The actin-binding protein drebrin disrupts NF2-LATS kinases complex assembly to facilitate liver tumorigenesis.

BACKGROUND AND AIMS: The Hippo signaling has emerged as a crucial regulator of tissue homeostasis, regeneration, and tumorigenesis, representing a promising therapeutic target. Neurofibromin 2 (NF2), a component of Hippo signaling, is directly linked to human cancers but has been overlooked as a target for cancer therapy. APPROACH AND RESULTS: Through a high-content RNA interference genome-wide screen, the actin-binding protein Drebrin (DBN1) has been identified as a novel modulator of YAP localization. Further investigations have revealed that DBN1 directly interacts with NF2, disrupting the activation of large tumor suppressor kinases (LATS1/2) by competing with LATS kinases for NF2 binding. Consequently, DBN1 knockout considerably promotes YAP nuclear exclusion and repression of target gene expression, thereby preventing cell proliferation and liver tumorigenesis. We identified three lysine residues (K238, K248, and K252) essential for DBN1-NF2 interaction and developed a mutant DBN1 (DBN1-3Kmut) that is defective in NF2 binding and incompetent to trigger NF2-dependent YAP activation and tumorigenesis both in vitro and in vivo. Furthermore, BTP2, a DBN1 inhibitor, successfully restored NF2-LATS kinase binding and elicited potent antitumor activity. The combination of sorafenib and BTP2 exerted synergistic inhibitory effects against HCC. CONCLUSIONS: Our study identifies a novel DBN1-NF2-LATS axis, and pharmacological inhibition of DBN1 represents a promising alternative intervention targeting the Hippo pathway in cancer treatment.

Upregulation of HOXA3 by isoform-specific Wilms tumour 1 drives chemotherapy resistance in acute myeloid leukaemia.

Upregulation of the Wilms' tumour 1 (WT1) gene is common in acute myeloid leukaemia (AML) and is associated with poor prognosis. WT1 generates 12 primary transcripts through different translation initiation sites and alternative splicing. The short WT1 transcripts express abundantly in primary leukaemia samples. We observed that overexpression of short WT1 transcripts lacking exon 5 with and without the KTS motif (sWT1+/- and sWT1-/-) led to reduced cell growth. However, only sWT1+/- overexpression resulted in decreased CD71 expression, G1 arrest, and cytarabine resistance. Primary AML patient cells with low CD71 expression exhibit resistance to cytarabine, suggesting that CD71 may serve as a potential biomarker for chemotherapy. RNAseq differential expressed gene analysis identified two transcription factors, HOXA3 and GATA2, that are specifically upregulated in sWT1+/- cells, whereas CDKN1A is upregulated in sWT1-/- cells. Overexpression of either HOXA3 or GATA2 reproduced the effects of sWT1+/-, including decreased cell growth, G1 arrest, reduced CD71 expression and cytarabine resistance. HOXA3 expression correlates with chemotherapy response and overall survival in NPM1 mutation-negative leukaemia specimens. Overexpression of HOXA3 leads to drug resistance against a broad spectrum of chemotherapeutic agents. Our results suggest that WT1 regulates cell proliferation and drug sensitivity in an isoform-specific manner.

Chromosome-level genome assembly of sea scallop Placopecten magellanicus provides insights into the genetic characteristics and adaptive evolution of large scallops.

Placopecten magellanicus (Gmelin, 1791), a deep-sea Atlantic scallop, holds significant commercial value as a benthic marine bivalve along the northwest Atlantic coast. Recognizing its economic importance, the need to reconstruct its genome assembly becomes apparent, fostering insights into natural resources and generic breeding potential. This study reports a high-quality chromosome-level genome of P. magellanicus, achieved through the integration of Illumina short read sequencing, PacBio HiFi sequencing, and Hi-C sequencing techniques. The resulting assembly spans 1778 Mb with a scaffold N50 of 86.71 Mb. An intriguing observation arises - the genome size of P. magellanicus surpasses that of its Pectinidae family peers by 1.80 to 2.46 times. Within this genome, 28,111 protein-coding genes were identified. Comparative genomic analysis involving five scallop species unveils the critical determinant of this expanded genome: the proliferation of repetitive sequences recently inserted, contributing to its enlarged size. The landscape of whole genome collinearity sheds light on the relationships among scallop species, enhancing our broader understanding of their genomic framework. This genome provides genomic resources for future molecular biology research on scallops and serves as a guide for the exploration of longevity-related genes in scallops.

