such as electrical and thermal conductivity, transparency to light, mechanical flexibility, and strength. Another critical feature of GR is the possibility to undergo controllable chemical doping with diverse atoms and/or functionalization with chemical groups in an out-of-plane geometry, allowing the desirable tuning of physical, chemical, magnetic, or/and optoelectronic properties for specific applications. Moreover, this broad set of properties opens the avenue for the design and development of novel and multifunctional (nano)materials via the integration of GR or GRms in composites and hybrids, further broadening the family and applicability of this exciting material.This Special Issue, “Graphene-Related Materials: Synthesis and Applications” aimed to collect selected, original and innovative articles presenting recent trends and advances on the design, synthesis, characterization, and applications of graphene-based materials and their composites in various fields of application. In particular, seven research articles were published. Arif et al. presented a successful one-pot blended reflux condensation route for the synthesis of nitrogen-doped reduced graphene oxide nickel-cobalt (N-rGO-Ni/Co) and nickel-silver (N-rGO-Ni/Ag) nanocomposites, which presented superior and stable electrocatalytic activity for Oxygen Reduction Reaction (ORR) . SedajovWe hope this Special Issue will stimulate further developments and new ideas via fruitful discussions between experts in academia and industry working in the field of Graphene-Related Materials.


Results show that the bacterial communities were generally similar to those observed in the cryoconite holes of other continents, but with few cyanobacteria (0.5% of sequences). The most abundant orders were Betaproteobacteriales, Cytophagales, Chitinophagales, Acetobacterales, Frankiales, Armatimonadales, Sphingobacteriales, Rhizobiales, Bacteroidales, Sphingomonadales, and Micrococcales. The bacterial communities differed between glaciers and both water pH and O2 concentration appeared to influence the bacterial community composition. This work thus provides the first description of the bacterial communities in cryoconite holes of South American glaciers.Cryoconite holes, ponds full of melting water with sediment on the bottom, are hotspots of biodiversity on glacier surfaces and host dynamic micro-ecosystems. They have been extensively investigated in different areas of the world , but so far no study has described the bacterial communities of the glaciers in the Andes, the world’s longest mountain range. In this study, we describe the bacterial communities of three small (< 2 km The supraglacial zone is the most biodiverse among glacier ecosystems1 and, for practical reasons, is also the most studied. Here, small holes of the glacier surface filled by meltwater, the cryoconite holes, form due to the atmospheric deposition of fine-grained sediment, the cryoconite, which locally decreases the albedo and melts the underlying ice. The main source of this sediment is the surrounding environment, but a small part of it is deposited after long-range atmospheric transport2. In the extreme glacier environment, cryoconite holes are protected habitats where general conditions are more favourable for life than in the other parts of a glacier, as they provide liquid water which also acts as a protective barrier against the intense UV radiation characteristic for glacier environments3.Glaciers and ice sheets have been recognized as a biome in their own right, mostly dominated by microorganisms that live in all parts of the glacier environment8. However, they do not show the same features in all the different geographic areas where they occur. For instance, on the Greenland Ice Sheet and in Antarctic regions, cryoconite holes are more stable than those that form on temperate mountain glaciers. On Himalayan glaciers, they can sometimes last for more than one year9, but on other temperate mountain glaciers as in the Alps, they are mostly ephemeral structures that can be destroyed by strong ablation that can quickly dismantle them washing away the sediment. The intense solar radiation can subsequently form new holes in a few days10.Cryoconite holes are present on glaciers in almost all areas of the world; so far they have been described with their microbiome in the Arctic, Antarctica, the Alps, Tien Shan, Karakoram, Himalaya and Caucasus11, mostly because of the difficulties and the costs of visiting glaciers repeatedly, as they usually occur in remote areas. Most often, the description of the bacterial communities of cryoconite holes is conducted with snapshot studies12, which allow a general description of their biotic communities, even if they cannot include the whole biodiversity of these environments13. These studies showed that filamentous cyanobacteria are the main organisms responsible for cryoconite grain formation8. Other abundant bacterial phyla found in these microhabitats are proteobacteria , actinobacteria, chloroflexi, acidobacteria and bacteroidetes14. Cryoconite hole bacterial communities change among glaciers16 probably according to several different