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Flexible and stretchable electronic systems for epidermal electronics
Provided herein are skin-mounted biomedical devices and methods of making and using biomedical devices for sensing and actuation applications. For example, flexible and/or stretchable biomedical devices are provided, including electronic devices useful for establishing conformal contact with the skin of a subject. Devices disclosed herein can comprise a plurality of sensing and/or actuating devices provided as part of a skin-mounted flexible or stretchable electronic circuit.
Extrapolation of systemic bioavailability assessing skin absorption and epidermal and hepatic metabolism of aromatic amine hair dyes in vitro
Approaches to assess the role of absorption, metabolism and excretion of cosmetic ingredients that are based on the integration of different in vitro data are important for their safety assessment, specifically as it offers an opportunity to refine that safety assessment. In order to estimate systemic exposure (AUC) to aromatic amine hair dyes following typical product application conditions, skin penetration and epidermal and systemic metabolic conversion of the parent compound was assessed in human skin explants and human keratinocyte (HaCaT) and hepatocyte cultures. To estimate the amount of the aromatic amine that can reach the general circulation unchanged after passage through the skin the following toxicokinetically relevant parameters were applied: a) Michaelis–Menten kinetics to quantify the epidermal metabolism; b) the estimated keratinocyte cell abundance in the viable epidermis; c) the skin penetration rate; d) the calculated Mean Residence Time in the viable epidermis; e) the viable epidermis thickness and f) the skin permeability coefficient. In a next step, in vitro hepatocyte K{sub m} and V{sub max} values and whole liver mass and cell abundance were used to calculate the scaled intrinsic clearance, which was combined with liver blood flow and fraction of compound unbound in the blood to give hepatic clearance. The systemic exposure in the general circulation (AUC) was extrapolated using internal dose and hepatic clearance, and C{sub max} was extrapolated (conservative overestimation) using internal dose and volume of distribution, indicating that appropriate toxicokinetic information can be generated based solely on in vitro data. For the hair dye, p-phenylenediamine, these data were found to be in the same order of magnitude as those published for human volunteers. - Highlights: • An entirely in silico/in vitro approach to predict in vivo exposure to dermally applied hair dyes • Skin penetration and epidermal conversion assessed in human skin explants and HaCaT • Systemic metabolism was modeled using hepatocyte cultures. • Toxicokinetically relevant parameters were applied to estimate systemic exposure. • There was a good agreement between in vitro and in vivo data.
Soft Core/Shell Packages for Stretchable Electronics
This paper presents materials and core/shell architectures that provide optimized mechanical properties in packages for stretchable electronic systems. Detailed experimental and theoretical studies quantitatively connect the geometries and elastic properties of the constituent materials to the overall mechanical responses of the integrated systems, with a focus on interfacial stresses, effective modulus, and maximum extent of elongation. Specific results include core/shell designs that lead to peak values of the shear and normal stresses on the skin that remain less than 10 kPa even for applied strains of up to 20%, thereby inducing minimal somatosensory perception of the device on the human skin. Additional, strain-limiting mesh structures embedded in the shell improve mechanical robustness by protecting the active components from strains that would otherwise exceed the fracture point. Demonstrations in precommercial stretchable electronic systems illustrate the utility of these concepts.
Patient Selection for Clinical Trials Eliminating Surgery for HER2-Positive Breast Cancer Treated with Neoadjuvant Systemic Therapy
Background: Patients with epidermal growth factor receptor 2-positive (HER2+) breast cancer and pathologic complete response (pCR) after neoadjuvant systemic therapy (NST) may be candidates for nonoperative clinical trials if residual invasive and in situ disease are eradicated. Methods: This study analyzed 280 patients with clinical T1-2N0-1 HER2+ breast cancer who underwent NST followed by surgical resection to determine key characteristics of patients with pCR in the breast and lymph nodes compared with those with residual disease. Results: Of the 280 patients, 102 (36.4%) had pCR in the breast and lymph nodes after NST, and 50 patients (17.9%) had residual ductal carcinoma in situ (DCIS) in the breast only. For 129 patients (46.1%), DCIS was present on the pretreatment biopsy, and NST failed to eradicate the DCIS component in 64.3%. Patients with residual disease were more likely to have hormone receptor-positive (HR+) tumors than those with negative tumors (73.4% vs. 50.8%; p < 0.0001). Radiologic response (odds ratio [OR], 5.62; p = 0.002) and HR+ status (OR, 2.56; p < 0.0001) were predictive of residual disease. Combined imaging methods after NST had a sensitivity of 97.1% and a negative predictive value of 70.6% for detection of residual disease. Patients with invasive disease and DCIS shown on the pretreatment core biopsy were less likely than those without DCIS to achieve pCR in the breast (31% vs. 43%; p = 0.038). Conclusion: The study results delineate and identify unique characteristics associated with HER2+ breast cancers that are important in selecting patients for inclusion in clinical trials assessing nonoperative management after NST, and the low negative predictive value of imaging mandates image-guided biopsy for selection.