Oxygenated Triazine-Heptazine Heterostructure Creates an Enormous Ascension to the Visible Light Photocatalytic Hydrogen Evolution Performance of Porous C3 N4 Nanosheets.

A highly efficient g-C3 N4 photocatalyst is developed by a novel one-pot thermal polymerization method under a salt fog environment generated by heating the aqueous solution of urea and mixed metal salts of NaCl/KCl, namely SF-CN. Thanks to the synergistic effect of the oxygenation and chemical etching of the salt fog, the obtained SF-CN is an oxygenated ultrathin porous carbon nitride with an intermolecular triazine-heptazine heterostructure, meanwhile, shows enlarged specific surface area, greatly enhanced absorption of visible light, narrowed band gap with a lower conduction band, and an increased photocurrent response due to the effective separation of photogenerated holes and electrons, comparing to those of pristine g-C3 N4 . The theoretical simulations further reveal that the triazine-heptazine heterostructure possesses better photocatalytic hydrogen evolution (PHE) capability than pure triazine and heptazine carbon nitrides. In turn, SF-CN demonstrates an excellent visible light PHE rate of 18.13 mmol h-1  g-1 , up to 259.00 times of that of pristine g-C3 N4 .

High glucose promotes the progression of colorectal cancer by activating the BMP4 signaling and inhibited by glucagon-like peptide-1 receptor agonist.

BACKGROUND: The detailed molecular mechanism between type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC) is still uncertain. Bone morphogenetic protein 4 (BMP4) dysregulation is implicated in T2DM and CRC, respectively. This study aims to investigate whether BMP4 can mediate the interaction of CRC with T2DM. METHODS: We firstly explored the expression of BMP4 in The Cancer Genome Altas (TCGA) databases and CRC patients with or without DM from the Shanghai Tenth People's Hospital. The diabetic model of CRC cell lines in vitro and the mice model in vivo were developed to explore the BMP4 expression during CRC with or without diabetes. Further inhibition of BMP4 to observe its effects on CRC. Also, glucagon-like peptide-1 receptor agonist (GLP-1RA) was used to verify the underlying mechanism of hypoglycemic drugs on CRC via BMP4. RESULTS: BMP4 expression was upregulated in CRC patients, and significantly higher in CRC patients with diabetes (P < 0.05). High glucose-induced insulin resistance (IR)-CRC cells and diabetic mice with metastasis model of CRC had increased BMP4 expression, activated BMP4-Smad1/5/8 pathway, and improved proliferative and metastatic ability mediated by epithelial-mesenchymal transition (EMT). And, treated CRC cells with exogenously BMP inhibitor-Noggin or transfected with lentivirus (sh-BMP4) could block the upregulated metastatic ability of CRC cells induced by IR. Meanwhile, GLP-1R was downregulated by high glucose-induced IR while unregulated by BMP4 inhibitor noggin, and treated GLP-1RA could suppress the proliferation of CRC cells induced by IR through downregulated BMP4. CONCLUSIONS: BMP4 increased by high glucose promoted the EMT of CRC. The mechanism of the BMP4/Smad pathway was related to the susceptible metastasis of high glucose-induced IR-CRC. The commonly used hypoglycemic drug, GLP-1RA, inhibited the growth and promoted the apoptosis of CRC through the downregulation of BMP4. The result of our study suggested that BMP4 might serve as a therapeutic target in CRC patients with diabetes.

Phase Separation of MAGI2-Mediated Complex Underlies Formation of Slit Diaphragm Complex in Glomerular Filtration Barrier.

