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Pathogenic structure variations (SVs) are associated with various types of cancer and rare genetic diseases. Recent studies have used Cas9 nuclease with paired guide RNAs (gRNAs) to generate targeted chromosomal rearrangements, focusing on producing fusion proteins that cause cancer, whereas research on precision genome editing for rectifying SVs is limited. In this study, we identified a novel complex genomic rearrangement (CGR), specifically an EYA1 inversion with a deletion, implicated in branchio-oto-renal/branchio-oto syndrome. To address this, two CRISPR-based approaches were tested. First, we used Cas9 nuclease and paired gRNAs tailored to the patient's genome. The dual CRISPR-Cas9 system induced efficient correction of paracentric inversion in patient-derived fibroblast, and effectively restored the expression of EYA1 mRNA and protein, along with its transcriptional activity required to regulate the target gene expression. Additionally, we used CRISPR activation (CRISPRa), which leads to the upregulation of EYA1 mRNA expression in patient-derived fibroblasts. Moreover, CRISPRa significantly improved EYA1 protein expression and transcriptional activity essential for target gene expression. This suggests that CRISPRa-based gene therapies could offer substantial translational potential for approximately 70% of disease-causing EYA1 variants responsible for haploinsufficiency. Our findings demonstrate the potential of CRISPR-guided genome editing for correcting SVs, including those with EYA1 CGR linked to haploinsufficiency.
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X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1, which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, very-long-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent ß-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fibroblasts at an efficiency of 7.4%. Adeno-associated virus (AAV)-mediated systemic delivery of NG-ABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction efficiency: â¼5.5%), spinal cord (â¼5.1%), and adrenal gland (â¼2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a significant step toward treating human ALD disease.
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Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Adrenoleucodistrofia , Dependovirus , Modelos Animais de Doenças , Edição de Genes , Terapia Genética , Animais , Adrenoleucodistrofia/terapia , Adrenoleucodistrofia/genética , Camundongos , Humanos , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Adenina , Mutação , Fibroblastos/metabolismo , Ácidos Graxos/metabolismo , Encéfalo/metabolismo , Encéfalo/patologiaRESUMO
Nucleotide repeat expansion disorders, a group of genetic diseases characterized by the expansion of specific DNA sequences, pose significant challenges to treatment and therapy development. Here, we present a precise and programmable method called prime editor-mediated correction of nucleotide repeat expansion (PE-CORE) for correcting pathogenic nucleotide repeat expansion. PE-CORE leverages a prime editor and paired pegRNAs to achieve targeted correction of repeat sequences. We demonstrate the effectiveness of PE-CORE in HEK293T cells and patient-derived induced pluripotent stem cells (iPSCs). Specifically, we focus on spinal and bulbar muscular atrophy and spinocerebellar ataxia type, two diseases associated with nucleotide repeat expansion. Our results demonstrate the successful correction of pathogenic expansions in iPSCs and subsequent differentiation into motor neurons. Specifically, we detect distinct downshifts in the size of both the mRNA and protein, confirming the functional correction of the iPSC-derived motor neurons. These findings highlight PE-CORE as a precision tool for addressing the intricate challenges of nucleotide repeat expansion disorders, paving the way for targeted therapies and potential clinical applications.