Local Thermal Ablation with Continuous EGFR Tyrosine Kinase Inhibitors for EGFR-Mutant Non-small Cell Lung Cancers that Developed Extra-Central Nervous System (CNS) Oligoprogressive Disease
Background: Most epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) patients treated with tyrosine kinase inhibitors (TKIs) experience oligoprogressive disease. Local ablation for isolated resistant sites continued with the original EGFR-TKI showed good efficacy in these patients. We conducted this multicenter retrospective study to investigate the potential benefit of thermal ablation in NSCLC patients that developed extra-central nervous system (CNS) oligoprogressive disease during TKI treatment. Methods: A total of 71 EGFR-mutant patients treated with EGFR-TKIs were identified. Progression-free survival 1 (PFS1) was calculated from the initiation of TKI treatment to first progression. Patients with metastatic sites ≤ 3 in less than 3 extra-CNS organs suitable for local ablation therapy received either radiofrequency ablation or microwave ablation to these sites and continued on the original TKIs. PFS2 was defined from the first progression to second progression after ablation. Results: The median PFS1 for all patients was 11.8 months. Eighty extra-CNS oligoprogressive lesions in 71 patients were ablated. Thirty-six of 71 patients progressed after thermal ablation and 31 of whom died during the study period. The median PFS2 after thermal ablation was 10.0 months, and the median overall survival was 26.4 months. PFS1 and PFS2 were highly correlated with OS, whereas PFS1 was not correlated with PFS2. The numbers of oligoprogressive lesions were significantly and independently associated with PFS2. Conclusion: Local thermal ablation for the oligoprogressive lesions with continuous EGFR-TKI treatment is associated with additional 10 months of disease control and should be recommended in TKI acquired resistant-NSCLC patients.
Laser System for Biological Tissues Final Report CRADA No. TSB-1549-98
The goal of this project was to demonstrate a laser-based system which can shape and resurface the epidermal plate as well as generate images on acrylic. The prototype system used a pulsed diode pumped laser to ablate and shape the surface using a high absorption pigment. A CCD camera will image the epidermal plate and computer software will analyze the image to guide laser ablation. . The deliverable was for LLNL to generate a final report upon completion of the project, which summarized the results of the project and the schematics for the prototype device. This project consisted of six (6) main tasks, all of which would be performed at LLNL over a seven (7) month period. Due to lack of manpower availability which caused delays in the project work, and minor redesigns which delayed manufacturing, the project was extended for an additional five months.
Protein expression and gene editing in monocots using foxtail mosaic virus vectors
Abstract Plant viruses can be engineered to carry sequences that direct silencing of target host genes, expression of heterologous proteins, or editing of host genes. A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9‐mediated gene editing in maize, Setaria viridis , and Nicotiana benthamiana . This was accomplished by duplicating the FoMV capsid protein subgenomic promoter, abolishing the unnecessary open reading frame 5A, and inserting a cloning site containing unique restriction endonuclease cleavage sites immediately after the duplicated promoter. The modified FoMV vectors transiently expressed green fluorescent protein (GFP) and bialaphos resistance (BAR) protein in leaves of systemically infected maize seedlings. GFP was detected in epidermal and mesophyll cells by epifluorescence microscopy, and expression was confirmed by Western blot analyses. Plants infected with FoMV carrying the bar gene were temporarily protected from a glufosinate herbicide, and expression was confirmed using a rapid antibody‐based BAR strip test. Expression of these proteins was stabilized by nucleotide substitutions in the sequence of the duplicated promoter region. Single guide RNAs expressed from the duplicated promoter mediated edits in the N. benthamiana Phytoene desaturase gene, the S. viridis Carbonic anhydrase 2 gene, and the maize HKT1 gene encoding a potassium transporter. The efficiency of editing was enhanced in the presence of synergistic viruses and a viral silencing suppressor. This work expands the utility of FoMV for virus‐induced gene silencing (VIGS), virus‐mediated overexpression (VOX), and virus‐enabled gene editing (VEdGE) in monocots.