BACKGROUND: Slit diaphragm is a specialized adhesion junction between the opposing podocytes, establishing the final filtration barrier to urinary protein loss. At the cytoplasmic insertion site of each slit diaphragm there is an electron-dense and protein-rich cellular compartment that is essential for slit diaphragm integrity and signal transduction. Mutations in genes that encode components of this membrane-less compartment have been associated with glomerular diseases. However, the molecular mechanism governing formation of compartmentalized slit diaphragm assembly remains elusive. METHODS: We systematically investigated the interactions between key components at slit diaphragm, such as MAGI2, Dendrin, and CD2AP, through a combination of biochemical, biophysical, and cell biologic approaches. RESULTS: We demonstrated that MAGI2, a unique MAGUK family scaffold protein at slit diaphragm, can autonomously undergo liquid-liquid phase separation. Multivalent interactions among the MAGI2-Dendrin-CD2AP complex drive the formation of the highly dense slit diaphragm condensates at physiologic conditions. The reconstituted slit diaphragm condensates can effectively recruit Nephrin. A nephrotic syndrome-associated mutation of MAGI2 interfered with formation of the slit diaphragm condensates, thus leading to impaired enrichment of Nephrin. CONCLUSIONS: Key components at slit diaphragm (e.g., MAGI2 and its complex) can spontaneously undergo phase separation. The reconstituted slit diaphragm condensates can be enriched in adhesion molecules and cytoskeletal adaptor proteins. Therefore, the electron-dense slit diaphragm assembly might form via phase separation of core components of the slit diaphragm in podocytes.

FBLN1 promotes chondrocyte proliferation by increasing phosphorylation of Smad2.

BACKGROUND: The role of fibulin-1 or FBLN1 in chondrocyte proliferation has not been reported so far. In this study, we aimed to verify whether FBLN1 promotes chondrocyte proliferation in elderly patients with knee osteoarthritis by phosphorylating Smad2. METHODS: Chondrocytes were isolated from cartilage samples collected from elderly patients with osteoarthritis (n = 6) and young patients (n = 6). The isolated chondrocytes were divided into the following three groups: control (medium only); cells transfected with adenovirus expressing green fluorescent protein (Ad-GFP); and those transfected with adenovirus expressing green fluorescent protein and FBLN1 (Ad-GFP-FBLN1). Furthermore, chondrocytes were divided into the following three groups in the mechanistic analysis: group 1, medium only; group 2, Ad-FBLN1; and group 3, Ad-FBLN1+pSmad2 inhibitor. The cells were analyzed for the relevant indicators after culturing for 48 h. RESULTS: There were more EdU-positive cells in the Ad-GFP-FBLN1 group than in the other two groups (both P < 0.05). Compared with the other two groups, the level of pSmad2 and Col2 in the Ad-GFP-FBLN1 group was significantly increased (P < 0.05). The gene expression level of each indicator was consistent with the protein expression level. There was no significant difference in the indicators between groups 1 and 3. The percentage of EdU-positive cells in group 2 was higher than that in the other two groups (P < 0.05). The expression of pSmad2 and Col2 in group 2 was higher than that in the other two groups (both P < 0.05). CONCLUSION: FBLN1 can promote chondrocyte proliferation in the knee cartilage in elderly patients by phosphorylating Smad2.

Genomic landscape of neutrophilic leukemias of ambiguous diagnosis.

Chronic neutrophilic leukemia (CNL), atypical chronic myeloid leukemia (aCML), and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are a group of rare and heterogeneous myeloid disorders. There is strong morphologic resemblance among these distinct diagnostic entities as well as a lack of specific molecular markers and limited understanding of disease pathogenesis, which has made diagnosis challenging in certain cases. The treatment has remained empirical, resulting in dismal outcomes. We, therefore, performed whole-exome and RNA sequencing of these rare hematologic malignancies and present the most complete survey of the genomic landscape of these diseases to date. We observed a diversity of combinatorial mutational patterns that generally do not cluster within any one diagnosis. Gene expression analysis reveals enrichment, but not cosegregation, of clinical and genetic disease features with transcriptional clusters. In conclusion, these groups of diseases represent a continuum of related diseases rather than discrete diagnostic entities.

Porphyrin-Based Metal-Organic Frameworks for Efficient Photocatalytic H2 Production under Visible-Light Irradiation.

