From static to dynamic: The influence of mechanotransduction on skin equivalents analyzed by bioimaging and RNAseq.

In this study, we explore the impact of mechanical stimuli on skin models using an innovative skin-on-a-chip platform, addressing the limitations of conventional transwell-cultured skin equivalents. This platform facilitates cyclic mechanical stimulation through compression and stretching, combined with automated media perfusion. Our findings, using bioimaging and bulk RNA sequencing, reveal increased expression of Keratin 10 and Keratin 14, indicating enhanced skin differentiation and mechanical integrity. The increase in desmosomes and tight junctions, observed through Claudin-1 and Desmoplakin 1 & 2 analysis, suggests improved keratinocyte differentiation due to mechanical stimulation. Gene expression analyses reveal a nuanced regulatory response, suggesting a potential connection to the Hippo pathway, indicative of a significant cellular reaction to mechanical stimuli. The results show the important influence of mechanical stimulation on skin model integrity and differentiation, demonstrating the potential of our microfluidic platform in advancing skin biology research and pharmaceutical testing.

Submolecular-scale control of phototautomerization.

Optically activated reactions initiate biological processes such as photosynthesis or vision, but can also control polymerization, catalysis or energy conversion. Methods relying on the manipulation of light at macroscopic and mesoscopic scales are used to control on-surface photochemistry, but do not offer atomic-scale control. Here we take advantage of the confinement of the electromagnetic field at the apex of a scanning tunnelling microscope tip to drive the phototautomerization of a free-base phthalocyanine with submolecular precision. We can control the reaction rate and the relative tautomer population through a change in the laser excitation wavelength or through the tip position. Atomically resolved tip-enhanced photoluminescence spectroscopy and hyperspectral mapping unravel an excited-state mediated process, which is quantitatively supported by a comprehensive theoretical model combining ab initio calculations with a parametric open-quantum-system approach. Our experimental strategy may allow insights in other photochemical reactions and proof useful to control complex on-surface reactions.

Novel Chip for Applying Mechanical Forces on Human Skin Models Under Dynamic Culture Conditions.

In recent years the need for in vitro skin models as a replacement for animal studies has resulted in significant progress in the development of skin-on-a-chip models. These devices allow the fine control of the microenvironment of the model and the incorporation of chemical and physical stimuli. In this study, we describe the development of an easy and low-budget open-top dynamic microfluidic device for skin-on-a-chip experiments using polydimethylsiloxane and a porous polyethylene terephthalate membrane. The chip allows the incorporation of compressive stimuli during the cultivation period by the use of syringe pumps. Proof-of-concept results show the successful differentiation of the cells and establishment of the skin structure in the chip. The microfluidic skin-on-a-chip models presented in this study can serve as a platform for future drug and feasibility studies.

Charge-state lifetimes of single molecules on few monolayers of NaCl.

In molecular tunnel junctions, where the molecule is decoupled from the electrodes by few-monolayers-thin insulating layers, resonant charge transport takes place by sequential charge transfer to and from the molecule which implies transient charging of the molecule. The corresponding charge state transitions, which involve tunneling through the insulating decoupling layers, are crucial for understanding electrically driven processes such as electroluminescence or photocurrent generation in such a geometry. Here, we use scanning tunneling microscopy to investigate the decharging of single ZnPc and H2Pc molecules through NaCl films of 3 to 5 monolayers thickness on Cu(111) and Au(111). To this end, we approach the tip to the molecule at resonant tunnel conditions up to a regime where charge transport is limited by tunneling through the NaCl film. The resulting saturation of the tunnel current is a direct measure of the lifetimes of the anionic and cationic states, i.e., the molecule's charge-state lifetime, and thus provides a means to study charge dynamics and, thereby, exciton dynamics. Comparison of anion and cation lifetimes on different substrates reveals the critical role of the level alignment with the insulator's conduction and valence band, and the metal-insulator interface state.

Portraits of Soot Molecules Reveal Pathways to Large Aromatics, Five-/Seven-Membered Rings, and Inception through π-Radical Localization.