environmental variables , as well as within glaciers among cryoconite holes19. Within-glacier variation was related to micro-environmental conditions of the single holes. For instance, on Forni Glacier  water pH and oxygen concentration, the amount of organic matter in the sediment, and the depth of cryoconite holes were related to the bacterial community structure13. On Baltoro Glacier (Pakistani Karakoram) the structure of bacterial communities appeared to vary according to the water pH of cryoconite holes4. Other variables related to within-glacier variation in cryoconite holes bacterial communities were temperature, solar radiation, wind exposition, electrical conductivity, ammonium, and phosphorous21.Investigations of the temporal variability of cryoconite hole bacterial communities of the same glacier along one or even more ablation seasons were seldom conducted22. Another study describing the gut microbiome of the glacier stonefly (Andiperla willinki) with 16S rRNA sequencing also reported some data about the composition of the bacterial communities of the glacier surface because these insects likely feed on supraglacial bacteria23, but no study specifically focused on the bacteria inhabiting cryoconite holes on glaciers in South America. This is a big gap of knowledge since the Andes host many glaciers  along a latitudinal range of ~ 8000 km from 11° N in Colombia and Venezuela to 55° S in Chile and Argentina25.To the best of our knowledge, no published information is currently available about cryoconite hole bacterial communities from South American glaciers. Only two studies described cryoconite characteristics of South American glaciers, but from a physico-chemical point of view, not reporting bacterial communities composition2) glaciers located at high altitudes (about 4000 m a.s.l.) in the Central Andes close to Santiago (Chile), while two (Exploradores and Perito Moreno) are large (> 8 km2) glaciers located respectively in Chilean and Argentinian Patagonia whose ablation zones (where we collected samples) reach low altitudes  are small  and oxygen concentration  in cryoconite holes differed significantly between glaciers. Tukey post-hoc tests highlighted that glaciers could be divided into two groups with respect to pH values, with Exploradores and Morado showing higher values than Perito Moreno, Iver, and East Iver  per sample and were discarded from the analyses of the alpha diversity. However, they were included in the analyses of beta diversity because they clustered with the other samples, and the results did not change when we re-ran the analyses excluding them (details not shown). Overall, these sequences clustered in 1467 Amplicon Sequences Variants (ASVs)4,62 = 42.56, P < 0.001; Gini index: F4,62 = 32.63, P < 0.001). Post-hoc tests showed that the Shannon index was higher in Exploradores samples, intermediate in Perito Moreno and Morado ones, and lower in Iver and East Iver ones, while the Gini index was higher in Iver and East Iver samples, intermediate in Perito Moreno and Morado ones, and lower in Exploradores ones . However, since the mean values of pH and [O2] differed between glaciers (see above), we mean-centred these variables within glacier before the analyses  thus obtaining ΔpH and Δ[O2] values for each sample. This procedure was necessary because mean-centred variables were not collinear with the glacier factor. According to this analysis, no trends were observed according to ΔpH  and Δ[O2] .Generalized least squares (GLS) models showed that alpha diversity indices varied significantly between glaciers .We used redundancy analysis (RDA) to investigate variation in bacterial communities between glaciers and with ΔpH and Δ[O2] Table ; Fig. 4.4,68 ≥ 6.181, PFDR < 0.001), without showing any consistent pattern. Iver was the glacier with the highest relative abundance of Betaproteobacteriales, Morado the one with the highest relative abundance of Frankiales, East Iver showed the highest abundance of Cytophagales and Micrococcales, and Perito Moreno had more Armatimonadales, Sphingobacteriales, and Acetobacterales, but fewer Betaproteobacteriales, Frankiales and Cytophagales than the other glaciers  corrected for overdispersion performed on the most abundant orders showed that the abundances of Betaproteobacteriales, Cytophagales, Chitinophagales, Acetobacterales, Frankiales, Armatimonadales, Sphingobacteriales, Rhizobiales, Bacteroidales, Sphingomonadales, and Micrococcales varied between glaciers   or [O2] .The five glaciers we considered showed different pH and  values we found in our samples. Indeed, Exploradores and Perito Moreno are located in Patagonia at low altitudes (< 200 m a.s.l.), while Iver and East Iver are the highest ones among those we investigated (samples were collected at about 4000 m a.s.l.) and Morado is at an intermediate value (3400 m a.s.l.).In this study, we provide the first description of the bacterial communities of cryoconite holes from South American glaciers, in particular from both small high-elevation glaciers of the Central Andes in the Santiago Metropolitan Region (Chile), and from the tongues of two large glaciers in Patagonian