Metal-organic frameworks (MOFs) are important photocatalytic materials for H2 production. To clarify the structure-function relationship and improve the photocatalytic activity, herein we explored a series of porphyrin-based zirconium MOFs (PCN-H2/Ptx:y, where x:y = 4:1, 3:2, 2:3, and 0:1) containing different ratios of H2TCPP and PtIITCPP [TCPP = tetrakis(4-carboxyphenyl)porphyrinate] as isostructural ligands and Zr6 clusters as nodes. Under visible-light irradiation, PCN-H2/Pt0:1 shows the highest average H2 evolution reaction rate (351.08 µmol h-1 g-1), which decreases along with lowering of the ratio of PtIITCPP in the PCN-H2/Ptx:y series. The differences in photocatalytic activity are attributed to more uniformly dispersed Pt2+ ions in PCN-H2/Pt0:1, which promotes charge transfer from porphyrins (photosensitizers) to PtII ions (catalytic centers), leading to efficient charge separation in the MOF materials. The bifunctional MOFs with photosensitizers and catalytic centers provide new insight for the design and application of porphyrin-based photocatalytic systems for visible-light-driven H2 production.

Gain-of-function mutations in granulocyte colony-stimulating factor receptor (CSF3R) reveal distinct mechanisms of CSF3R activation.

Granulocyte colony-stimulating factor (G-CSF or CSF3) and its receptor CSF3R regulate granulopoiesis, neutrophil function, and hematopoietic stem cell mobilization. Recent studies have uncovered an oncogenic role of mutations in the CSF3R gene in many hematologic malignancies. To find additional CSF3R mutations that give rise to cell transformation, we performed a cellular transformation assay in which murine interleukin 3 (IL-3)-dependent Ba/F3 cells were transduced with WT CSF3R plasmid and screened for spontaneous growth in the absence of IL-3. Any outgrowth clones were sequenced to identify CSF3R mutations with transformation capacity. We identified several novel mutations and determined that they transform cells via four distinct mechanisms: 1) cysteine- and disulfide bond-mediated dimerization (S581C); 2) polar, noncharged amino acid substitution at the transmembrane helix dimer interface at residue Thr-640; 3) increased internalization by a Glu-524 substitution that mimics a low G-CSF dose; and 4) hydrophobic amino acid substitutions in the membrane-proximal residues Thr-612, Thr-615, and Thr-618. Furthermore, the change in signaling activation was related to an altered CSF3R localization. We also found that CSF3R-induced STAT3 and ERK activations require CSF3R internalization, whereas STAT5 activation occurred at the cell surface. Cumulatively, we have expanded the regions of the CSF3R extracellular and transmembrane domains in which missense mutations exhibit leukemogenic capacity and have further elucidated the mechanistic underpinnings that underlie altered CSF3R expression, dimerization, and signaling activation.

A Method for Detecting Dynamic Mutation of Complex Systems Using Improved Information Entropy.

In this study, a nonlinear analysis method called improved information entropy (IIE) is proposed on the basis of constructing a special probability mass function for the normalized analysis of Shannon entropy for a time series. The definition is directly applied to several typical time series, and the characteristic of IIE is analyzed. This method can distinguish different kinds of signals and reflects the complexity of one-dimensional time series of high sensitivity to the changes in signal. Thus, the method is applied to the fault diagnosis of a rolling bearing. Experimental results show that the method can effectively extract the sensitive characteristics of the bearing running state and has fast operation time and minimal parameter requirements.

"Non-Naked" Gold with Glucose Oxidase-Like Activity: A Nanozyme for Tandem Catalysis.

It has been widely reported that "naked" gold nanoparticles (Au NPs) without protectors have glucose oxidase (GOx)-like activity, and the use of protectors can inhibit the GOx-like activity. Here, "non-naked" Au NPs with GOx-like activity are synthesized by using protein as protector. Although "naked" Au NPs have peroxidase-like activity and GOx-like activity, the optimal pH ranges of the both activities are obviously different. Fortunately, as-synthesized "non-naked" Au NPs show the dual enzyme-like activities at the same pH. So, "non-naked" Au NPs can be described as "tandem nanozyme." As another bonus, the participation of protein protector can stabilize the GOx-like activity and make Au NPs modifiable. Even though Au NPs are connected with graphene oxide (GO), the GOx-like activity is still not changed. Further, Au NPs-GO nanocomposites are applied on the one-pot nonenzymatic glucose colorimetric detection. The "non-naked" gold not only broadens the species of tandem nanozymes, but also facilitates the functionalization of nanozymes, which is promising for immunoassay, biosensor, and medical treatment.

Strong Coupling of MoS2 Nanosheets and Nitrogen-Doped Graphene for High-Performance Pseudocapacitance Lithium Storage.