Incipient soot early in the flame was studied by high-resolution atomic force microscopy and scanning tunneling microscopy to resolve the atomic structure and orbital densities of single soot molecules prepared on bilayer NaCl on Cu(111). We resolved extended catacondensed and pentagonal-ring linked (pentalinked) species indicating how small aromatics cross-link and cyclodehydrogenate to form moderately sized aromatics. In addition, we resolved embedded pentagonal and heptagonal rings in flame aromatics. These nonhexagonal rings suggest simultaneous growth through aromatic cross-linking/cyclodehydrogenation and hydrogen abstraction acetylene addition. Moreover, we observed three classes of open-shell π-radical species. First, radicals with an unpaired π-electron delocalized along the molecule's perimeter. Second, molecules with partially localized π-electrons at zigzag edges of a π-radical. Third, molecules with strong localization of a π-electron at pentagonal- and methylene-type sites. The third class consists of π-radicals localized enough to enable thermally stable bonds, as well as multiradical species such as diradicals in the open-shell triplet state. These π-diradicals can rapidly cluster through barrierless chain reactions enhanced by van der Waals interactions. These results improve our understanding of soot formation and the products formed by combustion and could provide insights for cleaner combustion and the production of hydrogen without CO2 emissions.

E96V Mutation in the Kdelr3 Gene Is Associated with Type 2 Diabetes Susceptibility in Obese NZO Mice.

Type 2 diabetes (T2D) represents a multifactorial metabolic disease with a strong genetic predisposition. Despite elaborate efforts in identifying the genetic variants determining individual susceptibility towards T2D, the majority of genetic factors driving disease development remain poorly understood. With the aim to identify novel T2D risk genes we previously generated an N2 outcross population using the two inbred mouse strains New Zealand obese (NZO) and C3HeB/FeJ (C3H). A linkage study performed in this population led to the identification of the novel T2D-associated quantitative trait locus (QTL) Nbg15 (NZO blood glucose on chromosome 15, Logarithm of odds (LOD) 6.6). In this study we used a combined approach of positional cloning, gene expression analyses and in silico predictions of DNA polymorphism on gene/protein function to dissect the genetic variants linking Nbg15 to the development of T2D. Moreover, we have generated congenic strains that associated the distal sublocus of Nbg15 to mechanisms altering pancreatic beta cell function. In this sublocus, Cbx6, Fam135b and Kdelr3 were nominated as potential causative genes associated with the Nbg15 driven effects. Moreover, a putative mutation in the Kdelr3 gene from NZO was identified, negatively influencing adaptive responses associated with pancreatic beta cell death and induction of endoplasmic reticulum stress. Importantly, knockdown of Kdelr3 in cultured Min6 beta cells altered insulin granules maturation and pro-insulin levels, pointing towards a crucial role of this gene in islets function and T2D susceptibility.

Influence of Von Willebrand Disease (VWD) and pregnancy on the expression of angiogenic factors in the porcine female reproductive tract.

Von Willebrand Disease (VWD) is a heritable disorder caused by defects of the Von Willebrand Factor (VWF), leading to deficiencies in coagulation and also angiogenesis. Women affected by VWD frequently show bleeding concerning the reproductive tract and may present with increased rates of miscarriages. We used a porcine model representing VWD type 1 and type 3 as well as the wildtype. Samples were obtained from the reproductive tract of non-pregnant sows and sows pregnant at time of placentation. Relative expression of the genes CALR, CCN2, CXCL8, ECE1, EDN1, F8, IGFBP7, and LGALS3 was analyzed. CCN2 and FVIII proteins were additionally analyzed using immunohistochemistry. In uterus and ovary significant upregulation of CCN2 was seen in non-pregnant pigs affected by VWD. This might be caused by the higher VEGFA-levels in these pigs and could have an influence angiogenesis. During pregnancy, CCN2 expression increased in wildtype pig uteri but hardly changed in those of pregnant pigs affected by VWD, presumably because the expression level in the latter pigs already was significantly increased before pregnancy. F8 expression was significantly reduced in uterus and ovary of VWD-affected pigs. VWF is known to protect FVIII from decomposition and a lack of VWF leads to lower levels of FVIII. Our results suggest that a reduced F8 expression primarily might contribute to those reduced FVIII levels in VWD-affected pigs. Additional significant results involving the pregnant pigs were detected for CALR, EDN1, and LGALS3. These genes are promising candidates for more detailed future studies.

Evaluation of native and non-native biomaterials for engineering human skin tissue.