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Edição de Genes , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Edição de Genes/métodos , Células HEK293 , Neurônios Motores/metabolismo , Diferenciação Celular/genética , Expansão das Repetições de DNA/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Inflammasomes are important sentinels of innate immune defense; they sense pathogens and induce the cell death of infected cells, playing key roles in inflammation, development, and cancer. Several inflammasome sensors detect and respond to specific pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively) by forming a multiprotein complex with the adapters ASC and caspase-1. During disease, cells are exposed to several PAMPs and DAMPs, leading to the concerted activation of multiple inflammasomes. However, the molecular mechanisms that integrate multiple inflammasome sensors to facilitate optimal host defense remain unknown. Here, we discovered that simultaneous inflammasome activation by multiple ligands triggered multiple types of programmed inflammatory cell death, and these effects could not be mimicked by treatment with a pure ligand of any single inflammasome. Furthermore, NLRP3, AIM2, NLRC4, and Pyrin were determined to be members of a large multiprotein complex, along with ASC, caspase-1, caspase-8, and RIPK3, and this complex drove PANoptosis. Furthermore, this multiprotein complex was released into the extracellular space and retained as multiple inflammasomes. Multiple extracellular inflammasome particles could induce inflammation after their engulfment by neighboring macrophages. Collectively, our findings define a previously unknown regulatory connection and molecular interaction between inflammasome sensors, which drives the assembly of a multiprotein complex that includes multiple inflammasome sensors and cell death regulators. The discovery of critical interactions among NLRP3, AIM2, NLRC4, and Pyrin represents a new paradigm in understanding the functions of these molecules in innate immunity and inflammasome biology as well as identifying new therapeutic targets for NLRP3-, AIM2-, NLRC4- and Pyrin-mediated diseases.
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Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pirina/metabolismo , Moléculas com Motivos Associados a Patógenos , Inflamação , Caspases/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
Heartworm (HW) disease, caused by Dirofilaria immitis, is a life-threatening ailment in dogs. HW disrupts blood flow and decreases cardiac output (CO). The accurate monitoring of CO during HW extraction is pivotal for patient survival and overall health. OBJECTIVE: This study aimed to assess the efficacy of using impedance cardiography (ICG) as a non-invasive approach for monitoring CO during interventional HW extraction. METHODS: Two cases of HW infections were treated via surgical extraction. The CO and mean arterial pressure (MAP) were monitored using the ICG technique during the anesthesia stabilization, extraction process, and post-extraction phases. RESULTS: In Case 1, the CO increased by 115% post-procedure, and in Case 2, the CO increased by 116%. In contrast, the MAP varied between the two cases. The ICG method provided real-time CO data without major disruptions during the extraction surgery. CONCLUSION: The ICG technique for CO monitoring during interventional HW extractions is effective.
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ß-arrestin 2 (ARRB2) is functionally implicated in cancer progression via various signaling pathways. However, its role in lung cancer remains unclear. To obtain clinical insight on its function in lung cancer, microarray data from lung tumor tissues (LTTs) and matched lung normal tissues (mLNTs) of primary non-small cell lung cancer (NSCLC) patients (n = 37) were utilized. ARRB2 expression levels were markedly decreased in all 37 LTTs compared to those in matched LNTs of NSCLC patients. They were significantly co-related to enrichment gene sets associated with oncogenic and cancer genes. Importantly, Gene Set Enrichment Analysis (GSEA) between three LTTs with highly down-regulated ARRB2 and three LTTs with lowly down-regulated ARRB2 revealed significant enrichments related to toll-like receptor (TLR) signaling and autophagy genes in three LTTs with highly down-regulated ARRB2, suggesting that ARRB2 was negatively involved in TLR-mediated signals for autophagy induction in lung cancer. Biochemical studies for elucidating the molecular mechanism revealed that ARRB2 interacted with TNF receptor-associated factor 6 (TRAF6) and Beclin 1 (BECN1), thereby inhibiting the ubiquitination of TRAF6-TAB2 to activate NF-κB and TRAF6-BECN1 for autophagy stimulated by TLR3 and TLR4, suggesting that ARRB2 could inhibit the TRAF6-TAB2 signaling axis for NF-κB activation and TRAF6-BECN1 signaling axis for autophagy in response to TLR3 and TLR4. Notably, ARRB2-knockout (ARRB2KO) lung cancer cells exhibited marked enhancements of cancer migration, invasion, colony formation, and proliferation in response to TLR3 and TLR4 stimulation. Altogether, our current data suggest that ARRB2 can negatively regulate lung cancer progression by inhibiting TLR3- and TLR4-induced autophagy.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , NF-kappa B/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Neoplasias Pulmonares/patologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores Toll-Like/metabolismo , Pulmão/metabolismo , Autofagia/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