ALD1 accumulation in Arabidopsis epidermal plastids confers local and non-autonomous disease resistance
The Arabidopsis plastid-localized ALD1 protein acts in the lysine catabolic pathway that produces infection-induced pipecolic acid (Pip), Pip derivatives, and basal non-Pip metabolite(s). ALD1 is indispensable for disease resistance associated with Pseudomonas syringae infections of naïve plants as well as those previously immunized by a local infection, a phenomenon called systemic acquired resistance (SAR). Pseudomonas syringae is known to associate with mesophyll as well as epidermal cells. To probe the importance of epidermal cells in conferring bacterial disease resistance, we studied plants in which ALD1 was only detectable in the epidermal cells of specific leaves. Local disease resistance and many features of SAR were restored when ALD1 preferentially accumulated in the epidermal plastids at immunization sites. Interestingly, SAR restoration occurred without appreciable accumulation of Pip or known Pip derivatives in secondary distal leaves. Our findings establish that ALD1 has a non-autonomous effect on pathogen growth and defense activation. We propose that ALD1 is sufficient in the epidermis of the immunized leaves to activate SAR, but basal ALD1 and possibly a non-Pip metabolite(s) are also needed at all infection sites to fully suppress bacterial growth. Thus, epidermal plastids that contain ALD1 play a key role in local and whole-plant immune signaling.
Spatial organization of cellulose microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy
Summary We used atomic force microscopy ( AFM ), complemented with electron microscopy, to characterize the nanoscale and mesoscale structure of the outer (periclinal) cell wall of onion scale epidermis – a model system for relating wall structure to cell wall mechanics. The epidermal wall contains ~100 lamellae, each ~40 nm thick, containing 3.5‐nm wide cellulose microfibrils oriented in a common direction within a lamella but varying by ~30 to 90° between adjacent lamellae. The wall thus has a crossed polylamellate, not helicoidal, wall structure. Montages of high‐resolution AFM images of the newly deposited wall surface showed that single microfibrils merge into and out of short regions of microfibril bundles, thereby forming a reticulated network. Microfibril direction within a lamella did not change gradually or abruptly across the whole face of the cell, indicating continuity of the lamella across the outer wall. A layer of pectin at the wall surface obscured the underlying cellulose microfibrils when imaged by FESEM , but not by AFM . The AFM thus preferentially detects cellulose microfibrils by probing through the soft matrix in these hydrated walls. AFM ‐based nanomechanical maps revealed significant heterogeneity in cell wall stiffness and adhesiveness at the nm scale. By color coding and merging these maps, the spatial distribution of soft and rigid matrix polymers could be visualized in the context of the stiffer microfibrils. Without chemical extraction and dehydration, our results provide multiscale structural details of the primary cell wall in its near‐native state, with implications for microfibrils motions in different lamellae during uniaxial and biaxial extensions.
Cancer stem cell-like population is preferentially suppressed by EGFR-TKIs in EGFR-mutated PC-9 tumor models
Highlights: • EGFR-TKIs strongly inhibited the β-catenin signal and decreased CSCs sorted out by SP cells. • The reduction of CSCs significantly reduced the migration capability. • Targeting β-catenin could be a potential strategy for EGFR-mutated NSCLC. Although the epidermal growth factor receptor (EGFR) and Wnt/β-catenin signaling systems synergistically regulate many essential developmental and regenerative processes in lung cancer, the mechanisms of their crosstalk remain poorly defined. Our study aimed to investigate an interaction between EGFR and the β-catenin signal.