A variety of human skin models have been developed for applications in regenerative medicine and efficacy studies. Typically, these employ matrix molecules that are derived from non-human sources along with human cells. Key limitations of such models include a lack of cellular and tissue microenvironment that is representative of human physiology for efficacy studies, as well as the potential for adverse immune responses to animal products for regenerative medicine applications. The use of recombinant extracellular matrix proteins to fabricate tissues can overcome these limitations. We evaluated animal- and non-animal-derived scaffold proteins and glycosaminoglycans for the design of biomaterials for skin reconstruction in vitro. Screening of proteins from the dermal-epidermal junction (collagen IV, laminin 5, and fibronectin) demonstrated that certain protein combinations when used as substrates increase the proliferation and migration of keratinocytes compared to the control (no protein). In the investigation of the effect of components from the dermal layer (collagen types I and III, elastin, hyaluronic acid, and dermatan sulfate), the primary influence on the viability of fibroblasts was attributed to the source of type I collagen (rat tail, human, or bovine) used as scaffold. Furthermore, incorporation of dermatan sulfate in the dermal layer led to a reduction in the contraction of tissues compared to the control where the dermal scaffold was composed primarily of collagen type I. This work highlights the influence of the composition of biomaterials on the development of complex reconstructed skin models that are suitable for clinical translation and in vitro safety assessment.

Visualization and identification of single meteoritic organic molecules by atomic force microscopy.

Using high-resolution atomic force microscopy (AFM) with CO-functionalized tips, we atomically resolved individual molecules from Murchison meteorite samples. We analyzed powdered Murchison meteorite material directly, as well as processed extracts that we prepared to facilitate characterization by AFM. From the untreated Murchison sample, we resolved very few molecules, as the sample contained mostly small molecules that could not be identified by AFM. By contrast, using a procedure based on several trituration and extraction steps with organic solvents, we isolated a fraction enriched in larger organic compounds. The treatment increased the fraction of molecules that could be resolved by AFM, allowing us to identify organic constituents and molecular moieties, such as polycyclic aromatic hydrocarbons and aliphatic chains. The AFM measurements are complemented by high-resolution mass spectrometry analysis of Murchison fractions. We provide a proof of principle that AFM can be used to image and identify individual organic molecules from meteorites and propose a method for extracting and preparing meteorite samples for their investigation by AFM. We discuss the challenges and prospects of this approach to study extraterrestrial samples based on single-molecule identification.

The ROC skin model: A robust skin equivalent for permeation and live cell imaging studies.

Rat epidermal keratinocyte (REK) Organotypic culture (ROC) is an epidermis model that is robust and inexpensive to develop and maintain, and it has in previous studies been shown to have permeability characteristics close to those of human skin. Here, we characterize the model further by structural comparison to native human and rat skin and by investigating functional characteristics of lipid packing, polarity, and permeability coefficients. We show that the ROC model has structural similarities to native human skin and observe human skin-like permeability coefficients for testosterone and mannitol. We develop a transwell device that allows live cell microscopy of the tissue at the air-liquid interface and establish transgenic cell lines expressing different fluorescently tagged proteins. This enables showing the migration of keratinocytes during the first days after seeding, finding that keratinocytes have a higher mean migration rate in the earlier days of development. Collectively, our results show that the ROC model is an inexpensive and robust epidermis model that works reproducibly across laboratories.

Comparative genomic analyses of multiple backcross mouse populations suggest SGCG as a novel potential obesity-modifier gene.

To nominate novel disease genes for obesity and type 2 diabetes (T2D), we recently generated two mouse backcross populations of the T2D-susceptible New Zealand Obese (NZO/HI) mouse strain and two genetically different, lean and T2D-resistant strains, 129P2/OlaHsd and C3HeB/FeJ. Comparative linkage analysis of our two female backcross populations identified seven novel body fat-associated quantitative trait loci (QTL). Only the locus Nbw14 (NZO body weight on chromosome 14) showed linkage to obesity-related traits in both backcross populations, indicating that the causal gene variant is likely specific for the NZO strain as NZO allele carriers in both crosses displayed elevated body weight and fat mass. To identify candidate genes for Nbw14, we used a combined approach of gene expression and haplotype analysis to filter for NZO-specific gene variants in gonadal white adipose tissue, defined as the main QTL-target tissue. Only two genes, Arl11 and Sgcg, fulfilled our candidate criteria. In addition, expression QTL analysis revealed cis-signals for both genes within the Nbw14 locus. Moreover, retroviral overexpression of Sgcg in 3T3-L1 adipocytes resulted in increased insulin-stimulated glucose uptake. In humans, mRNA levels of SGCG correlated with body mass index and body fat mass exclusively in diabetic subjects, suggesting that SGCG may present a novel marker for metabolically unhealthy obesity. In conclusion, our comparative-cross analysis could substantially improve the mapping resolution of the obesity locus Nbw14. Future studies will throw light on the mechanism by which Sgcg may protect from the development of obesity.