Layered material MoS2 is widely applied as a promising anode for lithium-ion batteries (LIBs). Herein, a scalable and facile dopamine-assisted hydrothermal technique for the preparation of strongly coupled MoS2 nanosheets and nitrogen-doped graphene (MoS2 /N-G) composite is developed. In this composite, the interconnected MoS2 nanosheets are well wrapped onto the surface of graphene, forming a unique veil-like architecture. Experimental results indicate that dopamine plays multiple roles in the synthesis: a binding agent to anchor and uniformly disperse MoS2 nanosheets, a morphology promoter, and the precursor for in situ nitrogen doping during the self-polymerization process. Density functional theory calculations further reveal that a strong interaction exists at the interface of MoS2 nanosheets and nitrogen-doped graphene, which facilitates the charge transfer in the hybrid system. When used as the anode for LIBs, the resulting MoS2 /N-G composite electrode exhibits much higher and more stable Li-ion storage capacity (e.g., 1102 mAh g-1 at 100 mA g-1 ) than that of MoS2 /G electrode without employing the dopamine linker. Significantly, it is also identified that the thin MoS2 nanosheets display outstanding high-rate capability due to surface-dominated pseudocapacitance contribution.

One-Step Hydrothermal Synthesis of Small TiO2 Porous Nanoparticles for Efficient Degradation of Organic Dyes.

In the work, small TiO2 porous nanoparticles with an average size of 60 nm have been prepared by a simple, eco-friendly, and one-step hydrothermal method. The TiO2 products are achieved by only using tetrabutyl titanate (TBOT), tetrapropylammonium hydroxide (TPAOH), and water as the starting materials. Various techniques such as SEM, TEM, HRTEM, XRD, Raman, and BET are used to characterize the surface morphology and structural features of products. The growth parameters including reaction time, titanium source and temperature of thermal treatment are systematically investigated. In the synthesis, TPAOH plays a decisive role as structure-directing agent for the formation of desirable TiO2 nanostructure. The photocatalytic tests manifest that the TiO2 nanoparticles annealed at 450 °C shows an outstanding photocatalytic activity for degradation of methyl orange (MO) and rhodamine B (RhB), which is comparable to Degussa P25. The superior performance may mainly come from the contributions of their small particle size and unique nanostructure.

A Feature Extraction Method Using Improved Multi-Scale Entropy for Rolling Bearing Fault Diagnosis.

A feature extraction method named improved multi-scale entropy (IMSE) is proposed for rolling bearing fault diagnosis. This method could overcome information leakage in calculating the similarity of machinery systems, which is based on Pythagorean Theorem and similarity criterion. Features extracted from bearings under different conditions using IMSE are identified by the support vector machine (SVM) classifier. Experimental results show that the proposed method can extract the status information of the bearing. Compared with the multi-scale entropy (MSE) and sample entropy (SE) methods, the identification accuracy of the features extracted by IMSE is improved as well.

Mesoporous Tungsten Oxides with Crystalline Framework for Highly Sensitive and Selective Detection of Foodborne Pathogens.

Foodborne pathogens like Listeria monocytogenes can cause various illnesses and pose a serious threat to public health. They produce species-specific microbial volatile organic compounds, i.e., the biomarkers, making it possible to indirectly measure microbial contamination in foodstuff. Herein, highly ordered mesoporous tungsten oxides with high surface areas and tunable pores have been synthesized and used as sensing materials to achieve an exceptionally sensitive and selective detection of trace Listeria monocytogenes. The mesoporous WO3-based chemiresistive sensors exhibit a rapid response, superior sensitivity, and highly selective detection of 3-hydroxy-2-butanone. The chemical mechanism study reveals that acetic acid is the main product generated by the surface catalytic reaction of the biomarker molecule over mesoporous WO3. Furthermore, by using the mesoporous WO3-based sensors, a rapid bacteria detection was achieved, with a high sensitivity, a linear relationship in a broad range, and a high specificity for Listeria monocytogenes. Such a good gas sensing performance foresees the great potential application of mesoporous WO3-based sensors for fast and effective detection of microbial contamination for the safety of food, water safety and public health.

The Transformation of Hybrid Silica Nanoparticles from Solid to Hollow or Yolk-Shell Nanostructures.