Optical Microscopy-Guided Laser Ablation Electrospray Ionization Ion Mobility Mass Spectrometry: Ambient Single Cell Metabolomics with Increased Confidence in Molecular Identification
Single cell analysis is a field of increasing interest as new tools are continually being developed to understand intercellular differences within large cell populations. Laser-ablation electrospray ionization mass spectrometry (LAESI-MS) is an emerging technique for single cell metabolomics. Over the years, it has been validated that this ionization technique is advantageous for probing the molecular content of individual cells in situ. Here, we report the integration of a microscope into the optical train of the LAESI source to allow for visually informed ambient in situ single cell analysis. Additionally, we have coupled this ‘LAESI microscope’ to a drift-tube ion mobility mass spectrometer to enable separation of isobaric species and allow for the determination of ion collision cross sections in conjunction with accurate mass measurements. This combined information helps provide higher confidence for structural assignment of molecules ablated from single cells. Here, we show that this system enables the analysis of the metabolite content of Allium cepa epidermal cells with high confidence structural identification together with their spatial locations within a tissue.
Ergothioneine alleviates senescence of fibroblasts induced by UVB damage of keratinocytes via activation of the Nrf2/HO-1 pathway and HSP70 in keratinocytes
Highlights: • EGT activated Nrf2/HO-1 pathway and HSP70 in UVB-irradiated keratinocytes. • EGT alleviated ROS production, apoptosis, and inflammation in UVB-irradiated keratinocytes. • EGT suppressed senescence and collagen imbalance in fibroblasts by inhibiting the keratinocyte-secreted factors. • EGT suppresses photoaging by UVB irradiation in a keratinocyte/fibroblast co-culture system. Ultraviolet B (UVB) irradiation induces skin damage and photoaging through several deleterious effects, including generation of reactive oxygen species (ROS), apoptosis of epidermal cells, inflammation, and collagen degradation in fibroblasts. Ergothioneine (EGT) is a naturally occurring amino acid with potential biological properties. We evaluated whether EGT protects against UVB-induced photoaging using a keratinocyte/fibroblast co-culture system. Keratinocytes were pretreated with EGT, irradiated with UVB, and co-cultured with fibroblasts. In keratinocytes, ROS production and apoptosis were assessed. We also analyzed the Nrf2/HO-1 pathway, HSP70, proapoptotic proteins, and paracrine cytokines by Western blotting and real-time PCR. Collagen degradation-related genes and senescence were also assessed in fibroblasts. EGT pretreatment of keratinocytes significantly inhibited downregulation of the Nrf2/HO-1 pathway and HSP70, and protected keratinocytes by suppressing production of ROS and cleavage of proapoptotic proteins, including caspase-8 and PARP. Furthermore, EGT significantly reduced the paracrine cytokines, including IL-1β, IL-6, and TNF-α. In co-cultures of fibroblasts with EGT-treated keratinocytes, the expression levels of collagen degradation-related genes and fibroblast senescence were significantly decreased; however, synthesis of procollagen type I was significantly increased. Our results confirm that EGT suppresses the modification of collagen homeostasis in fibroblasts by preventing downregulation of the Nrf2/HO-1 pathway and HSP70 in keratinocytes following UVB irradiation.
In vivo epidermal migration requires focal adhesion targeting of ACF7
Turnover of focal adhesions allows cell retraction, which is essential for cell migration. The mammalian spectraplakin protein, ACF7 (Actin-Crosslinking Factor 7), promotes focal adhesion dynamics by targeting of microtubule plus ends towards focal adhesions. However, it remains unclear how the activity of ACF7 is regulated spatiotemporally to achieve focal adhesion-specific guidance of microtubule. To explore the potential mechanisms, we resolve the crystal structure of ACF7's NT (amino-terminal) domain, which mediates F-actin interactions. Structural analysis leads to identification of a key tyrosine residue at the calponin homology (CH) domain of ACF7, whose phosphorylation by Src/FAK (focal adhesion kinase) complex is essential for F-actin binding of ACF7. Using skin epidermis as a model system, we further demonstrate that the phosphorylation of ACF7 plays an indispensable role in focal adhesion dynamics and epidermal migration in vitro and in vivo . Altogether, our findings provide critical insights into the molecular mechanisms underlying coordinated cytoskeletal dynamics during cell movement.