Nudix hydrolase NUDT19 regulates mitochondrial function and ATP production in murine hepatocytes.

Changes in intracellular CoA levels are known to contribute to the development of non-alcoholic fatty liver disease (NAFLD) in type 2 diabetes (T2D) in human and rodents. However, the underlying genetic basis is still poorly understood. Due to their diverse susceptibility towards metabolic diseases, mouse inbred strains have been proven to serve as powerful tools for the identification of novel genetic factors that underlie the pathophysiology of NAFLD and diabetes. Transcriptome analysis of mouse liver samples revealed the nucleoside diphosphate linked moiety X-type motif Nudt19 as novel candidate gene responsible for NAFLD and T2D development. Knockdown (KD) of Nudt19 increased mitochondrial and glycolytic ATP production rates in Hepa 1-6 cells by 41% and 10%, respectively. The enforced utilization of glutamine or fatty acids as energy substrate reduced uncoupled respiration by 41% and 47%, respectively, in non-target (NT) siRNA transfected cells. This reduction was prevented upon Nudt19 KD. Furthermore, incubation with palmitate or oleate respectively increased mitochondrial ATP production by 31% and 20%, and uncoupled respiration by 23% and 30% in Nudt19 KD cells, but not in NT cells. The enhanced fatty acid oxidation in Nudt19 KD cells was accompanied by a 1.3-fold increased abundance of Pdk4. This study is the first to describe Nudt19 as regulator of hepatic lipid metabolism and potential mediator of NAFLD and T2D development.

Nonbenzenoid High-Spin Polycyclic Hydrocarbons Generated by Atom Manipulation.

We report the on-surface synthesis of a nonbenzenoid triradical through dehydrogenation of truxene (C27H18) on coinage metal and insulator surfaces. Voltage pulses applied via the tip of a combined scanning tunneling microscope/atomic force microscope were used to cleave individual C-H bonds in truxene. The resultant final product truxene-5,10,15-triyl (1) was characterized at the single-molecule scale using a combination of atomic force microscopy, scanning tunneling microscopy, and scanning tunneling spectroscopy. Our analyses show that 1 retains its open-shell quartet ground state, predicted by density functional theory, on a two monolayer-thick NaCl layer on a Cu(111) surface. We image the frontier orbital densities of 1 and confirm that they correspond to spin-split singly occupied molecular orbitals. Through our synthetic strategy, we also isolate two reactive intermediates toward the synthesis of 1, derivatives of fluorenyl radical and indeno[1,2-a]fluorene, with predicted open-shell doublet and triplet ground states, respectively. Our results should have bearings on the synthesis of nonbenzenoid high-spin polycyclic frameworks with magnetism beyond Lieb's theorem.

π-Diradical Aromatic Soot Precursors in Flames.

Soot emitted from incomplete combustion of hydrocarbon fuels contributes to global warming and causes human disease. The mechanism by which soot nanoparticles form within hydrocarbon flames is still an unsolved problem in combustion science. Mechanisms proposed to date involving purely chemical growth are limited by slow reaction rates, whereas mechanisms relying on solely physical interactions between molecules are limited by weak intermolecular interactions that are unstable at flame temperatures. Here, we show evidence for a reactive π-diradical aromatic soot precursor imaged using non-contact atomic force microscopy. Localization of π-electrons on non-hexagonal rings was found to allow for Kekulé aromatic soot precursors to possess a triplet diradical ground state. Barrierless chain reactions are shown between these reactive sites, which provide thermally stable aromatic rim-linked hydrocarbons under flame conditions. Quantum molecular dynamics simulations demonstrate physical condensation of aromatics that survive for tens of picoseconds. Bound internal rotors then enable the reactive sites to find each other and become chemically cross-linked before dissociation. These species provide a rapid, thermally stable chain reaction toward soot nanoparticle formation and could provide molecular targets for limiting the emission of these toxic combustion products.

Recyclable Multilayer Packaging by Means of Thermoreversibly Crosslinking Adhesive in the Context of Food Law.