BACKGROUND: Free fatty acid receptors (FFARs) and toll-like receptors (TLRs) recognize microbial metabolites and conserved microbial products, respectively, and are functionally implicated in inflammation and cancer. However, whether the crosstalk between FFARs and TLRs affects lung cancer progression has never been addressed. METHODS: We analyzed the association between FFARs and TLRs using The Cancer Genome Atlas (TCGA) lung cancer data and our cohort of non-small cell lung cancer (NSCLC) patient data (n = 42), and gene set enrichment analysis (GSEA) was performed. For the functional analysis, we generated FFAR2-knockout (FFAR2KO) A549 and FFAR2KO H1299 human lung cancer cells and performed biochemical mechanistic studies and cancer progression assays, including migration, invasion, and colony-formation assays, in response to TLR stimulation. RESULTS: The clinical TCGA data showed a significant down-regulation of FFAR2, but not FFAR1, FFAR3, and FFAR4, in lung cancer, and a negative correlation with TLR2 and TLR3. Notably, GSEA showed significant enrichment in gene sets related to the cancer module, the innate signaling pathway, and the cytokine-chemokine signaling pathway in FFAR2DownTLR2UpTLR3Up lung tumor tissues (LTTs) vs. FFAR2upTLR2DownTLR3Down LTTs. Functionally, treatment with propionate (an agonist of FFAR2) significantly inhibited human A549 or H1299 lung cancer migration, invasion, and colony formation induced by TLR2 or TLR3 through the attenuation of the cAMP-AMPK-TAK1 signaling axis for the activation of NF-κB. Moreover, FFAR2KO A549 and FFAR2KO H1299 human lung cancer cells showed marked increases in cell migration, invasion, and colony formation in response to TLR2 or TLR3 stimulation, accompanied by elevations in NF-κB activation, cAMP levels, and the production of C-C motif chemokine ligand (CCL)2, interleukin (IL)-6, and matrix metalloproteinase (MMP) 2 cytokines. CONCLUSION: Our results suggest that FFAR2 signaling antagonized TLR2- and TLR3-induced lung cancer progression via the suppression of the cAMP-AMPK-TAK1 signaling axis for the activation of NF-κB, and its agonist might be a potential therapeutic agent for the treatment of lung cancer.
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Huntington's disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. CRISPR-Cas9 nuclease causes double-strand breaks (DSBs) in the targeted DNA that induces toxicity, whereas CRISPR interference (CRISPRi) using dead Cas9 (dCas9) suppresses the target gene expression without DSBs. Delivery of dCas9-sgRNA targeting CAG repeat region does not damage the targeted DNA in HEK293T cells containing CAG repeats. When this study investigates whether CRISPRi can suppress mutant HTT (mHTT), CRISPRi results in reduced expression of mHTT with relative preservation of the wild-type HTT in human HD fibroblasts. Although both dCas9 and Cas9 treatments reduce mHTT by sgRNA targeting the CAG repeat region, CRISPRi delays behavioral deterioration and protects striatal neurons against cell death in HD mice. Collectively, CRISPRi can delay disease progression by suppressing mHtt, suggesting DNA DSB-free CRISPRi is a potential therapy for HD that can compensate for the shortcoming of CRISPR-Cas9 nuclease.
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Doença de Huntington , Camundongos , Humanos , Animais , Doença de Huntington/genética , Doença de Huntington/terapia , Doença de Huntington/metabolismo , Quebras de DNA de Cadeia Dupla , Células HEK293 , Corpo Estriado/metabolismoRESUMO
This study provides experimental evidence regarding the mechanism of gallium nitride (GaN) selective-area growth (SAG) on a polished plateau-patterned sapphire substrate (PP-PSS), on which aluminum nitride (AlN) buffer layers are deposited under the same deposition conditions. The SAG of GaN was only observed on the plateau region of the PP-PSS, irrespective of the number of growth cycles. Indirect samples deposited on the bare c-plane substrate were prepared to determine the difference between the AlN buffer layers in the plateau region and silicon oxide (SiO2). The AlN buffer layer in the plateau region exhibited a higher surface energy, and its crystal orientation is indicated by AlN [001]. In contrast, regions other than the plateau region did not exhibit crystallinity and presented lower surface energies. The direct analysis results of PP-PSS using transmission electron microscopy (TEM) and electron backscattered diffraction (EBSD) are similar to the results of the indirect samples. Therefore, under the same conditions, the GaN SAG of the deposited layer is related to crystallinity, crystal orientation, and surface energy.