Hyperdimensional Imaging Contrast Using an Optical Fiber
Fluorescence properties of a molecule can be used to study the structural and functional nature of biological processes. Physical properties, including fluorescence lifetime, emission spectrum, emission polarization, and others, help researchers probe a molecule, produce desired effects, and infer causes and consequences. Correlative imaging techniques such as hyperdimensional imaging microscopy (HDIM) combine the physical properties and biochemical states of a fluorophore. Here we present a fiber-based imaging system that can generate hyper-dimensional contrast by combining multiple fluorescence properties into a single fluorescence lifetime decay curve. Fluorescence lifetime imaging microscopy (FLIM) with controlled excitation polarization and temporally dispersed emission can generate a spectrally coded, polarization-filtered lifetime distribution for a pixel. This HDIM scheme generates a better contrast between different molecules than that from individual techniques. This setup uses only a single detector and is simpler to implement, modular, cost-efficient, and adaptable to any existing FLIM microscope. We present higher contrast data from Arabidopsis thaliana epidermal cells based on intrinsic anthocyanin emission properties under multiphoton excitation. This work lays the foundation for an alternative hyperdimensional imaging system and demonstrates that contrast-based imaging is useful to study cellular heterogeneity in biological samples.
Proteomic Profiling of Radiation-Induced Skin Fibrosis in Rats: Targeting the Ubiquitin-Proteasome System
Purpose: To investigate the molecular changes underlying the pathogenesis of radiation-induced skin fibrosis. Methods and Materials: Rat skin was irradiated to 30 or 45 Gy with an electron beam. Protein expression in fibrotic rat skin and adjacent normal tissues was quantified by label-free protein quantitation. Human skin cells HaCaT and WS-1 were treated by x-ray irradiation, and the proteasome activity was determined with a fluorescent probe. The effect of proteasome inhibitors on Transforming growth factor Beta (TGF-B) signaling was measured by Western blot and immunofluorescence. The efficacy of bortezomib in wound healing of rat skin was assessed by the skin injury scale. Results: We found that irradiation induced epidermal and dermal hyperplasia in rat and human skin. One hundred ninety-six preferentially expressed and 80 unique proteins in the irradiated fibrotic skin were identified. Through bioinformatic analysis, the ubiquitin-proteasome pathway showed a significant fold change and was investigated in greater detail. In vitro experiments demonstrated that irradiation resulted in a decline in the activity of the proteasome in human skin cells. The proteasome inhibitor bortezomib suppressed profibrotic TGF-β downstream signaling but not TGF-β secretion stimulated by irradiation in HaCaT and WS-1 cells. Moreover, bortezomib ameliorated radiation-induced skin injury and attenuated epidermal hyperplasia. Conclusion: Our findings illustrate the molecular changes during radiation-induced skin fibrosis and suggest that targeting the ubiquitin-proteasome system would be an effective countermeasure.
Grasses use an alternatively wired bHLH transcription factor network to establish stomatal identity
Significance Plants both control and are controlled by the global climate. Grasses in natural and agricultural systems participate in the exchange of atmospheric CO 2 for biosphere-derived oxygen and water vapor via microscopic epidermal valves (stomata), but how these stomata are made in grasses is unknown. Using genetic screens and targeted genome editing, we identify and characterize master transcriptional regulators of stomatal initiation in the wheat relative Brachypodium . Surprisingly, the unique stomatal form and pattern of grasses is regulated by orthologs of Arabidopsis stomatal basic helix–loop–helix (bHLH) transcription factors, although the function of individual genes and regulation of their protein products have diverged. This finding suggests that the stomatal core bHLH transcription factors are excellent breeding targets to enhance performance in grasses.
Epidermal growth factor induced macropinocytosis directs branch formation of lung epithelial cells
Highlights: • EGF is essential for branch pattern formation of lung epithelial cells. • EGF receptor is highly expressed at the tip of branches to supply driving force to cell to collectively migrate. • Macropinocytosis is induced by EGFR expression and highly active at the tip of branches. • The Reaction-Diffusion model supports for our understanding of the molecular dynamics for the branch pattern formations. Lung branching morphogenesis is a complex system involving many molecular interactions to filling the three dimensional spaces; however, the underlying developmental mechanisms are still not fully understood. In this paper, we have investigated the effect of epidermal growth factor (EGF) on normal human bronchial epithelial cells and their three-dimensional (3D) branching pattern formation by using in vitro experiments and mathematical simulation. The results show that EGF is essential for 3D branch pattern formation and its receptor is highly expressed at the tip of branches to generate the drive force for cells to migrate. Macropinocytosis induced by EGFR expression is firmly contributed to the nutrition uptake at the tip of branches. Our findings for effective branching formation of human lung cells contribute to further understanding molecular mechanisms of organogenesis, and the important mechanisms also possibly participate in related lung disease such as malformation.