Lacking recyclability of multilayer packaging can be overcome by using a thermoreversible crosslinking adhesive consisting of maleimide- and furan-functionalized polyurethane-(PU-)prepolymers, reacting in a Diels-Alder-reaction. Here, the furan-functionalized PU-prepolymer carries furan-side-chains to avoid the usage of an additional crosslinking agent. Thus, N­(2­hydroxyethyl)maleimide and furfurylamine are the only two chemicals contained in the adhesive that are not listed in the appendix of EU Regulation 10/2011. Using migration modelling, it could be shown that, at 23 °C, both chemicals have lag-times of only a few minutes if 45 µm PE is used as a barrier. However, if the residual content is below 30 mg/kg, the legally specified maximum amount of 0.01 mg/kg food is not reached. After determining the diffusion coefficients and the activation energy of diffusion through ethylene-vinyl alcohol copolymer (EVOH), it could be determined that the lag-time of the migrants can be extended to at least 9 years by the use of 3 µm EVOH. From a food law point of view, the use of the described adhesive is possible if the above­mentioned measures are complied.

Synthesis of Cyclo[18]carbon via Debromination of C18Br6.

Cyclo[18]carbon (C18, a molecular carbon allotrope) can be synthesized by dehalogenation of a bromocyclocarbon precursor, C18Br6, in 64% yield, by atomic manipulation on a sodium chloride bilayer on Cu(111) at 5 K, and imaged by high-resolution atomic force microscopy. This method of generating C18 gives a higher yield than that reported previously from the cyclocarbon oxide C24O6. The experimental images of C18 were compared with simulated images for four theoretical model geometries, including possible bond-angle alternation: D18h cumulene, D9h polyyne, D9h cumulene, and C9h polyyne. Cumulenic structures, with (D9h) and without (D18h) bond-angle alternation, can be excluded. Polyynic structures, with (C9h) and without (D9h) bond-angle alternation, both show a good agreement with the experiment and are challenging to differentiate.

An sp-hybridized molecular carbon allotrope, cyclo[18]carbon.

Carbon allotropes built from rings of two-coordinate atoms, known as cyclo[n]carbons, have fascinated chemists for many years, but until now they could not be isolated or structurally characterized because of their high reactivity. We generated cyclo[18]carbon (C18) using atom manipulation on bilayer NaCl on Cu(111) at 5 kelvin by eliminating carbon monoxide from a cyclocarbon oxide molecule, C24O6 Characterization of cyclo[18]carbon by high-resolution atomic force microscopy revealed a polyynic structure with defined positions of alternating triple and single bonds. The high reactivity of cyclocarbon and cyclocarbon oxides allows covalent coupling between molecules to be induced by atom manipulation, opening an avenue for the synthesis of other carbon allotropes and carbon-rich materials from the coalescence of cyclocarbon molecules.

Elucidating the Geometric Substitution of Petroporphyrins by Spectroscopic Analysis and Atomic Force Microscopy Molecular Imaging.

Determination of the molecular structures of petroporphyrins has been crucial to understand the diagenetic pathways and maturation of petroleum. However, these studies have been hampered by their structural complexity and the challenges associated with their isolation. In comparison to the skeletal macrocyclic structures, much less is known about the substitutions, which are more sensitive to the maturation and diagenesis pathways. While these isolated vanadyl petroporphyrins largely consist of etioporphyrin and deoxophylloerythroetioporphyrin as expected, surprisingly, we find evidence that one or a few ß hydrogens are present in petroporphyrins of low carbon numbers using a combination of ultraviolet-visible spectroscopy, Fourier transform ion cyclotron resonance mass spectrometry, and non-contact atomic force microscopy. Petroporphyrins with ß hydrogens were not anticipated on the basis of their biological precursors. The data support dealkylation under catagenesis but not transalkylation or random alkylation of the ß and meso positions, despite the fact that more complex porphyrin structures are formed.

A Single-Molecule Chemical Reaction Studied by High-Resolution Atomic Force Microscopy and Scanning Tunneling Microscopy Induced Light Emission.

Atomic force microscopy (AFM) as well as scanning tunneling microscopy induced light emission (STM-LE) are, each on their own, powerful tools used to investigate a large variety of properties of single molecules adsorbed on a surface. However, accessing both structural information by AFM as well as optical information by STM-LE on the same molecule so far remains elusive. We present a combined high-resolution AFM and STM-LE study on single metal-oxide phthalocyanines. Using atomic manipulation, the molecules can be deliberately reduced. We demonstrate structure elucidation and adsorption geometry determination of single molecules with atomic resolution combined with optical characterization by STM-LE and the possibility of investigating the change in a molecule's exciton emission intensity by a chemical reaction.