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Digenome-seq is a powerful approach for determining the genome-wide specificity of programmable nuclease including CRISPR-Cas9 and CRISPR-Cpf1 (also known as Cas12a) and programmable deaminase including cytosine base editors (CBEs) and adenine base editors (ABEs). To define the genome-wide specificity of dLbCpf1-BE (also known as dLbCas12a-BE), genomic DNA is first incubated with dLbCpf1-BE, which induces C-to-U conversion at on-target and off-target sites, and then treated with a mixture of E. coli uracil DNA glycosylase (UDG) and Endonuclease VIII, which creates single-strand breaks (SSBs) by removing uracil in vitro. Digested genomic DNA is subjected to WGS, and then sequencing reads are aligned to the reference genome, resulting in straight alignments at on-target and off-target sites. The in vitro cleavage sites related to the straight alignments can be identified using the Digenome-seq computer tool.
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Citidina , Edição de Genes , Edição de Genes/métodos , Citidina/genética , Escherichia coli/genética , DNA/genética , Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-CasRESUMO
A variety of cancers have been found to have chromosomal rearrangements, and the genomic abnormalities often induced expression of fusion oncogenes. To date, a pair of engineered nucleases including ZFNs, TALENs, and CRISPR-Cas9 nucleases have been used to generate chromosomal rearrangement in living cells and organisms for disease modeling. However, these methods induce unwanted indel mutations at the DNA break junctions, resulting in incomplete disease modeling. Here, we developed prime editor nuclease-mediated translocation and inversion (PETI), a method for programmable chromosomal translocation and inversion using prime editor 2 nuclease (PE2 nuclease) and paired pegRNA. Using PETI method, we successfully introduced DNA recombination in episomal fluorescence reporters as well as precise chromosomal translocations in human cells. We applied PETI to create cancer-associated translocations and inversions such as NPM1-ALK and EML4-ALK in human cells. Our findings show that PETI generated chromosomal translocation and inversion in a programmable manner with efficiencies comparable of Cas9. PETI methods, we believe, could be used to create disease models or for gene therapy.
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Neoplasias , Translocação Genética , Humanos , Rearranjo Gênico , Genoma , Endonucleases , Genômica , Receptores Proteína Tirosina Quinases , Edição de Genes/métodos , Sistemas CRISPR-CasRESUMO
For the occupational adaptation and social integration of the intellectually disabled, it is helpful to improve their work performance and interpersonal skills. The purpose of the study was to evaluate the effectiveness of horticultural therapy (HT) programs to improve work performance and interpersonal relationships of persons with intellectual disabilities. Based on observations and analyses of how people with intellectual disabilities work, we have developed a 12-session HT program that includes upper limb movements and physical activities to improve hand function. We recruited, with the consent of their legal guardians, 14 (6 males, 8 females) participants who had intellectual disabilities and were working at a sheltered workshop in K-gu, Seoul, South Korea. The program consisted of twelve sixty-minute sessions that were conducted twice a week at a rooftop garden. For pre- and post-evaluation of the program, the survey of functional adaptive behavior (SFAB), interpersonal negotiation strategies, a horticultural job evaluation (self), hand function tests (pegboard, pinch gauge, fingertips), and blood sample tests for physiological indicators of exercise were conducted. Interpersonal negotiation strategies, functional adaptive behaviors, and physical abilities for job behaviors, including agility and grasping of the hand, improved significantly from before to after the program (p < 0.05). A positive result of VEGF (vascular endothermic growth factor) in blood sample tests implies the need for further research on cognitive changes caused by horticultural activities. This study has limitations due to the small number of participants, but the results suggest that low- to medium-intensity horticultural treatment programs using the upper body and hands could be effective for vocational rehabilitation of the intellectually disabled.