Here, a facile self-templating approach is presented for synthesis of hollow and yolk-shell mesoporous silica nanoparticles (HMSNs and YMSNs) through a selective etching of hybrid silica nanoparticles. The hybrid silica nanoparticles are from the co-condensation of tetraethylorthosilicate (TEOS) and N-[3-(trimethoxysilyl)propyl]ethylenediamine (TSD) by a simple one-step process. Two kinds of products including HMSNs and YMSNs can be easily prepared only by tuning the TSD amounts in the precursor. Significantly, the transformation of hollow structure does not use any sacrificial template and surface-protective agent. The etching mechanism and formation process are systematically investigated by SEM, TEM, TG, CHN elemental analysis and Si MAS NMR spectroscopy. The results reveal that the selective etching is mainly attributed to the discrepancy in density between the outer layer and inner area of hybrid silica, where its inner section is more readily dissolved while the outer shell is robust in hydrofluoric acid (HF) aqueous solution. Specifically, the new understanding is further extended to precisely prepare multi-shelled hollow/yolk-shell silica nanoparticles.

Shape-Controlled Hollow Mesoporous Silica Nanoparticles with Multifunctional Capping for In Vitro Cancer Treatment.

A series of multifunctional shape-controlled nonspherical hollow mesoporous silica nanoparticles (HMSNs) drug carriers have been prepared by employing Fe2 O3 with four morphologies (capsule, cube, rice, and rhombus) as a sacrificial template and a multifunctional cap as the encapsulating shell. The resulting shape-controlled nonspherical HMSNs perfectly replicate the original morphology of the Fe2 O3 templates, which possess a high specific surface area, good monodispersity, perpendicular mesoporous channels, and excellent biocompatibility. After modification of polyethylene glycol (PEG) and folic acid (FA), the shape-controlled HMSN core and functional shell can then be integrated into a single device (HMSNs-PEG-FA) to provide an efficient and tumor-cell-selective drug-delivery system. The shape-controlled HMSNs and HMSNs-PEG-FA all show controlled pH-responsive release behavior for the anticancer drug doxorubicin hydrochloride (DOX). The in vitro results indicate that HMSNs-PEG-FA is biocompatible and selectively targets HeLa cells (overexpressed folate receptors). Fluorescence images show that desirable surface modification and the nonspherical shape effectively facilitate cellular internalization of HMSNs. It is expected that the construction of these unique nanomaterials with controlled morphology through the hard-templating technique may also provide useful information for the design of nanoscale multifunctional systems.

miR-145 Contributes to Hypertrophic Scarring of the Skin by Inducing Myofibroblast Activity.

Hyperthrophic scarring of the skin is caused by excessive activity of skin myofibroblasts after wound healing and often leads to functional and/or aesthetic disturbance with significant impairment of patient quality of life. MicroRNA (miRNA) gene therapies have recently been proposed for complex processes such as fibrosis and scarring. In this study, we focused on the role of miR-145 in skin scarring and its influence in myofibroblast function. Our data showed not only a threefold increase of miR-145 levels in skin hypertrophic scar tissue but also in transforming growth factor ß1 (TGF-ß1)-induced skin myofibroblasts compared with healthy skin or nontreated fibroblasts (p < 0.001). Consistent with the upregulation of miR-145 induced by TGF-ß1 stimulation of fibroblasts, the expression of Kruppel-like factor 4 (KLF4) was decreased by 50% and α-smooth muscle actin (α-SMA) protein expression showed a threefold increase. Both could be reversed by miR-145 inhibition (p < 0.05). Restoration of KLF4 levels equally abrogated TGF-ß1-induced α-SMA expression. These data demonstrate that TGF-ß1 induces miR-145 expression in fibroblasts, which in turn inhibits KLF4, a known inhibitor of α-SMA, hence upregulating α-SMA expression. Furthermore, treatment of myofibroblasts with a miR-145 inhibitor strongly decreased their α-1 type I collagen expression, TGF-ß1 secretion, contractile force generation and migration. These data demonstrate that upregulation of miR-145 plays an important role in the differentiation and function of skin myofibroblasts. Additionally, inhibition of miR-145 significantly reduces skin myofibroblast activity. Taken together, these results suggest that miR-145 is a promising therapeutic target to prevent or reduce hypertrophic scarring of the skin.