Andes that reach low altitudes. These pieces of information fill a large geographical gap in our knowledge of glacier environments because this is the first description of the microbial communities of supraglacial environments in South America, a continent with about 30,000 km31. We note, however, that we measured water pH rather than that of the cryoconite because the first one was already reported to influence bacterial communities in cryoconite holes4.pH values seem to follow a different pattern: the highest values were recorded on Exploradores and Morado, while the lowest ones were on East Iver and Iver. However, all the mean pH values of the five glaciers were basic (between 8.57 and 10.47) and differences in pH among glaciers are not easily explained also considering the lithology of the surrounding environments. Indeed, Perito Moreno lies between rhyolitic and undifferentiated volcanic bedrock, Morado, Iver and East Iver are located between alkaline and andesitic igneous rocks, and Exploradores is characterized by a granitic bedrock. These orders belong to the phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Armatimonadota. Most of these orders are typical of cryoconite holes and dominate bacterial communities in these environments in all the geographical areas investigated so far: Arctic32, Antarctica5, Europe12, and Asia33. Betaproteobacteriales are a quite heterogeneous order where diverse members showed resistance to stressful conditions including oxidative stress and also degradation of aromatic compounds34. Interestingly, 48.12% of Betaproteobacteriales sequences in our samples belong to the genus Polaromonas, a bacterium well adapted to glacier environments36. According to our results, Polaromonas was quite abundant on all the glaciers but on Perito Moreno. This is an important genus because it is well adapted to harsh environments because of its ability to survive long-range atmospheric transport and thanks to its versatile metabolism (can use both organic and inorganic electron donors)35. Cytophagales are Gram-negative bacteria that are known to be less resistant to UV radiation than Gram-positive bacteria37. In addition, 91.68% of Cytophagales sequences belong to Hymenobacter, a heterotrophic genus composed of mostly heterotrophic, aerobic, Gram-negative bacteria which have been described in extreme environments like Antarctica, where a strain with extremely high resistance to solar radiation was isolated39. Armatimonadota and Frankiales were never reported as abundant inhabitants of cryoconite holes in other geographical areas. Interestingly, Armatimonadota were present with a high relative abundance on Perito Moreno only. This phylum includes both aquatic and terrestrial genera, surprisingly described in hot springs and geothermal soils40. Maybe, the presence of pigments, oligotrophy and the ability to degrade polysaccharides provide them with the ability to survive in such harsh environments40. Frankiales, together with Micrococcales, belong to the phylum of Actinobacteria and are Gram positive bacteria41. They were abundant on all glaciers, except for Perito Moreno and their high relative abundance may be related to their resistance to UV radiation42. cyanobacteria, which are considered important components of cryoconite hole bacterial communities and even ecosystem engineers in these environments9, were only 0.5% in our samples and no order was included among the most abundant ones. In summary, cryoconite holes of Andean glaciers seem to host bacterial communities dominated by the taxa typical of these environments worldwide, but with a low abundance of cyanobacteria, and a rather high abundance of Frankiales in all glaciers except for Perito Moreno, where a high abundance of Armatimonadota, another unusual taxon in cryoconite holes, was observed.The most abundant orders were (in decreasing order): Betaproteobacteriales, Cytophagales, Chitinophagales, Acetobacterales, Frankiales, Armatimonadales, Sphingobacteriales, Rhizobiales, Bacteroidales, Sphingomonadales, and Micrococcales3 as well as lower partial oxygen pressure and lower nutrient availability from aeolian deposition. Patagonian glaciers are 440 km from one another and c.ca 2000 km from the other glaciers we sampled, which, in turn, are less than 60 km from one another. In addition, it has been demonstrated that the main source of cryoconite bacterial communities is the local environment surrounding glacier2. The high-elevation glaciers we sampled are small and above the tree line, and generally in areas with sparse vegetation. In contrast, the Patagonian glaciers are large and their tongues (where we collected the samples) are well below the tree line and therefore exposed to very different potential sources of microorganisms with respect to the other glaciers.Despite this general similarity of the dominant orders of bacterial communities at a global scale, a closer inspection at the ASV level revealed that the bacterial communities of the different glaciers differed from one another. In addition, the RDA biplot showed that the three small and high-elevation glaciers clustered quite close to one another, whereas