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Pessoas com Deficiência , Horticultura Terapêutica , Deficiência Intelectual , Desempenho Profissional , Masculino , Feminino , Humanos , Deficiência Intelectual/reabilitação , Reabilitação Vocacional , Pessoas com Deficiência/reabilitaçãoRESUMO
RNA-guided CRISPR-Cas12a endonucleases are promising tools for genome engineering. Here we demonstrate that LbCas12a variants derived from Lachnospiraceae bacterium show a broad PAM preference, recognizing certain non-canonical PAMs with high efficiency. Furthermore, we engineered LbABE8e to carry G532R and/or K595R mutations, altering its original PAM specificities; these variants exhibited superior base editing activity in human cells compared with wild-type LbABE8e at sites with non-canonical PAMs. Based on this finding, we utilized the most effective LbCas12a and LbABE8e variants to demonstrate multiplexed and mutant-allele-specific gene editing in oncogenes, made possible by the variant's recognition of non-canonical PAMs. Importantly, LbCas12a-G532R/K595R and LbABE8e-G532R/K595R with optimized crRNA arrays targeted to triple oncogenic mutations inhibited colon cancer cell proliferation. Taken together, these results demonstrate the potential of engineered LbCas12a and LbABE8e as tools for targeting sites with alternative PAMs for genome engineering and therapeutic editing in cancer cells.
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The use of prime editing-a gene-editing technique that induces small genetic changes without the need for donor DNA and without causing double strand breaks-to correct pathogenic mutations and phenotypes needs to be tested in animal models of human genetic diseases. Here we report the use of prime editors 2 and 3, delivered by hydrodynamic injection, in mice with the genetic liver disease hereditary tyrosinemia, and of prime editor 2, delivered by an adeno-associated virus vector, in mice with the genetic eye disease Leber congenital amaurosis. For each pathogenic mutation, we identified an optimal prime-editing guide RNA by using cells transduced with lentiviral libraries of guide-RNA-encoding sequences paired with the corresponding target sequences. The prime editors precisely corrected the disease-causing mutations and led to the amelioration of the disease phenotypes in the mice, without detectable off-target edits. Prime editing should be tested further in more animal models of genetic diseases.
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Oftalmopatias , Edição de Genes , Animais , Edição de Genes/métodos , Fígado , Camundongos , Mutação , FenótipoRESUMO
Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5' terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA-crRNA complementary region, a penta(uridinylate) sequence, the 3' terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.
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Dependovirus , RNA Guia de Cinetoplastídeos , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Endonucleases/genética , Edição de Genes , Humanos , RNA , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Surgical procedures on large skin defects can be challenging in the short term due to the size of the lesion, infection, and tissue defect. A regenerative therapy for skin wounds has been applied to promote the healing process. An 8-month-old, Korean domestic short-haired female cat, weighing 3 kg, was rescued with extensive defects on the right flank to right inguinal region caused by bite wounds. In this case, amniotic membranes and adipose-derived mesenchymal stem cells were used as the regenerative therapy to treat the large skin defect rather than a surgical intervention alone. To the best of our knowledge, this is the first report of a case with of a large skin defect treated by applying allogeneic amniotic membranes and allogeneic mesenchymal stem cells to a cat.
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Electronic skin (E-skin) based on tactile sensors has great significance in next-generation electronics such as biomedical application and artificial intelligence that requires interaction with humans. To mimic the properties of human skin, high flexibility, excellent sensing capability, and sufficient spatial resolution through high-level sensor integration are required. Here, we report a highly sensitive pressure sensor matrix based on a piezoresistive cellulose/single-walled carbon nanotube-entangled fiber network, which forms its own porous structure enabling a superior pressure sensor with a high sensitivity (9.097 kPa-1), a fast response speed (<2 ms), and orders of magnitude detection range with a detection limit of 1 Pa. Furthermore, the remarkable device expandability based on the ease of patterning and scalability allows easy implementation of a large-area pressure sensor matrix which has 2304 (48 × 48) pixels. Combined with a real-time pressure distribution monitoring system, a flexible 3D touch sensor that simultaneously displays plane coordinates and pressure information and a scanning device that detects the morphology of the soft body 3D surface are successfully demonstrated.
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The surface migration of lubricants degrades the quality of thermoplastic polymer composites. In this study, the surface migration of lubricants in polypropylene composites were studied to improve the quality of the composites. Polypropylene (PP)/lubricant composites were manufactured using a co-rotating twin-screw extruder and injection molding, and the migration phenomena of the lubricant in the PP/lubricant composites were investigated under accelerated aging conditions with temperatures in the range of 20 to 90 °C and humidity of 100% for 72 h. The interrelation between the surface migration properties of PP/lubricant composites were investigated by considering their microstructural and morphological features, which were influenced by the thermal aging conditions. Further, the microstructural and morphological features were examined by contact angle, surface energy, attenuated total reflectance Fourier-transform infrared spectrometry, X-ray photoelectron spectroscopy, close-up digital imaging, and atomic force microscopy analyses. The polypropylene composites containing the magnesium stearate as the lubricant were found to exhibit a more stable migration behavior than the polypropylene composites containing a calcium stearate lubricant. This is attributed to multiple synergistic factors, such as interfacial tension and work of adhesion between PP and the lubricant. The findings of this study can be utilized to effectively manufacture high-quality thermoplastic composites for the fourth industrial revolution.
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Cracks typically deteriorate the structural and electrical properties of materials when not properly controlled. A few papers recently reported the controlling methods of crack formation in the brittle materials utilizing the lateral V-notch structure. For ductile materials, however, there have been few papers reporting cracking phenomenon, but full cracking control including predesigned initiation, propagation, and termination has not been reported yet. Therefore, we report a predesigned full cracking control in ductile conductive carbon nanotube (CNT) films by introducing inkjet-printed L-shape micronotch (LMN) structures inspired by directional stamp perforation marks. In spite of the high fracture toughness of CNT films, the LMNs determine locations of initial crack formation and guide crack propagation in a predesigned way. Selective connection of isolated cracks in the CNT film increases its resistance monotonically under tensile strain and thus tremendously well maintains high linearity (adj. R2 value > 0.99) in resistance change over record large strain ranges of 0.01-100%, which enables us to quantitatively classify strain values accurately for previously reported practical body signals for the first time. We believe that our facile printing-based crack control strategy not only provides a comprehensive solution to various stretchable sensor applications but also builds a new milestone for cracking mechanism studies in fracture mechanics.
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OBJECTIVE: Dysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of inflammation resolution, but the mechanisms underlying their mucosal healing capacity remain elusive. DESIGN: We investigated the role of the prostaglandin E2 (PGE2) receptor PTGER4 on the differentiation of intestinal macrophages in patients with IBD and mouse models of intestinal inflammation. We studied mucosal healing and intestinal epithelial barrier regeneration in Csf1r-iCre Ptger4fl/fl mice during dextran sulfate sodium (DSS)-induced colitis. The effect of PTGER4+ macrophage secreted molecules was investigated on epithelial organoid differentiation. RESULTS: Here, we describe a subset of PTGER4-expressing intestinal macrophages with mucosal healing properties both in humans and mice. Csf1r-iCre Ptger4fl/fl mice showed defective mucosal healing and epithelial barrier regeneration in a model of DSS colitis. Mechanistically, an increased mucosal level of PGE2 triggers chemokine (C-X-C motif) ligand 1 (CXCL1) secretion in monocyte-derived PTGER4+ macrophages via mitogen-activated protein kinases (MAPKs). CXCL1 drives epithelial cell differentiation and proliferation from regenerating crypts during colitis. Specific therapeutic targeting of macrophages with liposomes loaded with an MAPK agonist augmented the production of CXCL1 in vivo in conditional macrophage PTGER4-deficient mice, restoring their defective epithelial regeneration and favouring mucosal healing. CONCLUSION: PTGER4+ intestinal macrophages are essential for supporting the intestinal stem cell niche and regeneration of the injured epithelium. Our results pave the way for the development of a new class of therapeutic targets to promote macrophage healing functions and favour remission in patients with IBD.