Perito Moreno and Exploradores were separate from one another and the other glaciers. This pattern may be due to geographic distance, different altitudes, or other ecological and environmental differences among glaciers. Indeed, bacterial communities in high-elevation glaciers are exposed to higher UV radiation and, therefore, to high oxidative stress2]. We stress that these variables represent the difference between the pH and oxygen concentration of each cryoconite hole from the respective mean value of all the holes of that glacier. They therefore do not account per se for the difference in mean pH and oxygen concentration among glaciers highlighted in the analyses discussed above, because in this analysis this effect was accounted for by the glacier factor, which accounts for any difference between glaciers. Thus, this analysis suggests that bacterial communities vary consistently according to pH and oxygen concentration gradients present on each glacier, even if different glaciers show on average different pH and oxygen concentration values. In other words, for example, an increase of two pH units seems related to a similar variation in bacterial communities independently from the absolute pH value, at least in the range of variability of pH values recorded in this study. In addition, no effect of ΔpH on alpha diversity resulted from GLSs. pH is known to affect bacterial communities of cryoconite holes4, but little is known about the mechanisms underlying these relationships. Indeed, water pH may also be partially affected by the metabolic activities of the bacterial communities, so it is still unclear whether water pH affects cryoconite hole bacterial communities or, at least, partially, also the reverse occurs. Indeed, it has been reported that the photosynthetic activity of both bacteria and algae can alter CO2 concentration which, in turn, can alter pH43. In addition, in cryoconite holes, this activity can also be balanced by that of the heterotrophic bacterial community and other heterotrophic taxa 44. Furthermore, analyses of the most abundant orders within each glacier revealed that some varied according to pH. Different studies already investigated the effect of both water and sediment pH on bacterial community composition, proving that they influence the communities of different types of river sediments45. Nonetheless, so far only the decrease of Acidobacteria at basic pH values was already reported in literature data45 but was not detected in our study, probably because the pH values of our samples varied from 7.25 to 12.71 i.e. in a range where the abundance of Acidobateria is always low46.The RDA also showed that bacterial communities of cryoconite holes varied significantly with ΔpH and Δ concentrations within a glacier. Interestingly, no differences in cyanobacteria abundance were observed, indeed it would be expected that their abundance was higher when also [O2] was higher13, because of their photoautotrophic activity, but no evidence emerged about it. In any case, the mechanisms that link oxygen concentration in the water to bacterial community structure in the sediment are not easy to explain. Indeed, [O2] in the sediment can largely differ from that in the water, with the rapid formation of anoxic layers under a few millimetres of cryoconite49.Cryoconite holes are aerobic environments in the water, thanks to the high O2] values , and they show also similar pH and  was investigated through ANOVA followed by Tukey post-hoc tests.Variation among glaciers in pH and , respectively. Post-hoc tests (Tukey method) were also performed to assess pairwise differences in the structure of bacterial communities between glaciers, correcting P-values for multiple testing according to the false discovery rate (FDR) procedure59.Beta diversity analyses were conducted on a non-rarefied dataset, and were based on the Hellinger distance, which depends on the differences in the ASV proportion among samples, decreases the importance of ASV abundance over occurrence and avoids the double-zero problem when comparing ASV composition among samples2], and glacier (five-level factor). Variation in the abundance of the nine most abundant orders (see below) according to the variables that significantly affected the structure of bacterial communities identified by the RDAs was investigated by generalized linear models (GLMs) assuming a Poisson distribution and correcting for overdispersion. In these models, the dependent variable was the non-rarefied number of sequences assigned to that order per sample. We also included the log-transformed total number of sequences per sample as an offset. With this structure, the Poisson GLMs model the relative abundance of each bacterial order according to the predictors. Also in these cases, P-values were corrected using the same FDR procedure as above. Analyses were performed with R 3.5.160 with the vegan, BiodiversityR, multtest, and multcomp packages.Generalized least-square (GLS) models accounting for the heterogeneity of variance among glaciers were also used to investigate changes in alpha diversity indices according to ΔpH, Δ[OSupplementary Information.